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Current developments in SELDI Affinity technology
Abstract
The overall history and recent advancements in Surface-Enhanced Laser Desorption/Ionization (SELDI) affinity technology is reviewed. A detailed account of SELDI technology, utilizing Immobilized-Metal Affinity surfaces, pseudo-specific chromatographic surfaces, and biospecific interactive surfaces, is presented with particular emphasis placed upon examination of fundamental characteristics as well as specific applications for each. Finally, a detailed review of the specific use of such affinity surfaces in fundamental aspects of clinical, process, and research proteomics activity is presented.
... combines chromatography and mass spectrometry to provide a rapid protein expression profile (Issaq et al., 2002). It was originally described in 1993 by Hutchens and Yip and since then has undergone remarkable progress as an analytical technique (Tang et al., 2004). Proteins are applied to an array surface, which has been chemically or (Tang et al., 2004). ...
... It was originally described in 1993 by Hutchens and Yip and since then has undergone remarkable progress as an analytical technique (Tang et al., 2004). Proteins are applied to an array surface, which has been chemically or (Tang et al., 2004). Also, the mass spectrum should contain no array-mediated noise. ...
... in the production o f recombinant proteins from bench-top to high-volume scales (Tang et al., 2004). Studies have been carried out using SELDI as a method to identify biomarkers in urine samples (Vlahou et al., 2001;Dare et al., 2002). ...
The aim of this study was to identify novel, sensitive, and specific protein markers of hepatotoxicity in rat urine. Collection of urine is non-invasive compared to biopsy or serum analysis and therefore preferable when screening for toxicity. Carbon tetrachloride (CCI4) was used both acutely to produce hepatotoxicity and chronically to produce a rat model of liver fibrosis. An optimal dose of CCl4, and the time post-dosing for maximum acute liver injury, were established by histopathological examination and by assaying serum enzyme markers of liver injury. Urine was analysed using Surface Enhanced Laser Desorption/Ionisation (SELDI) ProteinChip® technology. SELDI revealed the appearance of a protein peak at 15.7 KDa in response to CCU-treatment, while one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) identified an 18.4 KDa protein in urine from CCl4-treated rats. This protein was identified by in-gel digestion and tandem mass spectrometry as Cu/Zn superoxide dismutase (SOD-1). SOD-1 catalyses the destruction of the superoxide anion and acts as a defence against oxidative damage. SOD-1 exists as a 32.5 KDa homodimer and it was concluded that the 15.7 KDa SELDI and 18.4 KDa SDS PAGE proteins are the SOD-1 subunit that runs at an anomalous MW on SDS PAGE. SOD-1 in the rat urine was confirmed by Western blotting with a commercial antibody and by measuring SOD activity. Further studies revealed that SOD-1 was increased in urine from CCU-treated rats between 12 hours and 60 hours post-dosing, at dose levels as low as 0.4 ml/kg CCI4. Western blots of homogenates showed that SOD-1 was being lost from rat liver presumably by necrosis. Although the enzyme SOD-1 is not specifically located in the liver, its appearance in the rat urine following hepatotoxicity is a novel finding. Since changes in SOD-1 levels were detected following low dose levels of CCI4 and the response was measured using non-invasive methods suggests that SOD-1 is thus a potential marker of hepatotoxicity. Liver fibrosis was induced by repeat dosing with CCI4 and confirmed by histopathological examination. Analysis of the urine samples by SDS-PAGE revealed an increase in SOD-1 in fibrotic rats but no other differences were evident. Examination of urine samples from rats with fibrosis, or acute hepatotoxicity, by two-dimensional gel electrophoresis revealed a number of proteins that were increased in these models. Two-dimensional gel electrophoresis of liver homogenates from the acute model showed a number of proteins that decreased. These results suggest that although the urinary proteins have not yet been identified it is highly probable that one or more will be a specific marker for the non-invasive identification of hepatotoxicity.
... SELDI technique eliminates the use of conventional organic matrices and utilizes an active probe for direct extraction, purification, enrichment, desorption and ionization of the targeted analyte, thereby reducing the time required for sample pretreatment. Furthermore, a SELDI probe can be manipulated and functionalized to enhance its specificity and selectivity for different applications [10]. ...
... Thus, the high absorption efficiency of the magnetic nanocomposite in the UV-Vis region leads to its potential applicability in SELDI analysis. The surface capacity of the probe plays an important role in the adsorption of the analyte, and a large surface area provides a high loading capacity of the analyte molecules, thereby improving the SELDI performance [10]. Therefore, to validate the surface properties of the magnetic nanocomposites, N 2 adsorption-desorption was employed, as shown in Fig. 2d. ...
... Of these properties, the scaffold chemistry of the substrate can promote different binding interactions with the analyte, including hydrophobic, van der Waals, hydrogen and electrostatic interactions. Furthermore, the surface capacity of the probe plays a vital role in SELDI analysis, and thus, a probe with a high surface capacity can explicitly bind to a large number of analytes; meanwhile, the functional groups present on the surface of the probe can stimulate selective and specific binding interactions with the analyte, thereby enhancing the extraction efficiency of the probe [10]. ...
The use of carbon black-Fe3O4 magnetic nanocomposite (CB-Fe3O4) as a probe for surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS) with a high extraction efficiency and sensitive detection is described. The magnetic nanocomposite was synthesized and fully characterized using X-ray photoelectron spectroscopy, X-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy, Ultraviolet-Visible spectroscopy, transmission electron microscopy and nitrogen sorption. The feasibility of the SELDI probe to extract and detect three classes of drugs (labetalol, metoprolol, doxepin, desipramine, triprolidine and methapyrilene) spiked in wine is demonstrated. All the drugs were successfully and reproducibly extracted and detected with high efficiency and with limits of detection (LOD) between 1 and 1000 pg mL⁻¹. The adsorption capacity of the nanocomposite for the drugs was evaluated by UV-Vis spectroscopy. The results showed that 27.8–36.1% of the drugs were adsorbed on the magnetic probe within 3 min. The nanocomposite was also applied for efficient analysis of amino acids and fatty acids. Both types of analytes can be extracted within a few minutes and then successfully quantified by SELDI-MS.
Graphical abstractA schematic presentation of carbon black-Fe3O4 magnetic probe for SELDI analysis of small molecules. The probe containing the analyte(s) is collected with the aid of a magnet and deposited on the target plate for mass spectrometry analysis.
... In general, EC is diagnosed in postmenopausal women (85% of cases) and its incidence is rising due to longer life expectancy and life style associated risk factors. Women with BMI above 35 have an odds ratio of 5.7 for developing EC, with an increase of 1.1 odds ratio per BMI unit (2, 3). Exposure to unopposed estrogens or tamoxifen or genetic aberrations associated with Lynch syndrome confer a cumulative risk up to 70% (4,5). ...
... ESI allows the creation of ions in solution, while in MALDI, ions are created by laser light pulsing on matrix embedded proteins. A variation of MALDI is SELDI (surface-enhanced laser desorption/ionization), where the proteins are applied on a modified matrix surface allowing binding of specific proteins or proteins classes (35). Mass analysis of proteins is primarily conducted using TOF (time-of-flight) or quadrupoles. ...
Endometrial cancer is the most common gynaecological malignancy in developed countries. Over 382,000 new cases were diagnosed worldwide in 2018, and its incidence and mortality are constantly rising due to longer life expectancy and life style factors including obesity. Two major improvements are needed in the management of patients with endometrial cancer, i.e., the development of non/minimally invasive tools for diagnostics and prognostics, which are currently missing. Diagnostic tools are needed to manage the increasing number of women at risk of developing the disease. Prognostic tools are necessary to stratify patients according to their risk of recurrence pre-preoperatively, to advise and plan the most appropriate treatment and avoid over/under-treatment. Biomarkers derived from proteomics and metabolomics, especially when derived from non/minimally-invasively collected body fluids, can serve to develop such prognostic and diagnostic tools, and the purpose of the present review is to explore the current research in this topic. We first provide a brief description of the technologies, the computational pipelines for data analyses and then we provide a systematic review of all published studies using proteomics and/or metabolomics for diagnostic and prognostic biomarker discovery in endometrial cancer. Finally, conclusions and recommendations for future studies are also given.
... Surface-enhanced laser desorption/ ionisation (SELDI) is another technique which is a relative variation of the MALDI technique allowing selection of proteins from crude mixtures before ionisation. Proteins are applied to a solid phase surface aimed to select for proteins according to their chemical properties (Tang et al. 2004). Bound proteins are then co-crystallised with the matrix and ionised utilising the MALDI technique. ...
... The mass analyser is, in large part, responsible for the performance of the mass spectrometer and the quality of the data output. The most commonly used mass analysers in proteomics are time-of-flight (TOF), quadrupole (q or Q) and ion traps (Tang et al. 2004). ...
Background: Pre-eclampsia (P-EC) is a major cause of maternal and neonatal mortality and morbidity. Alterations in the maternal vasculature and coagulation profile may predispose women with P-EC to subsequent deleterious cardiovascular consequences. Indeed women with a history of P-EC are known to have heightened risks of future cardiovascular disease and thromboembolic complications.Aims: To assess the relationship between circulating haemostatic factors and inflammatory cytokines in women with a previous history of P-EC.To investigate the relationship between haemostatic, angiogenic and anti-angiogenic factors in women with a past history of P-EC.To assess the plasma levels of Annexins A2 and A5 in pre-eclamptic women postpartum at different time intervals.To identify clusters of differentially expressed plasma proteins in P-EC associated with the susceptibility to developing future cardiovascular diseases.To examine miRNAs expression in P-EC post-delivery at different intervals time points.Study-I: 26 pre-eclamptic women and 14 age-matched to healthy women. Women were included within six months to 3 years post-delivery. Plasma TF, IL-6, IL-8 and IL-10 levels increased in the P-EC group, whereas plasma TFPI and TNF-α levels were reduced. Plasma TF/TFPI ratios and IL-10 values were significantly increased in the P-EC group (p<0.05, p<0.01, respectively). There were positive and significant correlations between TFPI(r= 0.5; p<0.01) and IL-10 and TF/TFPI ratio and IL-10 (r= 0.31; p<0.041).Study-II: 21 primiparous women after a pregnancy affected by P-EC and 21 women with a previously unaffected pregnancy. Blood samples were obtained at 6-12 months postpartum. Significant differences were not observed for VEGF, PlGF, sFlt-1, sEng, TF or TFPI between two groups.Study-III: 66 women who had P-EC at interval years starting from 2007, and then from 2012 till 2016 and five as a control group. Findings revealed that the level of ANXA5 was reduced in P-EC cohort, and there was an increase in levels of annexin A2, particularly in late post-delivery pre-eclamptic women, although these changes were statistically insignificant. The lack of statistical significance may be due to the small number of control compare to P-EC group.Study-IV: 5 women aged-matched with five women with normal pregnancy, results show that inflammation, immune response, blood coagulation and metabolism are dysregulated processes one-year post-delivery in women with a history of pre-eclampsia.Study-Study-V: Next-Generation Sequencing (NGS) technique was used during the discovery stage to identify miRNAs differentially expressed in P-EC postpartum (n=30) in comparison to five healthy control, then quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay used for confirmation and validation. 14 miRNAs that were significantly differentially expressed at a significance level of 0.05 (FDR) in the discovery experiment. The plasma miR-103a-3p were significantly differentially expressed in the validating experiment, which was downregulated in entire pre-eclamptic women with FC= -1.3; BH-adj P-value =0.033.Conclusions: Results suggest the presence of elevated inflammatory cytokines and an imbalance of the haemostatic system in women with a past-history of P-EC, which may contribute to the known increased risk of cardiovascular disease later in life. The proteomics findings provide insight into the dysregulated cardiometabolic profile in the P-EC group. The miRNAs novel marker pave the way for the importance of miR-103a-3p in monitoring P-EC women in the future and could be a useful tool in predicting the development of cardiovascular disease in P-EC.
... A special type of selective enrichment of the sample through adsorption of a specific group of substances based on nanomaterials is surface-enhanced laser desorption/ionization (SELDI). Surface-modified SELDI tiles are in different varieties, as substrates using hydrophobic interactions (e.g., C18, C4), hydrophilic, ionic, atomic (e.g., thiol), and antigen-antibody interactions that interact with proteins, nucleic acids, or other molecules with strictly defined physicochemical properties [86] (Figure 3). modified SELDI tiles are in different varieties, as substrates using hydrophobic interactions (e.g., C18, C4), hydrophilic, ionic, atomic (e.g., thiol), and antigen-antibody interactions that interact with proteins, nucleic acids, or other molecules with strictly defined physicochemical properties [86] ( Figure 3). ...
... Surface-modified SELDI tiles are in different varieties, as substrates using hydrophobic interactions (e.g., C18, C4), hydrophilic, ionic, atomic (e.g., thiol), and antigen-antibody interactions that interact with proteins, nucleic acids, or other molecules with strictly defined physicochemical properties [86] (Figure 3). modified SELDI tiles are in different varieties, as substrates using hydrophobic interactions (e.g., C18, C4), hydrophilic, ionic, atomic (e.g., thiol), and antigen-antibody interactions that interact with proteins, nucleic acids, or other molecules with strictly defined physicochemical properties [86] ( Figure 3). SELDI is a technique that allows for rapid identification of various cancer biomarkers (e.g., prostate [87], pancreas [88], epithelial ovarian cancer [89], melanoma [90]) in complex biological matrices, such as saliva [91], serum [92], urine [93], or cerebrospinal fluid [94]. ...
In recent years, matrix-assisted laser desorption/ionization (MALDI) has become the main tool for the study of biological macromolecules, such as protein nano-machines, especially in the determination of their molecular masses, structure, and post-translational modifications. A key role in the classical process of desorption and ionization of the sample is played by a matrix, usually a low-molecular weight weak organic acid. Unfortunately, the interpretation of mass spectra in the mass range of below m/z 500 is difficult, and hence the analysis of low molecular weight compounds in a matrix-assisted system is an analytical challenge. Replacing the classical matrix with nanomaterials, e.g., silver nanoparticles, allows improvement of the selectivity and sensitivity of spectrometric measurement of biologically important small molecules. Nowadays, the nanostructure-assisted laser desorption/ionization (NALDI) approach complements the classic MALDI in the field of modern bioanalytics. In particular, the aim of this work is to review the recent advances in MALDI and NALDI approaches.
... Prior to MS analysis, SELDI has the benefit of being able to quickly and efficiently separate complicated protein mixtures with minimal sample modification. This is achieved by selecting proteins for examination while they are still intact and have not been digested, utilising chromatographic chip surface technology (ProteinChipR) (Ellington and Szostak 1990;Tuerk and Gold 1990;Shevchenko et al. 1996;Merchant and Weinberger 2000;Huber 2003;Tang et al. 2004;Stasyk and Huber 2004;Aslam et al. 2017). ...
The main role for cancer disease treatment is to take care of mortality and morbidity rate by implementing all sorts of available options for health benefits of all patients with cancer including chemotherapy and or radiotherapy. But main obstacles are resistance and cancer patients’ compliance towards to these mentioned therapies depending upon altered cellular pathways. The type of therapy implemented for the cancer is majorly governed through typical biomarkers with its respected mutated genes. The proteomic approach is a useful technique for determining the reasons why anticancer drug resistance emerges. The proteomics deals with analysis of whole proteome simultaneously and involved with certain approaches which help physician to identify particular biomarkers utilized for clinical response evaluation during the cancer treatment. The tissue biomarker and serum biomarker analysis are one of main key step in selection of cancer treatment and proteomics are implemented for the same. Proteomics are also helpful for analysis of protein expression and given exploration of activity for signaling pathway. Proteomics are also used to identify illness signs in the body. But there are still need to explore more detail perfection into sample preparation before analysis to get accurate results. The involvement of artificial learning methods for generation of data for the expression of protein along with proteomics can give new horizons for treatment selection and fruitful options to patient for the same.
... It was developed in 1995 and has found application in the analysis of protein mixtures or proteins found in tissue, blood, urine, or other clinical samples. [123] In this method, the sample is applied to a modified surface, which allows specific binding of proteins from the sample to the surface. The binding acts as a solid phase chromatographic separation step, resulting in easier protein analysis. ...
In modern medical diagnostics, where analytical chemistry plays a key role, fast and accurate identification of pathogens is becoming increasingly important. Infectious diseases pose a growing threat to public health due to population growth, international air travel, bacterial resistance to antibiotics, and other factors. For instance, the detection of SARS-CoV-2 in patient samples is a key tool to monitor the spread of the disease. While there are several techniques for identifying
pathogens by their genetic code, most of these methods are too expensive or slow to effectively analyze clinical and environmental samples that may contain hundreds or even thousands of different microbes. Standard approaches (e.g., culture media and biochemical assays) are known to be very time- and labor-intensive. The purpose of this review paper is to highlight the problems associated with the analysis and identification of pathogens that cause many serious infections.
Special attention was paid to the description of mechanisms and the explanation of the phenomena and processes occurring on the surface of pathogens as biocolloids (charge distribution). This review also highlights the importance of electromigration techniques and demonstrates their potential for pathogen pre-separation and fractionation and demonstrates the use of spectrometric methods, such as MALDI-TOF MS, for their detection and identification.
... ProteinChip array® (Bio-Rad Laboratories, Inc., CA, USA) has been successfully applied to diagnose various malignancies, such as ovarian cancer and gastric cancer [52,53]. As shown in Figure 1B, ProteinChip array® is a convenient separation tool that utilizes chromatographic properties and its surfaces can be modified with various functional groups (hydrophobic, cationic, anionic, metal ion presenting and hydrophilic). ...
... And because of its high selectivity as well as high sensitivity, LC-MS/MS can detect very low amounts of biological substances. SELDI-TOF/MS (Tang et al., 2004;Li et al., 2002) involves the use of a chromatographic surface which is bound to a specific part of the desired protein, and the rest will be separated. In this technique, various chips are used to segregate proteins from the sample. ...
Plant-microbe relation has been an area of intensive research in plant science. It has far-reaching impacts, be it the plant pathogen interplay or the symbiotic relationships between plants and symbionts. A gene or gene product-centric approach has been used traditionally in the study of such relationships, which is time-consuming and less amenable for scaling up. Advances in modern molecular tools allow the comprehensive and all-encompassing analysis of the molecules present inside cells or tissue at a particular time and under a set of conditions. Such tools include genomics for the whole DNA analysis. It provides information about the potential of an organism. The transcriptome for the RNA analysis and proteome for the protein provide a snapshot of the informational molecules at work. Different gene(s) and their products involved in signaling and other pathways have been identified using this approach. Metabolomics deals with metabolite analysis, which is the ultimate product and representative of the current metabolic state. It often provides a lead into the effect of a change in levels of expression of a particular pathway. Integrating data generated from these is another emerging strategy to use data from all these “omics” tools. This book chapter aims to summarize the current technologies being used and highlights recent studies done using these tools.
... The obtained N 2 isotherm can be classified as type IV and the hysteresis loop can be assigned to H1/H2, indicating the mesoporous nature of the NPs [34]. The surface capacity of the MCNPs was investigated as high surface capacity of the substrate offers high loading number of the analytes molecules hence increasing the detection sensitivity [35]. The results revealed that the surface area of the prepared MCNPs was 129.2 m 2 g − 1 with pore volume and pore diameter of 0.32 cm 3 g − 1 and 10.01 nm, respectively. ...
For decades, the analysis of fingerprints (FP) has been used as the primary biometric mean of human identification. In parallel, the chemical analysis of latent fingermark (LFM) with particular reference to “touch chemistry”, offers additional intelligence to forensic examination; as such, continuous improvements in the versatility and sensitivity of detection of the molecular make up of FM is of obvious importance. In this light, we propose a facile approach to synthesize magnetic carbon nanoparticles (MCNPs) from candle soot for extraction and detection of endogenous and exogenous FM components. Initially, endogenous components of FM including fatty acids (FAs), squalene and triacylgycerols (TAGs), were successfully extracted and detected using the developed MCNPs and surface assisted laser desorption/ionization-mass spectrometry (SALDI-MS). Furthermore, the MCNPs enabled the detection of exogenous substances including antihistamine, β-blocker, antibiotic drugs and lead in contaminated FMs, whilst providing characteristic and unique fragmentation patterns for each drugs in the FM. The influence of environmental factors such as temperature on the stability of the exogenous substances in FM was investigated by exposing the contaminated FM to different temperatures over 24 hours, the findings revealed the drugs' instability at high temperatures and undergo different degrees of degradation whereas lead was more resilient to thermal stress. The detection of triprolidine, metoprolol and sulfamethoxazole from pharmaceutical tablets in FM was successfully achieved by gently touching the tablet powder. The limit of detection (LOD) values of the drugs in the tablet forms were 50, 200 and 750 ng mL⁻¹ for triprolidine, metoprolol and sulfamethoxazole, respectively and their recovery rates were 91.17% for triprolidine, 94.67% for metoprolol and 120.86% for sulfamethoxazole. Finally, to create a genuine casework scenario, contaminated FM was deposited on glass and metal surfaces then subjected to extraction using MCNPs and magnet without compromising the FM impression. Substrate control experiments revealed that the glass surface exhibiting some background signal, however, they did not interfere with the analysis and satisfactory extraction efficiency of endogenous and exogenous components of the FM on both surfaces was obtained using the MCNPs. Overall, this study proves the capability of MCNPs as new SALDI-MS substrate for both extraction and detection of FM components gathering more information pertaining to the donor’s lifestyle.
... The advanced computational techniques have also been used in other experimental studies performed on MM patients with innovative techniques. The introduction of surfaceenhanced laser desorption/ionization time-offlight mass spectrometry (SELDI-TOF-MS) has offered the tools to evaluate a wide range of proteins in patient samples [32,51,52]. This technique, in association with informatics data evaluation, is extremely advantageous in several medical conditions, such as in the study of the MBD in MM patients. ...
Simple Summary
Multiple myeloma is a malignant neoplasm of plasma cells with complex pathogenesis. With major progresses in multiple myeloma research, it is essential that we reconsider our methods for diagnosing and monitoring multiple myeloma disease. This fact needs the integration of serology, histology, radiology, and genetic data; therefore, multiple myeloma study has generated massive quantities of granular high-dimensional data exceeding human understanding. With improved computational techniques, artificial intelligence tools for data processing and analysis are becoming more and more relevant. Artificial intelligence represents a wide set of algorithms for which machine learning and deep learning are presently among the most impactful. This review focuses on artificial intelligence applications in multiple myeloma research, first illustrating machine learning and deep learning procedures and workflow, followed by how these algorithms are used for multiple myeloma diagnosis, prognosis, bone lesions identification, and evaluation of response to the treatment.
Abstract
Artificial intelligence has recently modified the panorama of oncology investigation thanks to the use of machine learning algorithms and deep learning strategies. Machine learning is a branch of artificial intelligence that involves algorithms that analyse information, learn from that information, and then employ their discoveries to make abreast choice, while deep learning is a field of machine learning basically represented by algorithms inspired by the organization and function of the brain, named artificial neural networks. In this review, we examine the possibility of the artificial intelligence applications in multiple myeloma evaluation, and we report the most significant experimentations with respect to the machine and deep learning procedures in the relevant field. Multiple myeloma is one of the most common haematological malignancies in the world, and among them, it is one of the most difficult ones to cure due to the high occurrence of relapse and chemoresistance. Machine learning- and deep learning-based studies are expected to be among the future strategies to challenge this negative-prognosis tumour via the detection of new markers for their prompt discovery and therapy selection and by a better evaluation of its relapse and survival.
... In 2004, ANN was introduced to detect the tumor in CRC [44]. 182 samples were detected by using Surface-Enhanced Laser Desorption/Ionization Mass Spectrometry (SELDI-MS) [45]. These samples include 55 samples from CRC patients, 35 samples from ColoRectal Adenoma (CRA) patients, and 92 samples from Healthy Persons (HP). ...
Human cell is a complex of interacting small molecules which work together to perform daily tasks of the cell. The reading and the measurements of this different molecules are called omics, where any dysfunction among these omics may cause different diseases, and cancer is not any exception. The advances in biomedical technology in general and in software development help to speed up, and reduce the cost of reading these omics. However, the challenge is how to integrate the big amount of data from these omics to help the predictions systems in cancer outcomes, where these outcomes include the diagnosis, development, and treatment of cancer.
... One solution to this issue is to incorporate on-surface analyte enrichment and cleanup directly on LDI surfaces. 8,16 Methods developed include surface modifications or affinity capture by various chemistries, followed by on-surface sample cleanup, and they have been applied to a number of MS surfaces, including DIOS, 17 SELDI, 18 SAMDI, 19 and NIMS. 20 On-surface capture chemistries that have been utilized include hydrophobic interactions, antibody-antigen, cysteine-maleimide, His 6 -Ni 2+ , biotin-streptavidin, and click chemistry. ...
High-throughput matrix-assisted laser desorption/ionization mass spectrometry (HT-MALDI-MS) has garnered considerable attention within the drug discovery industry as an information-rich alternative to assays using light-based detection methods. To date, these efforts have been primarily focused on assays using protein or peptide substrates. Methods for RNA or DNA analysis by HT-MALDI-MS have not been extensively reported due to the challenges associated with MALDI-MS of oligonucleotides, including the propensity to form multiple salt adducts, low ionization potential, and ease of fragmentation. The objective of this work was to develop a platform suitable for HT-MS analysis of RNA and DNA substrates that overcomes these hurdles by combining on-surface sample preparation with soft ionization. This has been accomplished through the selective immobilization of fluorous-tagged oligonucleotides on a fluorous-modified MS target plate, followed by on-surface enrichment, matrix addition, and direct laser desorption/ionization, a process dubbed fluorous HT-MS (F-HT-MS). The work has resulted in methods by which RNA and DNA substrates can be detected at nanomolar concentrations from a typical assay buffer system using procedures that are amenable to full automation. The protocols were applied to an miRNA biogenesis assay, demonstrating its potential for RNA processes and thereby filling a prominent gap in RNA drug discovery: the paucity of in vitro functional assays.
... Surface Enhanced Laser Desorption and Ionization Mass Spectrometry (SELDI MS) is a proteomic technique using chromatographic surfaces to retain proteins depending on their physicochemical properties, followed by direct analysis via TOF MS. [64] Several studies applying this technology have been carried out to analyze the protein profiles of biological fluids, especially serum samples [65,66] in order to find biomarkers. [66,67] Because this technique demands no more than a minute sample amount it is fit for the analysis of microdissected tissues. ...
The problem with cancer tissue is that its intratumoral heterogeneity and its complexity is extremely high as cells possess, depending on their location and function, different mutations, different mRNA expression and the highest intricacy in the protein pattern. Prior to genomic and proteomic analyses, it is therefore indispensable to identify the exact part of the tissue or even the exact cell. Laser‐based microdissection is a tried and tested technique able to produce pure and well‐defined cell material for further analysis with proteomic and genomic techniques. It sheds light on the heterogeneity of cancer or other complex diseases and enables the identification of biomarkers. This review aims to raise awareness for the reconsideration of laser‐based microdissection and seeks to present current state‐of‐the‐art combinations with omic techniques.
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... Mass-spectrometry is an umbrella term that describes a wide array of methods that ionize analytes and then detect and analyze ions in the gas phase. Different ionization methods (electrospray ionization (ESI) [258] or matrix-assisted laser desorption/ionization (MALDI) [259], surface-enhanced laser desorption/ ionization (SELDI) [259,260], etc.) can be coupled with different analyzers (quadrupoles, time-of-flight, orbitrap analyzers, etc.) to achieve different analytical characteristics [254]. Furthermore, MS instruments can be combined in tandem (MS/MS) to afford structural information on analyzed ions. ...
Precision oncology aims to tailor clinical decisions specifically to patients with the objective of improving treatment outcomes. This can be achieved by leveraging omics information for accurate molecular characterization of tumors. Tumor tissue biopsies are currently the main source of information for molecular profiling. However, biopsies are invasive and limited in resolving spatiotemporal heterogeneity in tumor tissues. Alternative non-invasive liquid biopsies can exploit patient’s body fluids to access multiple layers of tumor-specific biological information (genomes, epigenomes, transcriptomes, proteomes, metabolomes, circulating tumor cells, and exosomes). Analysis and integration of these large and diverse datasets using statistical and machine learning approaches can yield important insights into tumor biology and lead to discovery of new diagnostic, predictive, and prognostic biomarkers. Translation of these new diagnostic tools into standard clinical practice could transform oncology, as demonstrated by a number of liquid biopsy assays already entering clinical use. In this review, we highlight successes and challenges facing the rapidly evolving field of cancer biomarker research.
Lay Summary
Precision oncology aims to tailor clinical decisions specifically to patients with the objective of improving treatment outcomes. The discovery of biomarkers for precision oncology has been accelerated by high-throughput experimental and computational methods, which can inform fine-grained characterization of tumors for clinical decision-making. Moreover, advances in the liquid biopsy field allow non-invasive sampling of patient’s body fluids with the aim of analyzing circulating biomarkers, obviating the need for invasive tumor tissue biopsies. In this review, we highlight successes and challenges facing the rapidly evolving field of liquid biopsy cancer biomarker research.
... The principle is to characterize the analyte present in solution by retaining it on a surface derivatized with subsequent chemical probe, adding the matrix, drying and analyzing after laser desorption. The types of probes are as large as SAMs, dextran, antibody-and protein affinity probes and metal affinity or even hydrophobic interaction bio-probes [38]. When all these probes are arranged in surface arrays, it reduces considerably the time of analysis and contributes to the discovery of biomarkers of infection. ...
Adhesion of mammalian cells is mediated by a protein layer adsorbed from biological fluids or the extracellular matrix. For bacteria, this ‘conditioning film’ also exerts an influence even if the membrane receptors are not the same. In fact, in the early stage of adhesion, whatever the surface considered, cells and bacteria are somehow in contact with proteins. These assumptions shed light on the role of protein/surface interface in the early stage of cell or bacteria adhesion. In this article, we will first review some of the techniques currently used for the analysis of such a bio-interface from a surface science point of view. Then, we will focus on the possibility to chemically model such a bio-interface. To achieve this goal and simplify the numerous interactions between a protein and cells, the biomolecule can be probed at different scales: amino acid, peptide and protein as a whole. We will examine how the surface chemistry can help to graft these fragments, what are the strategies to graft a huge molecule on a surface and what are the relevant parameters to realize a biomimetic surface, taking into account the modifications undergone by the surface during the sterilization step.
... In this regard, the analyte of interest (usually proteins) is applied to a modified surface (chip) that plays an active role in the extraction, purification or modification of the analyte prior to desorption and ionization for MS analysis. A variety of materials can be used for this purpose, such as metals, cation/anion exchangers, hydrophobic compounds similar to C8 reverse phase chromatography and others [158].The samples spotted on the MALDI target or the SELDI surface are subjected further to MS analysis, typically using time-of-flight (TOF) mass analyzers, where the ions are separated based on their velocity differences. The m/z ratio is further determined by measuring the time needed for the formed ions to reach the detector. ...
Hepcidin-25, ein 2000 entdecktes Peptidhormon, das eine Schlüsselrolle im Eisenstoffwechsel spielt, hat das Verständnis von Eisenerkrankungen revolutioniert. In dieser Studie wurde LC gekoppelt mit einem Triple-Quadrupol MS in einer schnellen und robusten Methode zur Quantifizierung von Hepcidin-25 in menschlichem Serum verwendet, welche letztlich in Routine-Laboratorien genutzt werden soll. Zu diesem Zweck wurden zwei Probenvorbereitungsstrategien und zwei komplementäre LC Bedingungen untersucht, wobei eine saure mobile Phase (0,1% TFA) mit einem neuartigen Ansatz unter der Verwendung einer basischen mobilen Phase (0,1% NH3) verglichen wurde. In einem laborinternen Vergleich beider LC-MS/MS Methoden wurde Hepcidin-25 in humanen Proben unter Verwendung der gleichen Kalibrierstandards quantifiziert und eine sehr gute Korrelation der Ergebnisse ermittelt. Hierbei wurde die Analysestrategie mit saurer mobiler Phase als hochsensitiv (LOQ von 0,5 μg/L) und präzise (CV <15%) befunden und als Kandidat einer Referenzmethoden für die Hepcidin-25 Quantifizierung in realen Proben empfohlen. Einer der neuartigen Aspekte der Methodik war die Verwendung von Amino- und Fluor-silanisierten Autosampler-Fläschchen, um die Adsorption des 25 Reste umfassenden Peptids anOberflächen zu reduzieren. Darüber hinaus wurde diese LC-MS/MS-Methode in einer internationalen Ringversuchsstudie eingesetzt, bei der ein sekundäres Referenzmaterial als Kalibrierstandard verwendet wurde, und gemäß des International Consortium for Harmonization of Clinical Laboratory Results (ICHCLR) als optimal bewertet. In dieser Arbeit wurde die Bildung von Hepcidin-Komplexen mit Kupfer(II) untersucht. Die erste Umkehrphasen-chromatographische Trennung von Hepcidin-25/Cu(II) und Hepcidin-25 (Kupfer "frei") wurde unter Verwendung von mobilen Phasen mit 0,1% NH3 erreicht. LC-MS/MS und FTICR-MS wurden für die Charakterisierung der gebildeten Hepcidin-25-Cu(II)-Spezies bei pH-Werten von 11 bzw. 7,4 verwendet.
... Plasma proteomics holds immense potential to identify new markers [16]. The benefits of SELDI-TOF-MS include purification and isolation of small-molecular-weight proteins at high efficiency, stability, specificity and sensitivity [17,18]. Moreover, SELDI-TOF-MS is suitable for the semiquantification of small-molecularweight peptides and proteins, which could be potential diagnostic and prognostic markers that could facilitate the understanding of the pathogenesis of many diseases [19][20][21]. ...
Objective:
The isobaric tags for relative and absolute quantification (iTRAQ) technique for proteomic analysis was employed to identify diagnostic markers and therapeutic targets of Shenkangling intervention or prednisone tablets in rats with adriamycin nephropathy (AN).
Methods:
Fifty healthy, clean-grade Sprague-Dawley rats were selected, with 10 rats in the normal group and the remaining 40 rats receiving a tail vein injection of 5.5 mg/kg of adriamycin (ADR) to induce AN. Treatment began 1 week later. The normal group received gastric administration of normal saline. Forty rats with induced AN were further randomly divided into the AN modeling group (n = 10), AN modeling + prednisone treatment group (n = 10), AN modeling + Shenkangling intervention group (n = 10), and AN modeling + prednisone + Shenkangling intervention group (n = 10). iTRAQ was employed in combination with mass spectrometry to analyze the differentially expressed proteins in the urine after 3 weeks of treatment (in the fourth week of the experiment).
Results:
Compared with normal rats, AN rats had 6 down-regulated proteins and 1 upregulated protein. Compared with AN rats, prednisone rats had 2 down-regulated and 6 upregulated proteins. Compared with AN rats, combined treatment rats had 2 down-regulated and 8 upregulated proteins. Compared with the AN model group, the Shenkangling treatment group had 3 down-regulated and 9 upregulated proteins. Gro, Afamin, Cystatin-related protein 2, Afamin, and isoform CRA_a were considered diagnostic markers of primary nephrotic syndrome (PNS). Telomerase was considered the therapeutic target of prednisone. Urinary protein 2, Apolipoprotein A-II, 45 kDa calcium-binding protein, Vitronectin, and Osteopontin were the therapeutic targets of the Shenkangling intervention. Afamin, isoform CRA_a, Apolipoprotein A-IV, Coagulation factor XII, Prolactin-induced protein, and Coagulation factor XII were the therapeutic targets of the Shenkangling intervention combined with prednisone.
Conclusion:
The feasibility of urinary proteomics analysis in rats using a large number of proteins with finite molecular weights is controversial. The markers screened in this study may be of clinical value for the diagnosis and treatment of nephropathy. However, these findings should be confirmed in future cohort studies.
... SELDI TOF MS combined with antibody capture allows direct measurement of both the captured antigen's quantity and mass, whereby isoform, truncation and degradation can be evaluated. For a more detailed introduction to the technology the reader is referred to the literature (14,15). ...
Early detection of colorectal cancer (CRC) improves patient survival. Plasma tissue inhibitor of metalloproteinases 1 (TIMP-1) measurements by enzyme-linked immunosorbent assay (ELISA) have been suggested as a new method for the early detection of CRC. To further investigate the nature of TIMP-1 in plasma, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI TOF MS) was used. TIMP-1 measurements of plasma from 16 healthy donors and 14 CRC patients were performed using TIMP-1 monoclonal antibody in SELDI TOF MS and ELISA. SELDI TOF MS applying an antibody to TIMP-1 revealed that human plasma TIMP-1 has a mass of 25.1 kDa and exhibits several isoforms. Both methods showed increased plasma TIMP-1 values for cancer patients as compared to healthy individuals. The p values for the separation of the groups were 0.0019 for ELISA and <0.0001 for SELDI TOF MS. CRC did not fundamentally affect the appearance of TIMP-1 as evaluated by SELDI TOF MS.
... The MALDI ionizer generates predominantly singly charged ions (which may be positive or negative but generally only positive ions are analyzed by MS) and used most often in peptide mass fingerprinting because of its high accuracy in measuring peptide mass. An extension of the MALDI ionizer, termed surface-enhanced laser desorption/ionization or SELDI (Forde and McCutchen-Maloney, 2002;Tang et al., 2004;Wright, 2002;Yip and Lomas, 2002) has recently been developed for identifying proteins of different sizes on protein arrays. ...
Basic proteomic data acquisition involved protein separation, digestion and peptide mass fingerprinting to identify the proteins. This chapter outlines the main types of protein mass spectrometry, and charge deconvolution for computing the molecular mass from the distribution of multiple charged ions of the molecule of interest. Once we obtain a list of peptides with computed peptide mass, we can search them against a set of in silico digested peptides for matches. Statistical methods on how to identify proteins from such matches are presented.
... Several new techniques are still in incubation and beings foreseen as future proteomic diagnostic tools. Surface enhanced laser desorption ionization (SELDI) is based on the adsorption of charged peptide fragments on a metal wedge to enhance the sensitivity for protein detection using mass spectrometry [37]. The mass spectrometric data that has been accumulated over a period of time is now being used to train the machines (alternatively called as artificial intelligence) so that mass fingerprint recognition could be made easy, these techniques use several computation tools and therefore enable proteome pattern recognition [38]. ...
... Numerous reviews describe and critique SELDI-MS and its applications, eg, previous works. 117,119-122 A thorough review of SELDI affinity technology with specific attention to the history, technology, the nature of the different immobilized surfaces, and their applications has also been published 123 ). ...
The practice of laser desorption/ionization (LDI) mass spectrometry continues to evolve. In the most commonly adopted manifestation of LDI, matrix assisted laser desorption/ionization (MALDI), attention continues to be directed towards novel sample application strategies and modifications to the sample plate. Specifically, researchers continue to explore adaptations to the conventional, stainless steel sample plate that is the centerpiece of conventional LDI. Numerous variants of LDI-MS have been reported based on modifications of the plate surface, but none of these is widely adopted, either by end-users or instrument manufacturers. Further, at this time, advances in surface engineering have had only modest impact on day-to-day operation. In this article we review and discuss some of the numerous, but scattered reports on novel LDI strategies with an emphasis on modified sample support substrates and plates. We discuss and highlight innovations that have the potential to markedly enhance the utility of LDI-MS.
... SELDI TOF MS combined with antibody capture allows direct measurement of both the captured antigen's quantity and mass, whereby isoform, truncation and degradation can be evaluated. For a more detailed introduction to the technology the reader is referred to the literature (14,15). ...
Early detection of colorectal cancer (CRC) improves patient survival. Plasma tissue inhibitor of metalloproteinases 1 (TIMP-1) measurements by enzyme-linked immunosorbent assay (ELISA) have been suggested as a new method for the early detection of CRC. To further investigate the nature of TIMP-1 in plasma, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI TOF MS) was used. TIMP-1 measurements of plasma from 16 healthy donors and 14 CRC patients were performed using TIMP-1 monoclonal antibody in SELDI TOF MS and ELISA. SELDI TOF MS applying an antibody to TIMP-1 revealed that human plasma TIMP-1 has a mass of 25.1 kDa and exhibits several isoforms. Both methods showed increased plasma TIMP-1 values for cancer patients as compared to healthy individuals. The p values for the separation of the groups were 0.0019 for ELISA and <0.0001 for SELDI TOF MS. CRC did not fundamentally affect the appearance of TIMP-1 as evaluated by SELDI TOF MS.
... 9 While cleanup has historically been achieved through solid phase extraction, affinity capture has been previously used in conjunction with MALDI. 10,11 The SAMDI technology 12,13 pioneered by the Mrksich lab, enables specific capture of analytes for high-throughput sample preparation with a throughput of >100,000 compounds per day. 13 SAMDI uses self-assembled monolayers on gold arrays in 384 or 1536 formats that are compatible with state-of-the-art MALDI time-of-flight (TOF) instrumentation and liquid handling. ...
In recent years, the ability to unambiguously identify complex mixtures of analytes with high accuracy and resolving power in a label-free format continues to expand the application of mass spectrometry (MS) in the drug discovery process. This advantage combined with improved instrumentation makes MS suitable for targets with limited alternative assays for high-throughput screening (HTS). We describe a novel screening format using Self-Assembled Monolayers and matrix-assisted laser Desorption Ionization (SAMDI) technology. SAMDI enables affinity capture of a target protein for use in a small-molecule–binding assay format. Subsequent ionization enables the inferred identification of noncovalent compound interactions. SAMDI technology overcomes shot-to-shot variability by uniformly saturating the surface with captured protein, thereby minimizing matrix crystallization “hot spots.” Furthermore, the combination with high-resolution matrix-assisted laser desorption/ionization time of flight significantly reduces interference of small-molecule detection from salt, detergent, and matrix. By using a pooled library format, the SAMDI assay can significantly improve the throughput of MS-based screening irrespective of enzyme activity. Finally, we demonstrate binding affinity rank ordering from a pool of compounds that correlates with potency data from a biochemical assay.
Intercellular communication through secreted proteins is necessary in essential processes such as embryo and limb development, disease progression, and immune responses. There exist many techniques to study bulk solution protein concentrations, but there is a limited set of tools to study the concentrations of cell-secreted proteins in situ within diverse cell platforms while retaining spatial information. In this study, we have developed a microgel system that is able to quantitatively measure the cell-secreted protein concentration within defined three-dimensional culture configurations with single-cell spatial resolution, called μGeLISA (microgel-linked immunosorbent assay). This system, which is based on the surface modification of polyethylene glycol microgels, was able to detect interleukin 6 (IL-6) concentrations of 2.21-21.86 ng/mL. Microgels were also able to detect cell spheroid-secreted IL-6 and distinguish between low- and high-secreting single cells. The system was also adapted to measure the concentration of cell-secreted matrix metalloproteinase-2 (MMP-2). μGeLISA represents a highly versatile system with a straightforward fabrication process that can be adapted toward the detection of secreted proteins within a diverse range of cell culture configurations.
In the present study, we described the protein profile experimentally by 2D-PAGE and MALDI analysis to understand the stress mechanisms of cocoti sap and wine on E.coli Nissle 1917. We isolated one newly expressed protein from cocoti wine treated gel which is not present in both control and cocoti sap treated sample i.e. P21 prophage-derived head-stabilizing proteinVG03_ECOL6 (3n1) also called as Head protein gp3. This protein mainly activities related to the viral life cycle. It helps to attach the viral gene into host. The growth rate was delayed in cocoti wine treated E.coli Nissle 1917 when compared to control and cocoti sap treated samples. Stress mechanism induce many proteins they are involved in metabolic process, hydrolase activity, lyase activity, quinone binding, phosphotransferase system, carbohydrate metabolism, DNA binding, DNA repair, transferase activity, oxidoreductase, purine metabolism, transcription anti-termination, transcription regulation and other related activities.We proved that the predicted protein structure quality, resolution, density and error plot values by QMEAN analysis. Based on these results, only two differentially expressed proteins under sap stress showed that the significant results, which were N-acetylgalactosamine-specific phosphotransferase enzyme IIB component 1, PTPB1_ECOLI and DinI-like protein Z3305/ECs2939 in prophage CP-933VDINI1_ECO57. In case of wine stress, the differentially expressed proteins were Transcription anti-termination protein RFAH- ECO57 NusA and PUR7-eco24- phosphoribosylamidazole-succinocarboxamide synthase showed significant results. ProtParam analysis indicating that the multiple physico-chemical characters of differentially expressed proteins were differed and compared. The phylogenetic tree represents the relationship in-between the differentially expressed proteins, were showed siblings (related) as well as monophytic clade.
Mass spectrometry imaging (MSI) has provided a new perspective on acquiring spatial information of multiple molecules in various samples. Among the different ionization methods, matrix-assisted laser desorption/ionization (MALDI) has been widely utilized for detecting macromolecules, with difficulty for small molecules (m/z < 700 Da) due to the matrix interference or ionization suppression. In the past two decades, surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) gives rise to lots of attention on account of its unique performances, especially in untargeted analysis of small molecules. Selecting an appropriate substrate is a precondition for SALDI-MS and offers the possibility for SALDI-MS imaging (SALDI-MSI). In the last 5 years, different kinds of nanomaterials have been widely explored as substrates including metal/metal oxide-based, carbon-based, silicon-based, metal–organic frameworks-based, covalent organic frameworks-based substrates, with growing interests on composite materials and nanomaterials with homogeneous film structure. This review highlights recent advances of various nanomaterials as SALDI substrates, and their emerging imaging applications in botanic, forensic, metabolic and pathological fields. Finally, the merits and limitations of SALDI-MS are sketched out and some recommendations of this technique and its imaging are proposed.
This volume brings current knowledge of proteomics technologies and related developments with special reference to diseases caused by microbes. The editor has compiled chapters written by expert academicians which distill the information about useful methods in microbial proteomics for the benefit of readers. Chapters cover several methods used to investigate the microbial proteome and special topics such as antimicrobial drug resistance mechanisms, biomarker developments, post-translational modifications.
Key Features:
-Overview of several biochemical methods in proteomics
-Full-color, high-quality images of the most frequent technologies and applications
-Concise, well organized, and didactic format
-Updates in basic applied information
-Bibliographic references
-Information on proteomics for tuberculosis treatment
This reference work is intended for researchers seeking information on laboratory techniques applied in proteomics research and microbiology.
This unique introduction to the growing field of microfluidics applied to genomics provides an overview of the latest technologies and emphasizes its potential in answering important biological questions. Written by a physicist and a biologist, it offers a more comprehensive view than the previous literature. The book starts with key ideas in molecular biology, developmental biology and microtechnology before going on to cover the specifics of single cell analysis and microfluidic devices for single cell molecular analysis. Review chapters discuss the state-of-the art and will prove invaluable to all those planning to develop microdevices for molecular analysis of single cells. Methods allowing complete analysis of gene expression in the single cell are stressed - as opposed the more commonly used techniques that allow analysis of only a few genes at a time. As pioneers in the field, the authors understand how critical it is for a physicist to understand the biological issues and questions related to single cell analysis, as well for biologists to understand what microfluidics is all about. Aimed predominantly at graduate students, this book will also be of significant interest to scientists working in or affiliated with this field.
Proteome deals with complete set of proteins, expressed by a genome of a cell present inside of an organism at a specific time period. It includes expression study, information of modifications in proteins, interactions with other protein biomolecules, etc. The goal of proteomics is to know new protein information, whether a protein is over expressed or under expressed in specific situation and overall gives information of its effect on an organism. Thousands of proteins can be examined at a time as compared to other methodologies. Many advanced technologies have been evolved to investigate proteome in depth and generate a huge amount of data. The most common high throughput techniques such as polyacrylamide & agarose gel electrophoresis and their advanced versions in combination with mass spectrometry are being used in modern proteomics. The study of microbial proteome data helps us to know how bacteria get resistant to particular drug and which biomolecules are involved in that process. Database also gives the opportunity to develop better drugs that target new places on bacterial surface or new drugs on the same target. The advancement of proteomics technique and their applications in microbial research has granted a new hope to explore disease biomarkers and the development of diagnostic assays. Microbial protein's benefits are extensively used in the agricultural sector. Proteomics profiling has a key role in disease identification in humans and animals. Thanks to protein information as new targets are identified and more safer and effective drugs are produced.
Two-dimensional difference gel electrophoresis (2D-DIGE) remains to be one of the most popular and versatile methods of protein separation among many proteomics technologies. Similar to traditional two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), the proteins are separated based on their charges and molecular weight by 2D-DIGE. Different from 2D-PAGE, proteins are pre-labeled with different fluorescent dyes, and different protein samples are run in one gel by this method. Therefore, 2D-DIGE not only carries the advantages of 2D-PAGE but also eliminates gel-to-gel variation and achieves high resolution, sensitivity, and reproducibility.
Protein adsorption on biomaterial surfaces has been investigated in the development of protein-repellent implantable devices. While the study of the adsorption of a single protein have produced valuable insights of the role of specific proteins in the biological responses to biomaterials, a systematic high throughput screening method is needed to gain more comprehensive understanding of such a complex process, mainly because biomaterials are exposed to protein mixtures when implanted in the human body. To further advance our knowledge of the dynamics of protein adsorption/desorption at interfaces between proteins and solid surfaces, proteomic technologies have been explored to determine relationships between adsorbed proteins on the surfaces and subsequent biological responses. In this review, we will briefly describe the protein adsorption process and proteomics technologies and focus on subsequent biological responses to biomaterials such as blood/biomaterial interactions, biocompatibility, and cell behavior, to obtain more comprehensive understanding of the process for the development of improved biomaterials. We also highlight a number of challenges of contemporary proteomics technologies and future perspectives to advance our knowledge of protein adsorption/desorption dynamics on the surfaces of biomaterials.
Two winter triticale (x Triticosecale Wittmack) model cultivars: Hewo (tolerant to pink snow mould) and Magnat (sensitive) were used to test the effect of cold-hardening (4 weeks at 4°C) on soluble ≤50 kDa protein profiles of the seedling leaves. The presence and abundance of individual proteins were analysed via two-dimensional gel electrophoresis (2-DE) and Surface-Enhanced Laser Desorption/Ionization Time-of-Flight (SELDI-TOF). Up to now, no proteomics analysis of triticale response to hardening has been performed. Thus, the present paper is the first in the series describing the obtained results. In our experiments, the exposure to the low temperature-induced only quantitative changes in the leaves of both cultivars, causing either an increase or decrease of 4–50 kDa protein abundance. Among proteins which were cold-accumulated in cv. Hewo’s leaves, we identified two thioredoxin peroxidases (chloroplastic thiol-specific antioxidant proteins) as well as mitochondrial- β-ATP synthase subunit and ADP-binding resistance protein. On the contrary, in hardened seedlings of this genotype, we observed the decreased level of chloroplastic RuBisCO small subunit PW9 and epidermal peroxidase 10. Simultaneous SELDI-TOF analysis revealed several low mass proteins better represented in cold-hardened plants of tolerant genotype in comparison to the sensitive one and the impact of both genotype and temperature on their level. Based on those results, we suggest that indicated proteins might be potential candidates for molecular markers of cold-induced snow mould resistance of winter triticale and their role is worth to be investigated in the further inoculation experiments.
This chapter provides a discussion on various types of ionization methods. For years of mass spectrometer development, the instruments were constantly modified to reach better analytical capabilities, and their construction was supplied by new ion sources, analyzers, and vacuum technology. One of the most important, but usually undervalued part of mass spectrometry, is ion detector. This element is critical for sensitivity of the entire instrument. In majority of mass spectrometers, electron multipliers are used as basic detectors due to their cost efficiency, sensitivity, and reliability. Other commonly used detectors are similar in their principles of operation to a typical electron multiplier. Other interesting types of ion detection are solutions used in the ion cyclotron resonance (ICR) chambers and Orbitraps. In these instruments, the process occurs by the measurement of cyclotron frequencies of the ions traveling in the ICR chamber or by the measurement of oscillation frequencies of ions spinning around central electrode in the Orbitrap.
Early detection of graft injury after kidney transplantation is key to maintaining long-term good graft function. Graft injury could be due to a multitude of factors including ischaemia reperfusion injury, cell or antibody-mediated rejection, progressive interstitial fibrosis and tubular atrophy, infections and toxicity from the immunosuppressive drugs themselves. The current gold standard for assessing renal graft dysfunction is renal biopsy. However, biopsy is usually late when triggered by a change in serum creatinine and of limited utility in diagnosis of early injury when histological changes are equivocal. Therefore, there is a need for timely, objective and non-invasive diagnostic techniques with good early predictive value to determine graft injury and provide precision in titrating immunosuppression. We review potential novel plasma and urine biomarkers that offer sensitive new strategies for early detection and provide major insights into mechanisms of graft injury. This is a rapidly expanding field, but it is likely that a combination of biomarkers will be required to provide adequate sensitivity and specificity for detecting graft injury.
Graphene-based materials have emerged as a new class of matrix for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), but the factors governing the performance of graphene are not fully understood. In this study, we show two independent novel approaches to tune the performance of graphene as a dual-ion-mode MALDI matrix, i.e., chemical modification of graphene (e.g., oxidation, fluorination, amination, and carboxylation) and incubation of samples with graphene. We found that both approaches could significantly affect the MS signals, enabling a deep optimization of the performance of graphene in both positive and negative ion mode MALDI-TOF MS. Results showed that the performance of graphene in MALDI was dependent on its inherent chemical properties, self-assembly behavior, and interaction with targets. The two approaches were also validated in surface-enhanced laser desorption/ionization (SELDI). Under the optimized conditions, high sensitivity and good reproducibility were obtained for targets. To our knowledge, this is also the first report that aminated and carboxylated graphene were used as MALDI matrices. This work shows novel approaches to tailor graphene for its applications, and it also helps better understand the mechanism of the LDI process.
The last three chapters are on proteins which are the workhorses in the cell. The most fundamental property of a protein is its isoelectric point (pI) which determines what cellular components a protein can interact electrostatically (e.g., a positively charged substrate would demand a negatively charged enzyme). This chapter first explains the iterative approach to compute the theoretical protein pI and then apply it to a study of genomic adaptation of Helicobacter pylori, a bacterial gastric pathogen. Two research questions are addressed. How did H. pylori acquire its proteins with extraordinarily high pI? Are these proteins with high pI contribute to acid resistance of the pathogen? A detailed genomic analysis illustrates the hypothesis-testing approach in science.
Translation consists of three subprocesses: initiation, elongation, and termination, all involving signal motifs and their decoders. Shine-Dalgarno (SD) sequences in prokaryotic mRNA are decoded by anti-SD (aSD) sequences at the 3’ end of the small subunit rRNA (3’TAIL). Sense codons are decoded by tRNAs and stop codons by release factors. The signal motifs are stronger in highly expressed genes than lowly expressed genes. This chapter focuses on translation initiation and presents several new bioinformatics methods for characterizing evolution and co-evolution of signal motifs and their decoders. These methods are amply illustrated with real prokaryotic and eukaryotic data. The main factors determining translation initiation efficiency is the strength of the signal motif (e.g., location and pairing strength of SD and the nature of start codon), abundance of their decoder (e.g., number of ribosomes), and secondary structure that may embed SD and start codon rendering them inaccessible by their decoders. Mutations that result in SD/aSD incompatibility between a bacteriophage and a subset of its hosts leads to a more restricted host range.
Codon usage depends on mutation bias, tRNA-mediated selection, and the need for high efficiency and accuracy in translation. One codon in a synonymous codon family is often strongly over-used, especially in highly expressed genes, which often leads to a high dN/dS ratio because dS is very small. Many different codon usage indices have been proposed to measure codon usage and codon adaptation. Sense codon could be misread by release factors and stop codons misread by tRNAs, which also contribute to codon usage in rare cases. This chapter outlines the conceptual framework on codon evolution, illustrates codon-specific and gene-specific codon usage indices, and presents their applications. A new index for codon adaptation that accounts for background mutation bias (Index of Translation Elongation) is presented and contrasted with codon adaptation index (CAI) which does not consider background mutation bias. They are used to re-analyze data from a recent paper claiming that translation elongation efficiency matters little in protein production. The reanalysis disproves the claim.
Genomes change over time, so are the interactions of genes and gene products that breathe life into a genome. To have the most advantageous view of genomes, genes and their interactions, we need to see things from the very beginning. Substitution models enable us to trace history back to the very early forms of life, by reconstructing the genomic “books” erased and obliterated by billions of years of mutations. This chapter focuses on nucleotide-based substitution models, presenting three different ways of deriving, for various substitution models, transition probability matrices that are needed to compute evolutionary distances and to compute likelihood of a tree. These three different ways should allow students of different mathematical background to understand substitution models and their uses in phylogenetics. Almost all frequently used substitution models are nested models with the simple one being a special case of the more general one. The likelihood ratio test, as well as the information-theoretic indices as an alternative approach to model selection, is numerically illustrated in choosing the substitution model that best describes the aligned sequences.
The distance-based phylogenetic method is fast and remains the most popular one in molecular phylogenetics, especially in the big-data age when researchers often build phylogenetic trees with hundreds or even thousands of leaves. A distance-based method has two components: the evolutionary distance matrix typically derived from a substitution model, and the tree-building algorithm that constructs a tree from the distance matrix. Evolutionary distances differ not only by substitution models, but also by whether they are estimated independently (based on only information between two aligned sequences) or simultaneously (based on information from all pairwise comparisons). Simultaneous estimated distances perform much better in molecular phylogenetics than independently estimated distances. Frequently used tree-building algorithms include neighbor-joining which clusters two species by weighing two measures: how close they are to each other (which is used in a simpler method known as UPGMA) and how far they jointly are from all other species. An alternative method is based on the least-squares framework. Alternative trees first have their branches evaluated by the least-squares method, and then the best tree is chosen by either the least-square criterion or the minimum evolution criterion. Distance-based dating methods are also illustrated numerically, with both fossil-calibrated dating and tip-dating, together with the estimation of variation in the inferred dates by resampling.
As an important class of emerging chemical contaminants, short-chain chlorinated paraffins (SCCPs) are considered as one of the most challenging groups of compounds to analyze. In this paper, we report a new method for fast screening of SCCPs based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with graphene as a matrix and 2,5,6,9-tetrachlorodecane as an internal standard. We found that the use of graphene as MALDI matrix generated high peak intensities for SCCPs while producing few background noises. The ion fragmentation mechanisms of SCCPs in MALDI are discussed in detail. Under the optimized conditions, much lower detection limits of SCCP congeners (0.1–5 ng/mL) than those reported previously were obtained. Other distinct advantages such as short analysis time and simplified sample preparation procedures are also demonstrated. The method was successfully applied in fast screening of SCCPs in indoor dust samples and monitoring of human exposure levels to SCCPs, and the results were verified by gas chromatography coupled to negative chemical ionization quadrupole time-of-flight high-resolution mass spectrometry. This work not only offers a new promising tool for SCCP studies, but also further demonstrates the promise of graphene as a new generation of MALDI matrix.
Increased understanding of Chinese hamster ovary (CHO) cell physiology has been ushered in upon availability of the parental CHO-K1 cell line genome. Free and openly accessible sequence information has complemented transcriptomic and proteomic studies. The previous decade has also seen an increase in sensitivity and accuracy of proteomic methods due to technology development. In this genomic era, high-throughput screening methods, sophisticated informatic tools, and models continually drive major innovations in cell line development and process engineering. This review describes the various achievements in ‘omics techniques and their application to improve recombinant protein expression from CHO cell lines.
To study the metabolism of amyloid beta protein (A beta) in Alzheimer's disease, we have developed a new approach for analyzing the profile of soluble A beta and its variants, In the present method, A beta and its variants are immunoisolated with A beta-specific monoclonal antibodies. The identities of the Ap variants are determined by measuring their molecular masses using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The levels of A beta variants are determined by their relative peak intensities in mass spectrometric measurements by comparison with internal standards of known identities and concentrations. We used this method to examine the A beta species in conditioned media of mouse neuroblastoma cells transfected with cDNAs encoding wild type or mutant human amyloid precursor protein. In addition to human A beta-(1-40) and A beta-(1-42), more than 40 different human A beta variants were identified. Endogenous murine A beta and its variants were also identified by this approach. The present approach is a new and sensitive method to characterize the profile of soluble A beta in conditioned media and biological fluids. Furthermore, it allows direct measurement of each individual peptide in a peptide mixture and provides comprehensive information on the identity and concentration of A beta and A beta variants.
Biomolecular interaction analysis mass spectrometry (BIA/MS) is a multidimensional methodology for functional and structural protein analysis. Proteins are affinity captured and quantified from a solution via ligands covalently attached on the SPR sensor surface. Because the SPR detection is nondestructive, proteins retrieved on the SPR sensing surface can be further analyzed via mass spectrometry, either directly from the sensor/chip surface or separately, following elution and microrecovery. Detection of attomole amounts of proteins from complex biological mixtures is possible via the combined SPR-MS approach. A chip cutter with a circular heated cutter head is used for excising a chip or plastic mount of a defined circular shape that fits into an appropriately configured MALDI mass spectrometer target. The MALDI matrix is applied to the chip via an aerosol-spraying device. MALDI-TOF mass spectrometry analysis from the chip surface is performed on a custom-made MALDITOF mass spectrometer. The instrument consists of a linear translation stage or ion source capable of precise targeting of each of the flow cells under a focused laser spot.
Biomolecules can be rapidly identified and characterized using on-chip enzyme digestion. The chip platform provides an optimal podium for protein detection and can be expanded into a multiplexed, microscale proteomics tool.
-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two A peptide isoforms, A40 and A42. Here we report the detergent solubilization and partial characterization of -secretase. The activity of solubilized -secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the beta-secretase cleavage site. Cleavage of C100Flag by gamma-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the Abeta40 or Abeta42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the Abeta40 and Abeta42 termini. Recovery of catalytically competent, soluble gamma-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized gamma-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of Abeta40 and Abeta42 in mammalian cells. Upon gel exclusion chromatography, solubilized gamma-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 × 106. Anti-PS1 antibody immunoprecipitates gamma-secretase activity from the solubilized gamma-secretase preparation. These data suggest that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex.
ADDITION of the beta-hydroxy-beta-methylglutaryl-CoA (HmG-CoA) reductase inhibitor lovastatin to human HEK cells transfected with the amyloid precursor protein (APP) reduces intracellular cholesterol/protein ratios by 50%, and markedly inhibits beta-secretase cleavage of newly-synthesized APP. Exogenous water-solubilized cholesterol at 200 mu g/ml concentration increases newly synthesized beta-amyloidogenic products four-fold. These intracellular changes are detectable by immunoprecipitation and immunofluorescent labelling. Analyses of the fragments captured from culture medium by an N-terminal anti-beta-amyloid antibody on ProteinChip arrays and detected using surface-enhanced laser desorption/ionization (SELDI) mass spectrometry revealed that culture with cholesterol (200 mu g/ml) increased secretion of beta-amyloid 1-40 by 1.8-fold, and increased secretion of beta-amgloid 1-42. Changes in APP processing by cholesterol may mediate the way in which the ApoE4 allele increases risk of developing Alzheimer's disease (AD) in western populations. NeuroReport 10:1699-1705 (C) 1999 Lippincott Williams & Wilkins.
Factor B is a key component of the alternative pathway of the complement system. During complement activation, factor B complexed with activated C3 is cleaved into the Ba and Bb fragments by the protease factor D to form the C3 convertase from the complex between C3b and Bb. The Ba fragment contains three short consensus/complement repeat (SCR) domains, and the Bb fragment contains a von Willebrand factor type A (vWF-A) domain and a serine protease (SP) domain. Surface-enhanced laser desorption-ionization affinity mass spectrometry (SELDIAMS) was used to investigate the reaction of factor B with immobilised activated C3(NH3) in the presence of Mg(2+). A recombinant vWF-A domain (residues G229-Q448), the native Ba and Bb fragments and native factor B all demonstrated specific interactions with C3(NH3), while no interactions were detected using bovine serum albumin as a control. A mass analysis of the proteolysis of the vWF-A domain when this was bound to immobilised C3(NH3) identified two peptides (residues G229-K265 and T355-R381) that were involved with vWF-A binding to C3(NH3). A homology model for the vWF-A domain was constructed using the vWF-A crystal structure in complement receptor type 3. Comparisons with five different vWF-A crystal structures showed that large surface insertions were present close to the carboxyl and amino edges of the central beta-sheet of the factor B vWF-A structure. The peptides G229-K265 and T355-R381 corresponded to the two sides of the active site cleft at the carboxyl edge of the vWF-A structure. The vWF-A connections with the SCR and SP domains were close to the amino edge of this vWF-A beta-sheet, and shows that the vWF-A domain can be involved in both C3b binding and the regulation of factor B activity. These results show that (i) a major function of the vWF-A domain is to bind to activated C3 during the formation of the C3 convertase, which it does at its active site cleft; and that (ii) SELDIAMS provides an efficient means of identifying residues involved in protein-protein interactions.
Abstract: The risk from chemical carcinogens and environmental toxins is dependent on the metabolic balance between bioactivation and detoxification enzymes. Therefore, agents that alter enzyme expression are critical factors in toxicity. Enhancement or suppression of enzyme activities through gene expression is in part regulated by interactions between specific DNA promoter response elements and specific transcription proteins. DNA-protein interactions are dependent upon translocation of proteins from the cytoplasm to the nucleus and the affinity of proteins for binding to transcription promoter sequences. A key factor in both processes is the intracellular redox state, which influences protein-protein interactions and protein-DNA binding and can be altered by exposure to electrophiles, antioxidants and oxidative stress. Oxidative stress levels can be readily detected by measurable effects on the intracellular glutathione (GSH):glutathione disulfide redox potential, the major intracellularredox buffer. Alterations in the GSH redox pool can directly affect enzyme activity by altering disulfide bonds in the transcription factors regulating enzyme expression. These may affect: 1) specific DNA-protein and protein-protein interactions, 2) cyst(e)ine redox state within transcriptional proteins and 3) translocation of transcription proteins from cytoplasmic to nuclear compartments within the cell. The studies reported here are designed to investigate the relative changes in enzyme expression in response to cellular redox potential changes using the new proteomics technology of surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI). Treatment of HeLa and HT29 human cell lines to increase the expression of enzymes that are upregulated by oxidative stress was used as a model system to determine the efficacy of the SELDI technology in measuring changes in transcriptional protein binding to transcriptional response elements. An important goal is to determine whether the SELDI will allow simultaneous studies of multiple transcriptional protein-DNA interactions in response to controlled oxidative stress. This will provide a better understanding of the effect of electrophilic carcinogens and oxidants on the balance between activation and detoxification mechanisms in chemical carcinogenesis.
To study the metabolism of amyloid β protein (Aβ) in Alzheimer's disease, we have developed a new approach for analyzing the
profile of soluble Aβ and its variants. In the present method, Aβ and its variants are immuno-isolated with Aβ-specific monoclonal
antibodies. The identities of the Aβ variants are determined by measuring their molecular masses using matrix-assisted laser
desorption ionization time-of-flight mass spectrometry. The levels of Aβ variants are determined by their relative peak intensities
in mass spectrometric measurements by comparison with internal standards of known identities and concentrations. We used this
method to examine the Aβ species in conditioned media of mouse neuroblastoma cells transfected with cDNAs encoding wild type
or mutant human amyloid precursor protein. In addition to human Aβ-(1-40) and Aβ-(1-42), more than 40 different human Aβ variants
were identified. Endogenous murine Aβ and its variants were also identified by this approach. The present approach is a new
and sensitive method to characterize the profile of soluble Aβ in conditioned media and biological fluids. Furthermore, it
allows direct measurement of each individual peptide in a peptide mixture and provides comprehensive information on the identity
and concentration of Aβ and Aβ variants.
Affinity mass spectrometry (AMS) was used to evaluate the structural diversity of the E2 component of pyruvate dehydrogenase complex (PDC) in normal and diseased liver cells, including those from patients with the autoimmune disease primary biliary cirrhosis (PBC). Two different antibodies to PDC-E2, the immunodominant mitochondrial autoantigen in patients with PBC, were used. AMS was performed directly on frozen liver sections and purified bile duct epithelial cells. Mass spectrometric signals associated with the molecular recognition of PBC-specific antigenic determinants were enhanced by an in situ enzyme-linked signal amplification process. Samples from patients with PBC gave strong positive signals for the antigen(s) recognized by the monoclonal antibody C355.1. Conversely, tissues from normal and disease controls showed only a minimal signal. AMS was used to identify specific antigenic determinants within the E2 component of PDC for comparison with unknown antigenic determinants observed by affinity capture with C355.1 monoclonal antibody from PBC samples. PDC components bound to C355.1 were mapped and identified by mass before dissociation from the E2 component. A similar approach was used to identify unknown antigenic determinants associated with PBC. We believe AMS may be an important new approach with wide application to the identification of molecules associated with a number of disease states.
Effective MALDI-TOF MS of salt-, detergent-, and glycerol-contaminated peptide and protein samples is accomplished by loading samples onto synthetic membranes, washing away contaminants, adding matrix, and desorbing samples directly from the membrane surface. The method easily removes contaminants which prevent effective MALDI of peptides and proteins from stainless steel surfaces, obviating the need for laborious further purification and associated sample losses. Polyethylene, polypropylene, C8, and C18 surfaces all proved effective at removing contaminants. Scanning electron microscope images of sample surfaces reveal that significantly smaller matrix crystals form on polyethylene and polypropylene surfaces than on stainless steel, C8, or C18 surfaces. Desorption from polyethylene and polypropylene surfaces generates consistently reproducible spectra with better mass resolution than observed for samples desorbed from stainless steel. Improved resolution, combined with reduced intensity of product ion spectra, indicate that peptide and protein molecular ions have less internal energy when desorbed from polyethylene and polypropylene surfaces than from stainless steel. MALDI of contaminated samples can be accomplished by straightforward, on-probe purification, resulting in higher resolution spectra than observed in samples desorbed from stainless steel.
Rapid advances in genomic sequencing, bioinformatics, and analytical instrumentation have created the field of proteomics, which at present is based largely on two-dimensional electrophoresis (2-DE) separation of complex protein mixtures and identification of individual proteins using mass spectrometry. These analyses provide a wealth of data, which upon further evaluation leads to many questions regarding the structure and function of the proteins. The challenge of answering these questions create a need for high-specificity approaches that may be used in the analysis of biomolecular recognition events and interacting partners, and thereby places great demands on general protein characterization instrumentation and the types of analyses they need to perform. Over the past five years we have been actively involved in interfacing two general, instrumental techniques, surface plasmon resonance-biomolecular interaction analysis (SPR-BIA) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, into a single concerted approach for use in the functional and structural characterization of proteins. Reviewed here is the recent progress made using biomolecular interaction analysis - mass spectrometry (BIA-MS) in the detailed characterization of proteins and protein-protein interactions and the development of biosensor chip mass spectrometry (BCMS) as a new chip-based proteomics approach.
Biomolecular interaction analysis mass spectrometry (BIA/MS) is a multiplexed analytical technique that utilizes a unique combination of surface plasmon resonance (SPR) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection and analysis of small amounts of proteins residing in complex biological systems. In order to achieve high sensitivity during BIA/MS, certain experimental parameters and sequences of events need to be optimized and maintained. Immobilized ligand density, flow rate and biosensor control (in SPR-BIA) and matrix choice and application (in MALDI-TOF MS) have significant influence on the final outcome of the BIA/MS analysis and, consequently, need to be optimized and carefully controlled. In addition, chip washing and cutting are essential in converting the SPR-active sensor chips into target surfaces amenable to MALDI-TOF MS. Reviewed here are the prerequisites for successfully interfacing SPR-BIA with MALDI-TOF MS.
Two β-secretases, BACE1 and BACE2, are involved in generation of Alzheimer's disease Aβ peptides 1–3. We report that secretion of Aβ peptides (Aβ1–40/42 and Aβ11–40/42) is abolished in cultures of BACE1-deficient embryonic cortical neurons, and that whereas both human and murine BACE1 can cleave either human or murine β-amyloid precursor protein (APP) at the +1 site of Aβ, cleavage at the +11 site is species specific. We establish that BACE1 is the principal neuronal protease required to cleave APP at +1 and +11 sites that generate N-termini of Aβ. Alzheimer's disease, a progressive neurodegenerative disorder, is characterized by deposition of Aβ-amyloid and neurofibrillary tangles in many brain regions, particularly the hippocampus and cerebral cortex 4,5. Endoproteolytic cleavage of APP by β-and γ-sec-retase generates toxic Aβ peptides. Both β-secretases are type I trans-membrane aspartyl proteases 1–3,6 .
Biomolecular interaction analysis mass spectrometry (BIA/MS) is a two-dimensional chip-based analytical technique geared toward quantitative and qualitative analysis of small volumes of biological samples. Interactions between surface-immobilized ligands and solute-borne analytes are quantitatively viewed in real time through surface plasmon resonance sensing, followed by qualitative matrix-assisted laser desorption/ionization time-of-flight MS analysis of the analyte(s) affinity-retained on the sensor surface. In this work, BIA/MS was used in the detection of a number of protein biomarkers from human urine. Small volumes of human urine were analyzed for cystatin C, beta(2)-microglobulin, urinary protein 1, and retinol-binding protein (RBP). Multiaffinity sensor surfaces were created to simultaneously and rapidly detect all four proteins in a single BIA/MS analysis on a two-flow cell sensor chip configuration. Furthermore, RBP was analyzed separately from both urine and plasma samples. Results indicate that BIA/MS can be used successfully in rapid screening of a number of urinary proteins indicated as putative biological markers for renal dysfunction.
An automated biomolecular interaction analysis instrument (BI-Acore) based on surface plasmon resonance (SPR) has been used to determine human immunoglobulin G (IgG) in real time. Polyclonal anti-human IgG antibody was covalently immobilized to a carboxymethyldextran modified gold film surface. The samples of human IgG prepared in HBS buffer were poured over the immobilized surface. The signal amplification antibody was applied to amplify the response signal. After each measurement, the surface was regenerated with 0.1 mol/L H3PO4. The assay was rapid, requiring only 30 min for antibody immobilization and 20 min for each subsequent process of immune binding, antibody amplification and regeneration. The antibody immobilized surface had good response to human IgG in the range of 0.12-60 nmol/L with a detection limit of 60 pmol/L. The same antibody immobilized surface could be used for more than 110 cycles of binding, amplification and regeneration. The results demonstrate that the sensitivity, specificity and reproducibility of amplified immunoassay using real-time BIA technology are satisfactory.
Background: Surface-enhanced laser desorption/ionization (SELDI) is an affinity-based mass spectrometric method in which proteins of interest are selectively adsorbed to a chemically modified surface on a biochip, whereas impurities are removed by washing with buffer. This technology allows sensitive and high-throughput protein profiling of complex biological specimens.
Methods: We screened for potential tumor biomarkers in 169 serum samples, including samples from a cancer group of 103 breast cancer patients at different clinical stages [stage 0 (n = 4), stage I (n = 38), stage II (n = 37), and stage III (n = 24)], from a control group of 41 healthy women, and from 25 patients with benign breast diseases. Diluted serum samples were applied to immobilized metal affinity capture Ciphergen ProteinChip® Arrays previously activated with Ni2+. Proteins bound to the chelated metal were analyzed on a ProteinChip Reader Model PBS II. Complex protein profiles of different diagnostic groups were compared and analyzed using the ProPeak software package.
Results: A panel of three biomarkers was selected based on their collective contribution to the optimal separation between stage 0–I breast cancer patients and noncancer controls. The same separation was observed using independent test data from stage II–III breast cancer patients. Bootstrap cross-validation demonstrated that a sensitivity of 93% for all cancer patients and a specificity of 91% for all controls were achieved by a composite index derived by multivariate logistic regression using the three selected biomarkers.
Conclusions: Proteomics approaches such as SELDI mass spectrometry, in conjunction with bioinformatics tools, could greatly facilitate the discovery of new and better biomarkers. The high sensitivity and specificity achieved by the combined use of the selected biomarkers show great potential for the early detection of breast cancer.
Biomolecular interaction analysis mass spectrometry (BIA/MS) is a multidimensional methodology for functional and structural protein analysis. Proteins are affinity captured and quantified from a solution via ligands covalently attached on the SPR sensor surface. Because the SPR detection is nondestructive, proteins retrieved on the SPR sensing surface can be further analyzed via mass spectrometry, either directly from the sensor/chip surface or separately, following elution and microrecovery. Detection of attomole amounts of proteins from complex biological mixtures is possible via the combined SPR-MS approach. A chip cutter with a circular heated cutter head is used for excising a chip or plastic mount of a defined circular shape that fits into an appropriately configured MALDI mass spectrometer target. The MALDI matrix is applied to the chip via an aerosol-spraying device. MALDI-TOF mass spectrometry analysis from the chip surface is performed on a custom-made MALDITOF mass spectrometer. The instrument consists of a linear translation stage or ion source capable of precise targeting of each of the flow cells under a focused laser spot.
To extend the analytical capabilities of immobilized metal ion affinity chromatography (IMAC) for evaluation of biologically relevant peptide-metal ion interactions, we have prepared synthetic peptides representing metal-binding protein surface domains from the human plasma metal transport protein known as histidine-rich glycoprotein (HRG). Three synthetic peptides, representing multiples of a 5-residue repeat sequence (Gly-His-His-Pro-His) from within the histidine- and proline-rich region of the C-terminal domain were prepared. Prior to immobilization, the synthetic peptides were evaluated for identity and sample homogeneity by matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS), a method developed recently for the mass determination of high-molecular-mass biopolymers. 2,5-Dihydroxybenzoic acid was evaluated as a matrix to facilitate the laser desorption and ionization of intact peptides and was found to be ideally suited for determinations of mass within the low-mass region of interest (641.7 to 1772.8 dalton). We observed minimal chemical noise from photochemically generated peptide-matrix adduct signals, clustering, and multiply-charged peptide species. Peptides with bound sodium and potassium ions were observed; however, these signal intensities were reduced by immersion of the sample probe tip in water. Mixtures of the three different synthetic peptides were also evaluated by LDTOF-MS after their elution through a special immobilized peptide-metal ion column designed to investigate metal ion transfer. We found LDTOF-MS to be a useful new method to verify the presence of peptide-bound metal ions. Thus, LDTOF-MS is ideally suited for the rapid (3−5 min), sensitive (<1 pmol), accurate (±0.05%), and relatively high resolution (m/Δm = 300−500, full width at half maximum, where m = mass) evaluation of synthetic peptides. Further, LDTOF-MS was found to be an important tool for the characterization of peptide mixtures and peptide-metal ion interactions.
Pathologic states within the prostate may be reflected by changes in serum proteomic patterns. To test this hypothesis, we analyzed serum proteomic mass spectra with a bioinformatics tool to reveal the most fit pattern that discriminated the training set of sera of men with a histopathologic diagnosis of prostate cancer (serum prostate-specific antigen [PSA] ≥4 ng/mL) from those men without prostate cancer (serum PSA level <1 ng/mL). Mass spectra of blinded sera (N = 266) from a test set derived from men with prostate cancer or men without prostate cancer were matched against the discriminating pattern revealed by the training set. A predicted diagnosis of benign disease or cancer was rendered based on similarity to the discriminating pattern discovered from the training set. The proteomic pattern correctly predicted 36 (95%, 95% confidence interval [CI] = 82% to 99%) of 38 patients with prostate cancer, while 177 (78%, 95% CI = 72% to 83%) of 228 patients were correctly classified as having benign conditions. For men with marginally elevated PSA levels (4–10 ng/mL; n = 137), the specificity was 71%. If validated in future series, serum proteomic pattern diagnostics may be of value in deciding whether to perform a biopsy on a man with an elevated PSA level.
Nitrocellulose is examined as a membrane substrate for peptide mapping by CNBr digestion using MALDI MS analysis. Using this methodology, proteins electroblotted from SDS-PAGE can be purified on the membrane by washing and further characterized by in situ chemical digestion followed by MALDI MS analysis. It is demonstrated that nitrocellulose is versatile in such analysis due to its excellent retention of both small and large fragments and the wide range of fragments that appear in MALDI MS. This is particularly advantageous in cases where multiple reactions and washing steps are involved. The analysis is found to be improved by first eluting the digest from the membrane, since interaction of the matrix with nitrocellulose may degrade the MALDI MS performance.
Identification of ligands separated with affinity chromatography has been facilitated by direct analysis of the bound ligand using matrix-assisted laser desorption time-of-flight mass spectrometry. Tbe mass spectral detection of analytes separated by immunoaffinity chromatography and immobilized metal ion affinity chromatography is shown. For example, cytochrome c is used as an affinity support to purify the anti-cytochrome c monoclonal antibody from ascites, and the mass spectrum of the anti-cytochrome c monoclonal antibody was obtained by direct analysis of an aliquot of the column bed. Direct analyses of metal binding proteins and phosphopeptides bound to immobilized metal ion affinity columns are also demonstrated. The method is characterized by minimal sample manipulation and high sensitivity with low picomoles and high femtomoles of analytes being readily observed.
A 337-nm laser has been used to desorb and ionize proteins from poly(vinylidene difluoride) membranes. Proteins separated by electrophoresis and electroblotted onto 0.5 X 7 cm membranes are located by scanning laser desorption, and their molecular weights are determined by mass spectrometry. Overlapping protein bands are easily deconvoluted. Conditions have been developed to carry out trypsin proteolysis of protein bands directly on the membrane. The same membrane is then reintroduced into the mass spectrometer, and peptide products are mapped by matrix-assisted laser desorption.
Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) of proteins, electroblotted onto polymer membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation is demonstrated. The proteins are desorbed directly from the blot membranes after matrix application. Desorption with 2.94-mu m infrared radiation and succinic acid as matrix was found superior to 355-nm UV desorption using 2,5-dihydroxybenzoic acid as matrix. Several commercially available membranes tested resulted in protein signals after matrix incubation of the membrane. Systematic investigations for five different poly(vinylidene fluoride) (PVDF) membranes showed improved results for membranes exhibiting high specific surfaces. The matrix should be applied immediately after the blotting procedure while the blot is still wet. Lateral resolution of the protein band is preserved after the MALDI preparation procedure. Staining with organic dyes results in broad protein signals shifted in mass due to addition of several dye molecules; staining with colloidal stains such as colloidal gold or India ink somewhat deteriorates the quality of the spectre, but renders the correct protein mass.
An automated biomolecular interaction analysis instrument (BI-Acore) based on surface plasmon resonance (SPR) has been used to determine human immunoglobulin G (IgG) in real time. Polyclonal and-human IgG antibody was covalently immobilized to a carboxymethyldextran-modified gold film surface. The samples of human IgG prepared in HBS buffer were poured over the immobilized surface. The signal amplification antibody was applied to amplify the response signal. After each measurement, the surface was regenerated with 0.1 mol/L H3PO4. The assay was rapid, requiring only 30 min for antibody immobilization and 20 min for each subsequent process of immune binding, antibody amplification and regeneration. The antibody immobilized surface had good response to human IgG in the range of 0.12–60 nmol/L with a detection limit of 60 pmol/L. The same antibody immobilized surface could be used for more than 110 cycles of binding, amplification and regeneration. The results demonstrate that the sensitivity, specificity and reproducibility of amplified immunoassay using real-time BIA technology are satisfactory.
We present two new desorption strategies for the mass spectrometric analysis of macromolecules. These desorption strategies are based on the molecular design and construction of two general classes of sample ‘probe’ surfaces. The first class of surfaces is designed to enhance the desorption of intact macromolecules presented alone (neat) to the surface; we call this surface-enhanced neat desorption (SEND). The availability of probe surfaces derivatized with, or composed of, multiple types and defined numbers of energy-absorbing molecules will facilitate investigations of energy transfer and desorption/ionization mechanisms. The second class of probe surfaces is designed to enhance the desorption of specific macromolecules captured directly from unfractionated biological fluids and extracts; we call this surface-enhanced affinity capture (SEAC). Use of these new probe surfaces as chemically defined solid-phase reaction centers will facilitate protein discovery through molecular recognition in situ, and also macromolecular structure analysis through the sequential chemical and/or enzymatic modification of the adsorbed analyte in situ. Specific examples of laser-assisted SEND and SEAC time-of-flight mass spectrometry are presented to illustrate the potential for increased selectivity, analyte detection sensitivity, and mass measurement accuracy.
Matrix-assisted laser desorption/ionization mass spectrometry (LDI MS) has shown its potential for desorbing ions of biomolecules with relative molecular masses (Mr) up to about 300 000 daltons. Determinations of Mr with an accuracy of 0.01–0.2% from about 1 pmol of sample are possible. Examples are shown for proteins, glycoproteins, oligonucleotides and carbohydrates. Examples of the combination of LDI MS with biochemical methods are shown, including the determination of the carbohydrate content of glycoproteins, the identification of disulphide-bonded subunits by cleavage of the light and heavy chains of a monoclonal antibody, the detection of the time course of enzymatic reactions and the analysis of proteins bound to a PVDF membrane.
Methods are described for the purification of proteins prior to analysis by matrix-assisted laser-desorption mass spectrometry. Contaminated protein samples were immobilized onto the surfaces of sample targets and rinsed. In general, a layer of electrosprayed nitrocellulose gave better results than the roughened gold surface of an untreated target. Using this approach, spectra could be obtained from low picomole quantities of protein in the presence of contaminants which did not inhibit the binding of the protein to the substrate.
This preliminary investigation tests the premise that biologically relevant (1) peptide-metal ion interactions, and (2) metal ion-dependent macromolecular recognition events (e.g., peptide-peptide interactions) may be modeled by biomimetic affinity chromatography. Divinylsulfone-activated agarose (6%) was used to immobilize three different synthetic peptides representing metal-binding protein surface domains from the human plasma metal transport protein histidine-rich glycoprotein (HRG). The synthetic peptides represented 1-3 multiple repeat units of the 5-residue sequence (Gly-His-His-Pro-His) found in the C-terminal of HRG. By frontal analyses, immobilized HRG peptides of the type (GHHPH)nG, where n = 1-3, were each found to have a similar binding capacity for both Cu(II) ions and Zn(II) ions (31-38 mumol/ml gel). The metal ion-dependent interaction of a variety of model peptides with each of the immobilized HRG peptide affinity columns demonstrated differences in selectivity despite the similar internal sequence homology and metal ion binding capacity. The immobilized 11-residue HRG peptide was loaded with Cu(II) ions and used to demonstrate selective adsorption and isolation of proteins from human plasma. These results suggest that immobilized metal-binding peptides selected from known solvent-exposed protein surface metal-binding domains may be useful model systems to evaluate the specificity of biologically relevant metal ion-dependent interaction and transfer events in vitro.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of high mass proteins (> 100,000 Da), directly deposited on polyethylene membranes, is demonstrated. The spectral quality obtained, using standard sample preparation conditions, is equal or superior to that obtained with metal sample stages. Compared to the use of poly(vinylidene difluoride) transfer membranes, this material allows the acquisition of excellent quality MALDI mass spectra from high-mass proteins with a standard UV laser. This gain in capability is not at the expense of either mass accuracy or signal reproducibility; both approach that obtained with standard sample preparations on stainless steel. In addition, for the applications shown, the use of PE as a sample support reduces the severe ion suppression effects typically observed in the MALDI analysis of high-mass mixtures. This permits more precise mass measurements to be made via the use of internal calibration and is illustrated by the mass measurement of a chimeric mouse/human antibody (MW approximately 150,000 Da) by coaddition of bovine albumin dimer (MW approximately 130,000 Da).
A new technique has been developed that provides affinity separations directly on the surface of a matrix-assisted laser desorption/ionization mass spectrometer (MALDI-MS) probe. This strategy provides both in situ identification of biomolecules through biospecific antibody/antigen interactions and molecular weight information in a rapid and facile manner. Our technique, which we call probe affinity MS, directly couples the high selectivity of immobilized affinity chromatography with the sensitivity of MALDI-MS. As this technique permits the rapid identification of antigens, ligands, and other compounds from complex biological mixtures, we believe that it will be useful in a wide range of applications in virtually all fields of the life sciences.
Molecular recognition based on the high affinity of avidin for biotin has been combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The rich chemistry of biotinylation reagents underlies the development of a mass tracer method for the rapid and sensitive analysis of biotinylated analyte in complex mixtures. In this method, the biotinylated analyte is captured with immobilized avidin agarose beads. The beads with the bound complex are deposited on the MALDI sample probe, followed by elution of the retained compounds and removal of the beads. The use of MALDI matrix solutions in conjunction with drying of the beads is shown to be sufficient to overcome the avidin-biotin interaction. The studies of several different avidin-biotin interaction schemes tailored to the MALDI analysis are presented. It is demonstrated that, with an optimized scheme, the extremely high selectivity of the avidin-biotin interaction is preserved, generating species represented in the MALDI spectra that arise only from the avidin-biotin interaction. Biotinylated species in the nanomolar range can be isolated and analyzed with this technique.
Probe affinity mass spectrometry (PAMS) is a technique that combines affinity separations directly with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this approach, a binding molecule, such as an antibody, lectin or receptor, is covalently attached to the surface of a MALDI probe. This permits the analyte of interest to be selectively captured and concentrated on the probe surface prior to MALDI-MS analysis. A major limitation of our initial PAMS immobilization chemistry was that it produced only a relatively small number of binding sites on the probe, as it was based on forming a single monolayer of the binding molecule. Because of this limitation, we have investigated new immobilization chemistries for PAMS that are not confined by monolayer formation and thus allow a larger number of analyte molecules to be captured by the probe. We have developed a new PAMS chemistry that first attaches very high molecular weight (approximately 500,000) dextrans to the MALDI probe, followed by immobilization of the binding molecules to the probe-bound dextrans. Because the size of each dextran molecule is significantly larger than the binding molecule, multiple binding molecules can be linked to the same dextran chain. We have demonstrated that these surfaces possess approximately 500 times more analyte binding sites than probes prepared with our original PAMS chemistry. This chemistry is applicable to any binding molecule that contains a primary amine and is suitable, therefore, for a wide range of applications.
A simple and effective method has been proposed in this work for combination of immunoaffinity extraction with MALDI MS. In this method, an antibody is attached to the surface of a MALDI probe tip via a thin nitrocellulose film. This allows the corresponding antigen to be selectively captured and concentrated on the probe tip from complex plasma solution for MALDI MS analysis. The whole procedure can be completed within 1 h. This combination offers several excellent performance features in the analysis of SNX-111, a therapeutic peptide. It combines the high specificity of affinity chromatography with the high sensitivity of mass spectrometry in a rapid analysis. Direct mass detection provides unambiguous determination by the observation of signals at characteristic m/z values. This method has been used successfully to determine the therapeutic peptide at relevant doses.
Lactoferricin is a bioactive peptide fragment (3196 Da) derived from lactoferrin (80 kDa) that contains the bactericidal domain and the lymphocyte receptor-binding domain of lactoferrin. Although lactoferricin has been produced from lactoferrin by proteolytic digestion in vitro, its natural occurrence and distribution in vivo are still not clear, in part because of the absence of a suitable detection means. Surface-enhanced laser desorption/ionization (SELDI) was used to detect and characterize lactoferricin by affinity mass spectrometry. Human, porcine, and bovine lactoferricin in unfractionated serum samples were found to bind specifically to ligands presenting a terminal n-butyl group. SELDI was used to detect and quantify each species of lactoferricin in a manner that was independent of the presence of intact lactoferrin, partially degraded lactoferrin, and lactoferrin peptides containing the lactoferricin peptide sequence. The limit of detection of bovine lactofericin in serum was as low as 200 pg/ml. The FKCRRWQWR-homoserine/-homoserine lactone moiety of bovine lactoferricin, which includes the complete antimicrobial center (i.e., RRWQWR), was shown to be responsible for interaction with the n-butyl group. The SELDI procedure defined here is the only molecular recognition tool known to date that is capable of distinguishing the multi-functional lactoferricin domain located within structurally related but distinct forms of lactoferrin and its metabolic fragments. Enabling the direct quantitation of lactoferricin produced in vivo opens new opportunities to evaluate lactoferrin function.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has in the past decade found routine use in the biological sciences. With this use has evolved several mass spectrometric-based methods directed at the intricate investigation of biomolecular structure and function. One such methodology involves the enzymatic modification of a protein prior to the mass spectrometric readout of the resulting products. The enzyme-modification/mass spectrometric approach has a definite use in a number of applications, including: the verification/identification of protein sequence, elucidation of post-translational modifications, the investigation of protein higher-order structure, and even the characterization of the modifying enzyme. To avoid the potentials of sample loss and autolytic interferences in the mass spectrum, mass spectrometer targets can be covalently derivatized with enzymes for use in the characterization procedures. The enzymatically active, or bioreactive, probes are used by application of the analyte to the activated surface, followed by application of a suitable MALDI matrix and mass analysis from the surface of the probe. Limited transfer and handling steps eliminate sample losses, and surface-tethered enzymes (and autolytic fragments) are prohibited from interfering with analytical signals in the mass spectra. In addition, the probes are rapid and easy to use. Reviewed here are issues of concern during the manufacture and use of the bioreactive probes, and application of the probes to investigate protein structure and function.
The ability to define specific alterations in the structure and function of proteins as they are introduced and processed in vivo remains an important goal. We have evaluated the generation, in vivo, of an antimicrobial peptide (lactoferricin) derived from ingested bovine lactoferrin by surface-enhanced laser desorption/ionization (SELDI). SELDI was used in the affinity mass spectrometry operational mode to detect and quantify lactoferricin directly from unfractionated gastric contents using a chemically defined ligand with a terminal n-butyl group as the lactoferricin affinity capture device. By this method, we were able to detect and quantify lactoferricin directly upon examination of unfractionated gastric contents recovered from an adult subject 10 min after ingestion of bovine lactoferrin (200 ml of 10 mg/ml (1.2 x 10(-4) mol/l) solution). Lactoferricin produced in vivo was directly captured by a surface-enhanced affinity capture (SEAC) device composed of molecules with a terminal n-butyl group and analyzed by laser desorption/ionization time-of-flight mass spectrometry. The recovery of standard lactoferricin or lactoferrin added to an aliquot of the gastric contents was determined to be nearly 100%, confirming the efficiency of this method. The amount of lactoferricin detected in the gastric contents was 16.9+/-2.7 microg/ml (5.4+/-0.8 x 10(-6) mol/l). However, a large proportion of ingested lactoferrin was found to be incompletely hydrolyzed. Lactoferrin fragments containing the lactoferricin region were analyzed by in situ pepsin hydrolysis after being captured on the SEAC device. Partially degraded lactoferrin fragments containing the lactoferricin region, including fragments corresponding to positions 17-43, 17-44, 12-44, 9-58 and 16-79 of the bovine lactoferrin sequence, were found to be present at concentrations as high as 5.7+/-0.7 x 10(-5) mol/l. These results suggest that significant amounts of bovine lactoferricin would be produced in the human stomach following ingestion of food, such as infant formula, supplemented with bovine lactoferrin. We propose that physiologically functional quantities of human lactoferricin could be generated in the stomach of breast-fed infants, and possibly, in the case of adults, from lactoferrin secreted into saliva.
Immobilized lectins have now been incorporated into affinity surfaces that can be used to isolate broad classes of samples for mass spectrometric analysis. A carbohydrate and a bacterial species that displays the carbohydrate binding motif were isolated and concentrated out of solutions containing salt, urea, buffers, and other contaminants that are deleterious to MALDI mass spectrometry. Concanavalin A was immobilized to a gold foil via a self-assembled monolayer. Samples in phosphate buffer or urine were applied to the capture surface and allowed to interact. The capture surface was then washed to remove salts and other unbound components and subjected to matrix-assisted laser desorption/ionization on a time-of-flight mass spectrometer. The lectin-derivatized surface allowed samples to be concentrated and readily characterized at relatively low levels.
A bioaffinity probe based on the idea of immobilizing avidin on the probe surface to extract biotinylated oligosaccharide is described. The probe is produced by taking advantage of the natural affinity of proteins for hydrophobic polymer films. The avidin is immobilized by simply drying the solution on a polymer film surface. This produces a bioaffinity probe that shows enhanced activity for biotin-labeled oligosaccharides. The probe is produced in a matter of minutes but is highly effective for concentrating biotinylated oligosaccharide on the surface. The best matrix for the analysis is DHB, and the best film for the probe is a polyester material commonly used for transparency film. The efficacy of the probe is illustrated with neutral and anionic oligosaccharides. Oligosaccharides derivatized with biotin are retained while those that are unlabeled are washed away. No trace of the unlabeled oligosaccharide is observed in the mass spectrum.
The profile of amyloid beta (A beta) peptide variants secreted into the media of human cultured cells that express the amyloid precursor protein was examined by Surface Enhanced Laser Desorption/Ionization (SELDI) ProteinChip technology from Ciphergen Biosystems using biologically active ProteinChip Arrays. An anti-A beta polyclonal antibody (anti-NTA4) was used to capture and purify multiple immunoreactive A beta fragments from a single microliter of media onto the ProteinChip Array. Fragments retained on the surface of the ProteinChip Array were detected directly by mass in the ProteinChip System to provide detailed information on the identity of different A beta variants secreted from the cultured cells. We discuss existing and potential applications of this immunoassay for the detection and relative quantitation of A beta species from both cultured cell systems and clinical samples.
Gas chromatography (GC)/surface ionization organic mass spectrometry (SIOMS) has been found to give much higher sensitivity for measurements of phencyclidine (PCP) than the conventional GC/electron impact (EI)-mass spectrometry (MS). Thus, we have established a detailed procedure for measurements of PCP in body fluids by both mass chromatography and selected-ion monitoring (SIM) of SIOMS using pethidine as an internal standard (IS). Good linearity was found in the range of 0.25-10 ng/mL of whole blood or urine, when measured by mass chromatography, and in the range of 0.025-1.0 ng/mL of whole blood by SIM. The recoveries of PCP and IS spiked to whole blood were 106 +/- 17% at 1 ng/mL and 113 +/- 11% at 5 ng/mL; that of IS was 97.8 +/- 10.4% at 5 ng/mL. The detection limits (signal-to-noise ratio = 3) were estimated to be 0.05 ng/mL of whole blood or urine by mass chromatography and 0.01 ng/mL of whole blood by SIM. The coefficients of intraday and interday variations were not greater than 10.3%. We could detect PCP from rat whole blood 2 h after subcutaneous injection of PCP (1 mg/kg) by mass chromatography. The mean PCP concentration in rat blood was 47.7 +/- 6.2 ng/mL (mean +/- SD, n = 4).
The overall history and recent advances in surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) technology is reviewed herein. Fundamentals of SELDI-TOF analysis are presented while drawing comparisons with other laser-based mass spectrometry techniques. The application of SELDI-TOF-MS to functional genomics and biomarker discovery is discussed and exemplified by elucidating a biomarker candidate for prostatic carcinoma. Finally, a short discussion regarding future SELDI requirements and developments is supplied.
beta-Amyloid (Abeta), a 39-43 residue peptide, is the principal component of senile plaques found in the brains of patients with Alzheimer's disease (AD). There are two main lines of evidence that its deposition is the cause of neurodegeneration. First, mutations found in three genes in familial Alzheimer's cases give rise to increased production of the longest, most toxic, form, Abeta 1-42. Second. aggregated Abeta is toxic to neuronal cells in culture. Inhibitors of the proteases involved in its release from the amyloid precursor protein are, therefore, of major therapeutic interest. The best candidates for the releasing proteases are both aspartyl proteases, which are integrated into the membranes of the endoplasmic reticulum and Golgi network. A sensitive assay using Ciphergen's Seldi system has been developed to measure all the variants of Abeta in culture supernatants, which will be of great value in screening inhibitors of these proteases. With this assay, it has been shown that increasing intracellular cholesterol increases the activities of both beta-secretase, and gamma-secretase 42. Moreover, changing the intracellular targeting of amyloid precursor glycoprotein (APP) yields increased alpha-secretase cleavage, and increases in the amounts of oxidized/nitrated forms of Abeta.
The determinants of binding of a peptide lacking C-termini-exposed lysine residues to a kringle domain were investigated using an up-regulated lysine binding kringle (K2Pg[C4G/E56D/K72Y]) of plasminogen and a peptide (a1-PAM) with a sequence derived from a surface-exposed M-like streptococcal protein. Significant kringle-induced chemical shifts in a His side-chain of a1-PAM were revealed by two-dimensional NMR. Further studies using isothermal titration calorimetry (ITC) provided support for the involvement of His12 in the peptide/ protein complex. In an effort to screen a1-PAM-derived truncation peptides, a combinatorial mixture, a1deltaa2-PAM[H12X] (where X=Pro, Arg, His, Trp, Lys, Ala, Phe, Asp and Gly), was analyzed using the surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI) platform. The major peptide that remained bound to the surface of the K2Pg[C4G/ E56D/K72Y]-containing chip was that containing His12, corresponding to the wild-type sequence. Minor peaks, representing binding, were obtained for Lys12-, Arg12- and Trp12-containing peptides. Individual peptides containing these amino acids were then examined using ITC and the binding constants obtained correlated with the relative strengths of binding estimated from the SELDI-based screen.
In a preliminary report (Bundy, J. L.; Fenselau, C. Anal. Chem 1999, 71, 1460-1463), we demonstrated the use of lectin-derivatized surfaces to capture and concentrate complex carbohydrates as well as microorganisms from sample matrixes unamenable to direct MALDI mass spectrometry. Here, we extend the work to include samples representative of a wider variety of microorganisms of importance to human health and of enveloped viruses. In this study, lectins were immobilized directly to a membrane surface via primary amines. A complementary approach was also explored, using immobilized carbohydrates to capture bacteria via microbial lectins expressed on their surfaces. The carbohydrate-based surfaces were constructed by first immobilizing streptavidin to the membrane, followed by attachment of a commercially produced biotin/carbohydrate polymer. Acid treatment of the sample prior to mass spectrometric analysis permits the observation of protein biomarkers from the captured microbial samples in the 5-20 kDa mass range. Bacteria samples were detected from physiological buffers, urine, milk, and processed chicken samples using the biocapture probes. Viral samples were detected from culture based on glycoprotein moieties desorbed directly from the surface. The carbohydrate-based system provided greater sensitivity than the lectin system, possibly due to the larger number of accessible saccharide ligands on the polymer.
This article presents current trends and advances in protein biochip technologies that rely upon extraction and retention of target proteins from liquid media. Analytical strengths as well as technical challenges for these evolving platforms are presented with particular emphasis on selectivity, sensitivity, throughput and utility in the post-genome era. A general review of protein biochip technology is provided, which delineates approaches for protein biochip format and operation, as well as protein detection. A focused discussion of three protein biochip technologies, Biomolecular Interaction Analysis (Biacore, Uppsala, Sweden), Surface Enhanced Laser Desorption/Ionisation (SELDI) ProteinChip Arrays (Ciphergen Biosystems, Fremont, CA, USA) and Fluorescent Planar Wave Guide (Zeptosens, Witterswil, Switzerland), follows along with examples of relevant applications.
A transgenic mouse expressing the human beta-amyloid precursor protein with the "Swedish" mutation, Tg2576, was used to investigate the mechanism of amyloid-beta peptide (Abeta) deposition. We characterized Abeta deposits in the cerebral cortex biochemically and pathologically. A surface-enhanced laser desorption/ionization affinity mass spectrometric study using the 6E10 monoclonal antibody demonstrated that the major species of Abeta in a formic acid-extracted fraction of the cortex were Abeta(1-38), Abeta(1-40) and Abeta(1-42). Immunohistochemistry using antibodies to the carboxy-terminal epitopes of Abeta(1-40) and Abeta(1-42), as well as 6E10, showed that plaques containing Abeta(1-42) were more numerous than those containing Abeta(1-40) throughout the cortex. Laser confocal analysis of the immunoreactivities in the plaques demonstrated that Abeta(1-40) was preferentially located in the central part of the Abeta(1-42) positive plaques. Enzyme-linked immunosorbent assay measurements of Abeta(1-40) and Abeta(1-42) showed that Abeta(1-40) was several-fold more abundant than Abeta(1-42). From these data we suggest that Abeta(1-42) deposition may precede Abeta(1-40) deposition, while Abeta(1-40) begins to deposit in the central part of the plaques and accumulates there. Furthermore, localization of Abeta(1-40) corresponded almost exactly to congophilic structures, which were associated with aberrant swollen synapses detected with antibodies to synaptophysin and alpha-synuclein. Thus, Abeta deposits in Tg2576 mice have similar characteristics to those in Alzheimer's disease.
The lack of a sensitive immunoassay for quantitating serum prostate-specific membrane antigen (PSMA) hinders its clinical utility as a diagnostic/prognostic biomarker. An innovative protein biochip immunoassay was used to quantitate and compare serum PSMA levels in healthy men and patients with either benign or malignant prostate disease. PSMA was captured from serum by anti-PSMA antibody bound to ProteinChip arrays, the captured PSMA detected by surface-enhanced laser desorption/ionization mass spectrometry, and quantitated by comparing the mass signal integrals to a standard curve established using purified recombinant PSMA. The average serum PSMA value for prostate cancer (623.1 ng/ml) was significantly different (P < 0.001) from that for benign prostate hyperplasia (117.1 ng/ml) and the normal groups (age <50, 272.9 ng/ml; age >50, 359.4 ng/ml). These initial results suggest that serum PSMA may be a more effective biomarker than prostate-specific antigen for differentiating benign from malignant prostate disease and warrants additional evaluation of the surface-enhanced laser desorption/ionization PSMA immunoassay to determine its diagnostic utility.
Cortical granule lectin (CGL) is released by the egg of the South African toad Xenopus laevis upon fertilization. The lectin binds to oligosaccharides in the extracellular matrix of the egg to form a physical block to prevent additional sperm penetration or polyspermy. To identify the oligosaccharides that bind to CGL, the lectin was immobilized on the surface of a matrix-assisted laser desorption/ionization probe. This bioaffinity probe was used to determine oligosaccharides that bind preferentially to CGL. Structural analyses based on collision-induced dissociation was used to determine that oligosaccharides with the sulfate esters at the nonreducing ends preferentially bind to the lectin.
Human beta-defensins are antimicrobial peptides that may be critical in the innate immune response to infection. hBD1 and hBD2 are expressed in oral epithelial cells and are detected near the surface of oral tissue, consistent with a role in the epithelial protective barrier function. In this report, we examine secretion of beta-defensins in vitro and in biological fluid using ProteinChip(R) Array, surface enhanced laser desorption/ionization (SELDI) technology combined with time-of-flight mass spectrometry. We show that the 47-amino acid form of hBD1 and the 41-amino acid form of hBD2 are the major secreted forms. These forms are both expressed and secreted under conditions anticipated from previous analysis of beta-defensin mRNAs; specifically, hBD1 is detected in culture supernatant from both unstimulated and stimulated cells, and hBD2 is detected only in stimulated cells. Identity of hBD1 and hBD2 was confirmed by immunocapture on the ProteinChip surface. Both peptides are also present in gingival crevicular fluid that accumulates between the tissue and tooth surface, although hBD1 is also found in several smaller forms suggesting extracellular proteolysis. This methodology offers several technical advantages for detection of defensins in biological fluids, including ease and speed of screening, no need for HPLC preliminary processing, and small sample size.