Article

Novel Application of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) in Shotgun Proteomics: Comprehensive Profiling of Rat Kidney Proteome

Article

Novel Application of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) in Shotgun Proteomics: Comprehensive Profiling of Rat Kidney Proteome

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Abstract

In shotgun proteomics, multidimensional liquid chromatography (MDLC) is commonly used to reduce sample complexity and increase dynamic range of protein identification. Since reversed-phase chromatography is mostly used as the second-dimensional separation before mass spectrometric analysis, the improvement of MDLC primarily depends on the first dimension of separation. Here, we present a novel whole proteome analysis method that separates peptides based on ERLIC. Tryptic peptides were retained on a weak anion exchange column through ERLIC with a high organic mobile phase. They were then distributed into multiple fractions based on both pI and polarity through the simultaneous effect of electrostatic repulsion and hydrophilic interaction when eluted using a salt-free pH gradient of increasing water content. Applying this to rat kidney tissue, we identified 4821 proteins and 30 659 unique peptides with high confidence from two replicates using LTQ-FT. This was 36.2% and 64.3% higher, respectively, than was obtained with the widely used SCX separation mode. Notably, the identification of both highly hydrophobic and basic peptides increased over 120% using the ERLIC method. The results indicate that ERLIC is a promising alternative to SCX as the first dimension of MDLC. In total, 5499 proteins and 35 847 unique peptides of rat kidney tissue are characterized.

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... Each type of HPLC, either conventional or UHPLC, has undergone significant developments lately and exciting new separation combinations of different stationary phases or the use of unexpected mobile phases for conventional ion-exchange or HILIC stationary phases have been introduced. [60][61][62] However, reversed-phase chromatography (RPC) remains the most widely used separation technique for sample preparation or separation for both proteins and peptides. Reversed phase chromatography is, based on interaction between the sample and the hydrophobic stationary phase. ...
... Hao et al. [60] compared the analysis of kidney proteome using the multidimensional peptide separation with SCX and RP-LC-MS=MS and the newly developed ERLIC and RP-LC-MS=MS. [62] Authors showed that ''Compared with the widely used SCX-RP sequence, the ERLIC-RP sequence identified significantly higher numbers of proteins and unique peptides from rat kidney tissue. Interestingly, many more basic and hydrophobic peptides were identified in this method than in SCX-RP, which is encouraging for potential use in the analysis of membrane proteins.'' ...
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During the last ten to fifteen years a huge development in the field of analytical techniques used for deciphering the complex nature of the proteome took place. Instrumentation and methods development for sample cleanup, fractionation, preconcentration, chromatographic separation, and detection become affordable to a broad range of laboratories and new, exciting methods for both separation and detection were published and applied. Newly developed techniques and equipment for separation and detection, such as high pressure nano-HPLC and new techniques for multidimensional HPLC separation, enabled proteomics to experience dynamic growth and enter new paths. Furthermore, proteomics starts entering the “real life” and it is increasingly applied for clinical diagnostics and follow-up of patient's status during the treatment.For any proteomic analysis, one of the most important and sometimes the most difficult task is the separation of the complex mixtures of proteins or peptides prior to their detection and data analysis. This review describes some aspects and limitations of HPLC, both multidimensional and one-dimensional, in proteomics research without attempting to discuss all available HPLC methods, their benefits and shortcomings which would need far more space than available here.
... Reducing protein sample complexity in two-dimensional liquid chromatography (2D-LC) investigations is a crucial issue. To this effect, various techniques of peptide separation prior to reverse phase liquid chromatography (RPLC) hyphenated to mass spectrometry (MS) detection are employed: electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) [18,19], hydrophilic interaction chromatography (HILIC) [20], ion exchange chromatography (IEX) (e.g., strong cation exchange [SCX]) [21], and RPLC with alkaline and acidic mobile phases [22,23]. In addition, recently http://dx. ...
... Reducing protein sample complexity in two-dimensional liquid chromatography (2D-LC) investigations is a crucial issue. To this effect, various techniques of peptide separation prior to reverse phase liquid chromatography (RPLC) hyphenated to mass spectrometry (MS) detection are employed: electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) [18,19], hydrophilic interaction chromatography (HILIC) [20], ion exchange chromatography (IEX) (e.g., strong cation exchange [SCX]) [21], and RPLC with alkaline and acidic mobile phases [22,23]. In three-dimensional sample separations were also envisaged and developed for different purposes: reverse phase (RP)-SCX-RP approach allowing higher detection of hydrophilic peptides and consequently enhancing the quality of protein identification and quantification [24,25], three-dimensional (3D)-LC/MS setup for enrichment and identification of biotinylated proteins [26], and combinatorial use of SCX (ERLIC-SCX) chromatographic separations for phosphoproteome studies [27]. ...
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In the frame of proteins identification from mouse adipose tissue, two strategies were compared for the off-line elution of peptides from the SCX column in 2D-LC-MS/MS analyses. First, the salt gradient (using K(+) as displacing agent) was evaluated from 25 to 500 mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptides digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. Fifteen fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC-MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptides quality characteristics (molecular weight, isoelectric point or GRAVY index distributions) were observed between the two datasets, The pH gradient approach was found superior with 27.5% more unique peptides and 10% more distinct proteins identifications compared to the salt-based elution method. In conclusion, our data imply the pH gradient SCX fractionation more desirable for proteomic analysis of entire adipose tissue. Copyright © 2015. Published by Elsevier Inc.
... Given that the stationary phase used as the second dimension separation in nano-LC-MS/MS setups is almost exclusively comprised of reversed-phase (RP) material, the first peptide separation dimension should ideally be orthogonal to RP and offer high chromatographic resolution. Many different techniques have been employed for this purpose such as HILIC [11], ZIC-HILIC [12], ERLIC [13], WAX [14], high-pH reversed-phase [15], SAX [16], or SCX [17]. Recently Ritorto et al. have demonstrated the merits of hSAX chromatography [18], which separates peptides primarily based on the number of acidic residues and the stationary phase is characterized by ultralow hydrophobicity. ...
Chapter
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The bottom-up proteomic analysis of cell line and tissue samples to a depth > 10,000 proteins still represents a considerable challenge because of the sheer number of peptides generated by proteolytic digestions and the high dynamic range of protein expression. As a result, comprehensive protein coverage requires multidimensional peptide separation. Recently, off-line hydrophilic strong cation exchange (hSAX) chromatography has proven its merits for high resolution separation of peptides due to its high degree of orthogonality to reversed-phase liquid chromatography. Here we describe the use of hSAX for the deep analysis of tissue proteomes. The protocol includes optimized sample preparation steps (lysis with the aid of mechanical disruption, one-step disulfide bridge reduction and alkylation), setup and operation of hSAX columns and gradients, desalting of hSAX fractions prior to LC-MS/MS analysis, and suggestions for the choice of data acquisition parameters and data analysis using MaxQuant. Application of the protocol to the fractionation of 300 μg human brain tissue digest led to the identification of more than 100,000 unique peptide sequences representing over 10,195 proteins and 9,500 genes in 3 days of measurement time on a Q Exactive Plus mass spectrometer.
... For quantitative protein profiling, tryptic peptides have been labeled with iTRAQ reagents which are available in 4-plex or 8-plex kits (Adav et al., 2011a(Adav et al., , 2012a. To improve protein identification, quantification and total coverage of proteome/secretome, an electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) technique has been developed and adopted for iTRAQ labeled peptides (Adav et al., 2010bHao et al., 2010. The peptide elution profile using ERLIC technique together with high-performance liquid chromatography gradient is shown in Fig. 8.4. ...
Chapter
Due to the depleting finite fossil fuel, the impact of energy crisis, global warming and environmental deterioration; interest has been kindled for sustainable bioresources. Chemicals and biofuel derived from lignocellulosic biomass are clean, sustainable, renewable and have a great potential in the future chemical and energy market. The main obstruction so far has been the suitable biomass hydrolysis technology. Due to the potential to produce copious amount of cellulases and their potential biotechnological application, especially with respect to lignocellulosic biofuels; Trichoderma sp. is comprehensively studied. The protein composition of the secretome by Trichoderma reesei and its mutant on lignocellulosic biomasses was explored using proteomics approach with high throughput isobaric tag for relative and absolute quantification technique. This chapter exploits state-of-the art proteomic profiling of secretome and their quantitative expressions during lignocellulosic biomass utilization. This technique highlighted diverse enzymes which are dependent on nature and complexity of different biomasses. The comparative substrate-dependent expressions of cellulolytic, hemicellulolytic, ligninolytic and proteolytic enzymes, their quantitative expressions and regulation during biomass degradation facilitated underlying synergetic mechanism for lignocellulose hydrolysis and further its application to lignocellulosic biofuel and sustainable development. The attempts have been made to correlate lignocellulosic biomass complexity and quantitative composition of T. reesei secretome with particular interest in economically attractive lignocellulosic biorefinery
... The suspension was sonicated (UP200H, Hielscher) on ice for 3 cycles of 60 kHz, each lasting 30 s, incubated for 1 hr at room temperature, and centrifuged (14000×g at 4°C for 15 min). After 1 hr of incubation at room temperature, the homogenate was centrifuged (3-30 K, Sigma) at 20,000×g at 4°C for 30 min (8). Protein concentration was measured using the modified Bradford assay with BSA standards (9). ...
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Transforming Growth Factor-beta (TGF-β) activation appears to be crucial for tissue injury in Diabetic Nephropathy (DN). Fibromodulin, the small leucine-rich proteoglycan, has been proposed to be the potent TGF-β modulator. In this study, the therapeutic effects of fibromodulin in the kidneys of streptozotocin (STZ)-induced diabetic rats were investigated. Diabetic rats received intraperitoneal (IP) injections of recombinant adenovirus expression vectors (RAd5) containing fibromodulin (RAd-FMOD) and were killed after 10 weeks. Proteins were isolated from the rat kidney and separated using two-dimensional gel electrophoresis. The differentially expressed proteins were analyzed using Matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Ten spots were identified using MALDI-TOF-MS. The identified proteins were primarily responsible for cell metabolism, cytoskeleton formation, and oxidative stress. RAd-FMOD treatment markedly attenuated the albuminuria in diabetic rats. Taken together, these results provide a valuable clue in exploring the mechanism underlying the therapeutic effects of fibromodulin in diabetic nephropathy suggesting that it can be a potential agent in the treatment of this disease.
... Two hundred micrograms of peptides from each condition were individually labeled with respective isobaric tags (control sample, 114; iberin-treated sample, 115), followed by 2 h of incubation, quenching by water, desalting using C 18 solid-phase extraction cartridge, and then vacuum centrifugation to dryness. The iTRAQ-labeled peptides were reconstituted in buffer A (10 mM ammonium acetate, 85% acetonitrile, 0.1% formic acid) and fractionated using an electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) column (200 by 4.6 mm, 5-m particle size, 200-Å pore size) by a high-performance liquid chromatography (HPLC) system (Shimadzu, Japan) at flow rate of 1.0 ml/min, using our previously optimized protocol (20). The HP liquid chromatograms were recorded at 280 nm, and fractions were collected online using an automated fraction collector. ...
Article
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Food is now recognized as a natural resource of novel antimicrobial agents including those that target the virulence mechanisms of bacterial pathogens. Iberin, an isothiocyanate compound from horseradish, was recently identified as a quorum sensing inhibitor (QSI) of the bacterial pathogen Pseudomonas aeruginosa. Here, we used a comparative systems biology approach to unravel the molecular mechanisms of iberin's effects on QS and virulence factor expression of P. aeruginosa. Our study shows that both of the systems biology methods used (i.e. RNA-sequencing and proteomics) complement each other and provide a thorough overview of the impact of iberin on P. aeruginosa. RNA sequencing-based transcriptomics showed that iberin inhibits the expression of the GacA-dependent small regulatory RNAs RsmY and RsmZ; this was verified by using gfp-based transcriptional reporter fusions with the rsmY or rsmZ promoter regions. iTRAQ proteomics showed that iberin reduces the abundance of the LadS protein, an activator of GacS. Taken together, the findings suggest that iberin's mode of QS inhibition is through down-regulation of the Gac/Rsm QS network which in turn leads to repression of QS-regulated virulence factors such as pyoverdine, chitinase and protease IV. Lastly, as expected from the observed repression of small regulatory RNA synthesis, we also show that iberin effectively reduces biofilm formation. This suggests that small regulatory RNAs might serve as potential targets for the future development of therapies against pathogens that use QS for controlling virulence factor expression and assume the biofilm mode of growth in the process of causing disease.
... To reduce sample complexity and enhance separation, protein identification, and its confidence, we reconstituted the iTRAQ labeled peptides in buffer A (10 mM ammonium acetate, 85% acetonitrile, 0.1% formic acid) and fractionated it by ERLIC method using a PolyWAX LP column (4.6 × 200 mm, 5 μm, 300 Å) (PolyLC, Columbia, MD) adopting the HPLC system (Shimadzu) at a flow rate of 1.0 mL/min using our optimized laboratory protocol. 30,31 The HPLC mobile phase consisted of buffer A (10 mM ammonium acetate, 85% acetonitrile, 0.1% acetic acid) and buffer B (30% acetonitrile, 0.1% formic acid), while 60 min gradient was 100% buffer A for 5 min, 0−36% buffer B for 25 min, 36−100% buffer B for 20 min, and 100% buffer B for 10 min at 1 mL/min flow rate. The HPLC chromatograms were recorded at 280 nm, and fractions were collected using automated fraction collector, concentrated using vacuum centrifuge, and reconstituted in 0.1% formic acid for LC−MS/MS analysis. ...
Article
Dementia is a major public health burden characterized by impaired cognition and loss of function. There are limited treatment options due to inadequate understanding of its pathophysiology and underlying causative mechanisms. In order to elucidate the perturbed pathways contributing to pathophysiology of Vascular Dementia (VaD), discovery-driven iTRAQ-based quantitative proteomics techniques were applied on frozen brain samples to profile the proteome from VaD and age-matched non-dementia controls. The iTRAQ quantitative data revealed significant up-regulation of Protein-L-isoaspartate O-methyltransferase (PIMT) and sodium-potassium transporting ATPase, while post-translational modification analysis suggested deamidation of catalytic and regulatory subunits of sodium-potassium transporting ATPase. Spontaneous protein deamidation of labile asparagines, generating abnormal L-isoaspartyl residues, is associated with cell aging, dementia due to Alzheimer's Disease (AD) and may be a cause of neurodegeneration. As ion channel proteins play important roles in cellular signaling processes, alterations in their function by deamidation may lead to perturbations in membrane excitability and neuronal function. Structural modeling of sodium-potassium transporting ATPase revealed close proximity of these deamidated residues to the catalytic site during E2P confirmation. The deamidated residues may disrupt electrostatic interaction during E1 phosphorylation which may affect ion transport and signal transduction. Our findings suggest impaired regulation and compromised activity of ion channel proteins contribute to the pathophysiology of VaD.
... The digests were quenched by the addition of formic acid to 1%, followed by desalting by sep-pak (Waters, Milford, MA). Peptides were then fractionated with electrostatic repulsion-hydrophilic interaction chromatography 57 . Fractions were assayed for protein concentration using a BCA assay and pooled into 18 fractions of equal protein concentration, evaporated to dryness and resuspended in 100 ml of 0.2% formic acid. ...
Article
Mass spectrometry (MS) instruments and experimental protocols are rapidly advancing, but the software tools to analyse tandem mass spectra are lagging behind. We present a database search tool MS-GF+ that is sensitive (it identifies more peptides than most other database search tools) and universal (it works well for diverse types of spectra, different configurations of MS instruments and different experimental protocols). We benchmark MS-GF+ using diverse spectral data sets: (i) spectra of varying fragmentation methods; (ii) spectra of multiple enzyme digests; (iii) spectra of phosphorylated peptides; and (iv) spectra of peptides with unusual fragmentation propensities produced by a novel alpha-lytic protease. For all these data sets, MS-GF+ significantly increases the number of identified peptides compared with commonly used methods for peptide identifications. We emphasize that although MS-GF+ is not specifically designed for any particular experimental set-up, it improves on the performance of tools specifically designed for these applications (for example, specialized tools for phosphoproteomics).
... The concentrated iTRAQ-labeled peptides from the three sets were then desalted separately using Sep-Pak C18 cartridges and then ERLIC fractionated by high-performance liquid chromatography (HPLC). The iTRAQ-labeled peptides were reconstituted in buffer A (10 mM ammonium acetate, 85% acetonitrile, 0.1% formic acid) and fractionated on a PolyWAX LP column (4.6 × 200 mm, 5 μm, 300 Å) (PolyLC, Columbia, MD, USA) adopting the HPLC system (Shimadzu, Kyoto, Japan) at a flow rate of 1 ml/min (Hao et al., 2010). The 60 min gradient HPLC mobile phases consisted of buffer A (10 mM ammonium acetate, 85% acetonitrile, 0.1% acetic acid) and buffer B (30% acetonitrile, 0.1% formic acid) as follows: 100% buffer A for 5 min, 0-36% buffer B for 25 min, 36-100% buffer B for 20 min, and 100% buffer B for 10 min at 1 ml/min flow rate. ...
... Thus our SCX-SPE step separated peptides by charge, a mechanism exploited in several SCX-based techniques [38][39][40]. In ERLIC-2 the average pI decreases with the retention time since acidic peptides are well retained (Fig. S-4) as reported previously [41,42]. Thus, ERLIC-2 is an ideal technique for separation along the entire pI range of tryptic peptides. ...
Article
Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~ 75% more proteins and even ~ 134% more peptides compared to the 2D strategy. Additionally, we could quantify ~ 50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research.
... In this way, ERLIC elutes peptides in order of increasing hydrophobicity and acidity. ERLIC has proven effective at separating and identifying modified and unmodified proteins [119][120][121][122]. Smaller-diameter columns with lower loading capacities and smaller stationary phase particles offer an advantage in microproteomics. ...
Article
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Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed.
... The iTRAQ ratios of 114/113 (cellulose/glucose), 115/113 (xylan/ glucose), and 116/113 (starch/glucose) are presented in this study. The iTRAQ labeled peptides were reconstituted in buffer A (10 mM ammonium acetate, 85% acetonitrile, 0.1% formic acid) and fractionated using ERLIC column (200 × 4.6 mm, 5 μm particle size, 300 Å pore size) by a HPLC system (Shimadzu, Japan) at a flow rate of 1.0 mL min −1 using our previously optimized protocol [36]. The HPLC chromatograms were recorded at 280 nm, and fractions were collected using an automated fraction collector and vacuum dried. ...
... The advantage of iTRAQ labeling is that the signal obtained from combined peptides enhances the sensitivity of detection in MS/MS. However, the variability in labeling efficiencies and the costly reagents are major limitations of this method [74]. Labeling also increases complexity of the samples and can reduce number of the identified peptides during MS/MS run. ...
Article
The effectiveness of treatment of renal diseases is limited because the lack of diagnostic, prognostic and therapeutic markers. Despite the more than a decade of intensive investigation of urinary biomarkers, no new clinical biomarkers were approved. This is in part because the early expectations toward proteomics in biomarkers discovery were significantly higher than the capability of technology at the time. However, during the last decade, proteomic technology has made dramatic progress in both the hardware and software methods. In this review we are discussing modern quantitative methods of mass-spectrometry and providing several examples of their applications for discovery and validation of renal disease biomarkers. We are optimistic about future prospects for the development of novel of specific clinical urinary biomarkers.
... Zwitterionic HILIC (ZIC-HILIC) was developed and evaluated as a multidimensional separation approach and was found to resemble the 2D SCX separation [107]. Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), a variation of HILIC, has been shown to perform better than SCX as an orthogonal separation technique [108]. ERLIC has also been applied to explore the phosphoproteome-as well as glycoproteome-from rat kidneys [109]. ...
Article
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Alzheimer’s disease (AD) is an irreversible neurodegenerative disease characterized by progressive cognitive decline. The two cardinal neuropathological hallmarks of AD include the buildup of cerebral β amyloid (Aβ) plaques and neurofibrillary tangles of hyperphosphorylated tau. The current disease-modifying treatments are still not effective enough to lower the rate of cognitive decline. There is an urgent need to identify early detection and disease progression biomarkers that can facilitate AD drug development. The current established readouts based on the expression levels of amyloid beta, tau, and phospho-tau have shown many discrepancies in patient samples when linked to disease progression. There is an urgent need to identify diagnostic and disease progression biomarkers from blood, cerebrospinal fluid (CSF), or other biofluids that can facilitate the early detection of the disease and provide pharmacodynamic readouts for new drugs being tested in clinical trials. Advances in proteomic approaches using state-of-the-art mass spectrometry are now being increasingly applied to study AD disease mechanisms and identify drug targets and novel disease biomarkers. In this report, we describe the application of quantitative proteomic approaches for understanding AD pathophysiology, summarize the current knowledge gained from proteomic investigations of AD, and discuss the development and validation of new predictive and diagnostic disease biomarkers.
... When comparing the pI values for the early to intermediate/late fractions, a trend for decreasing (from 5.2 to 2.7) and increasing (from 4.0 to 6.1) values were observed for ERLIC and SCX, respectively. This agrees with earlier observations that basic peptides elute early in ERLIC [39] and that the addition of basic groups in general increases the retention in SCX [40]. Furthermore, the values for direct analysis after in-solution digestion and for insolution digestion with subsequent TiO 2 enrichment were compared but did not differ significantly. ...
Article
Full-text available
Phosphorylation of proteins is important for controlling cellular signaling and cell cycle regulatory events. The process is reversible and phosphoproteins normally constitute a minor part of the global proteome in a cell. Thus, sample preparation techniques tailored for phosphoproteome studies are continuously invented and evaluated. This paper aims at evaluating the performances of the most popular techniques for phospho-enrichments in sub-proteome analysis, such as viral proteomes expressed in human cells during infection. A two-species sample of Adenovirus type 2 infected human cells was used, and in-solution digestion, strong cation exchange (SCX), and electrostatic repulsion hydrophilic interaction chromatography (ERLIC) fractionation, and subsequent enrichment by TiO2, were compared with SDS-PAGE fractionation and in-gel digestion. Evaluation was focused on phosphopeptide detection in the sub-proteome. The results showed that the SCX+TiO2 or ERLIC+TiO2 combinations had the highest enrichment efficiencies, but SDS-PAGE fractionation and in-gel digestion resulted in the highest number of identified proteins and phosphopeptides. Furthermore, the study demonstrates the usefulness of applying as many orthogonal techniques as possible in deep phosphoproteome analysis, since the overlap between approaches was low. Graphical Abstract The phosphoproteome originating from Adenovirus type 2 infected cells was investigated with different phosphopeptide-selective techniques
... Fractionation of peptides by high-pressure liquid chromatography (HPLC) was performed as previously described in [16] and [53]. Desalted peptides were re-dissolved in 200 μL mobile phase A (85 % acetonitrile (ACN), 0.1 % acetic acid). ...
Article
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Background Extracellular vesicles (EVs) act as key mediators of intercellular communication and are secreted and taken up by all cell types in the central nervous system (CNS). While detailed study of EV-based signaling is likely to significantly advance our understanding of human neurobiology, the technical challenges of isolating EVs from CNS tissues have limited their characterization using ‘omics’ technologies. We therefore developed a new Protein Organic Solvent Precipitation (PROSPR) method that can efficiently isolate the EV repertoire from human biological samples. ResultsIn the current report, we present a novel experimental workflow that outlines the process of sample extraction and enrichment of CNS-derived EVs using PROSPR. Subsequent LC-MS/MS-based proteomic profiling of EVs enriched from brain homogenates successfully identified 86 of the top 100 exosomal markers. Proteomic profiling of PROSPR-enriched CNS EVs indicated that > 75 % of the proteins identified matched previously reported exosomal and microvesicle cargoes, while also expanded the known human EV-associated proteome with 685 novel identifications. Similarly, lipidomic characterization of enriched CNS vesicles not only identified previously reported EV-specific lipid families (PS, SM, lysoPC, lysoPE) but also uncovered novel lipid isoforms not previously detected in human EVs. Finally, dedicated flow cytometry of PROSPR-CNS-EVs revealed that ~80 % of total microparticles observed were exosomes ranging in diameter from ≤100 nm to 300 nm. Conclusions These data demonstrate that the optimized use of PROSPR represents an easy-to-perform and inexpensive method of enriching EVs from human CNS tissues for detailed characterization by ‘omics’ technologies. We predict that widespread use of the methodology described herein will greatly accelerate the study of EVs biology in neuroscience.
... LC-MS/MS and Data Analysis-LC-MS/MS was carried out as described previously (29). Briefly, peptides were separated and analyzed on a liquid chromatograph (Dionex UltiMate 3000 Nano-LC Systems, Dionex, Singapore) at a 300 nl/min flow rate coupled to an LTQ-FT Ultra mass spectrometer (Thermo Electron, Bremen, Germany). ...
Article
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Progesterone receptor (PR) exists in two isoforms, PRA and PRB, and both contain activation functions AF-1 and AF-2. It is believed that AF-1 is primarily responsible for the ligand-independent activity, whereas AF-2 mediates ligand-dependent PR activation. Although more than a dozen post-translational modifications of PR have been reported, no post-translational modification on AF-1 or AF-2 has been reported. Using LC-MS/MS-based proteomic analysis, this study revealed AF-1 monomethylation at Lys-464. Mutational analysis revealed the remarkable importance of Lys-464 in regulating PR activity. Single point mutation K464Q or K464A led to ligand-independent PR gel upshift similar to the ligand-induced gel upshift. This upshift was associated with increases in both ligand-dependent and ligand-independent PR phosphorylation and PR activity due to the hyperactivation of AF-1. In contrast, mutation of Lys-464 to the bulkier phenylalanine to mimic the effect of methylation caused a drastic decrease in PR activity. Importantly, PR-K464Q also showed heightened ligand sensitivity, and this was associated with increases in its functional interaction with transcription co-regulators NCoR1 and SRC-1. These results suggest that monomethylation of PR at Lys-464 probably has a repressive effect on AF-1 activity and ligand sensitivity.
... The tryptic peptides derived from the atherosclerotic plaque samples were prepared as previously described, with minor modifications 45 . Peptides generated from approximately 1 mg of proteins were fractionated using ERLIC on a HPLC system and then combined into 20 separate fractions 67 . Peptides from each fraction were separated and analyzed in triplicate on a Dionex Ultimate 3000 RSLCnano system coupled to a Q Exactive instrument (Thermo Scientific, San Jose, USA). ...
Article
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Atherosclerosis arises from leukocyte infiltration and thickening of the artery walls and constitutes a major component of vascular disease pathology, but the molecular events underpinning this process are not fully understood. Proteins containing an Asn-Gly-Arg (NGR) motif readily undergo deamidation of asparagine to generate isoDGR structures that bind to integrin αvβ3 on circulating leukocytes. Here we report the identification of isoDGR motifs in human atherosclerotic plaque components including extracellular matrix (ECM) proteins fibronectin and tenascin C, which have been strongly implicated in human atherosclerosis. We further demonstrate that deamidation of NGR motifs in fibronectin and tenascin C leads to increased adhesion of the monocytic cell line U937 and enhanced binding of primary human monocytes, except in the presence of a αvβ3-blocking antibody or the αv-selective inhibitor cilengitide. In contrast, under the same deamidating conditions monocyte-macrophages displayed only weak binding to the alternative ECM component vitronectin which lacks NGR motifs. Together, these findings confirm a critical role for isoDGR motifs in mediating leukocyte adhesion to the ECM via integrin αvβ3 and suggest that protein deamidation may promote the pathological progression of human atherosclerosis by enhancing monocyte recruitment to developing plaques.
... Moreover, co-eluting components, non-observed during the chromatography, could obscure the analysis, since they might lead to suppression or enhancement of the analyte [12]. Modern and accurate approaches in proteomic analysis, combine RPC and chromatographic techniques such as ERLIC-RP [13], HILIC-RP [14] and SCX [15,16]. In the present study, quantification of peptide was performed by RP-HPLC after gradient elution [17]. ...
Article
The recovery of high molecular weight peptides from complex biological samples is a challenging task. Herein, a reliable, cost effective and rapid methodology was developed for the recovery and quantification of a myelin oligodendrocyte glycoprotein epitope namely (LysGly)5MOG35-55 from rat plasma. Removal of plasma proteins before quantification of the peptide was achieved after precipitation by an acetonitrile/water/formic acid solution. Using the developed protocol, average recoveries of the peptide from plasma ranged between 83.3 and 90.3%.
... Therefore, orthogonal fractionation of the peptide mixture is beneficial for increased sensitivity. "Offline" electrostatic repulsion hydrophilic interaction chromatography (ERLIC) fractionates peptides based on charge (Alpert, 2008;Hao et al., 2010) prior to reversed-phase chromatography of each ERLIC fraction during LC-MS/MS. It is expected that fractions at the end of the ERLIC gradient will contain relatively fewer peptides than initial fractions, so fraction volume is recommended to be scaled accordingly (see step 5 below). ...
Method
Small open reading frames (smORFs) encode previously unannotated polypeptides or short proteins that regulate translation in cis (eukaryotes) and/or are independently functional (prokaryotes and eukaryotes). Ongoing efforts for complete annotation and functional characterization of smORF‐encoded proteins have yielded novel regulators and therapeutic targets. However, because they are excluded from protein databases, initiate at non‐AUG start codons, and produce few unique tryptic peptides, unannotated small proteins cannot be detected with standard proteomic methods. Here,, we outline a procedure for mass spectrometry‐based detection of translated smORFs in cultured human cells from protein extraction, digestion, and LC‐MS/MS, to database preparation and data analysis. Following proteomic detection, translation from a unique smORF may be validated via siRNA‐based silencing or overexpression and epitope tagging. This is necessary to unambiguously assign a peptide to a smORF within a specific transcript isoform or genomic locus. Provided that sufficient starting material is available, this workflow can be applied to any cell type/organism and adjusted to study specific (patho)physiological contexts including, but not limited to, development, stress, and disease. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Protein extraction, size selection, and trypsin digestion Alternate Protocol 1: In‐solution C8 column size selection Support Protocol 1: Chloroform/methanol precipitation Support Protocol 2: Reduction, alkylation, and in‐solution protease digestion Support Protocol 3: Peptide de‐salting Basic Protocol 2: Two‐dimensional LC‐MS/MS with ERLIC fractionation Basic Protocol 3: Transcriptomic database construction Alternate Protocol 2: Transcriptomics database generation with gffread Basic Protocol 4: Non‐annotated peptide identification from LC‐MS/MS data Basic Protocol 5: Validation using isotopically labeled synthetic peptide standards and siRNA Basic Protocol 6: Transcript validation using transient overexpression
... It has been shown that deamidated peptides or protein subunits can be well separated from their native counterparts by hydrophilic interaction liquid chromatography (HILIC) [56,57]. To improve the separation even further, the Sze group developed an electrostatic repulsion hydrophilic interaction chromatography (ERLIC) method that is capable of separating peptides based on their isoelectric points and hydrophilicities [58][59][60][61]. In this method, RPLC was used in the first dimensional separation, the co-eluted deamidation-related peptides were then collected and subjected to ERLIC separation in the second dimension based on both their isoelectric point (pI) and polarity. ...
Article
Deamidation is a nonenzymatic and spontaneous posttranslational modification (PTM) that introduces changes in both structure and charge of proteins, strongly associated with aging proteome instability and degenerative diseases. Deamidation is also a common PTM occurring in biopharmaceutical proteins, representing a major cause of degradation. Therefore, characterization of deamidation alongside its inter-related modifications, isomerization and racemization, is critically important to understand their roles in protein stability and diseases. Mass spectrometry (MS) has become an indispensable tool in site-specific identification of PTMs for proteomics and structural studies. In this review, we focus on the recent advances of MS analysis in protein deamidation. In particular, we provide an update on sample preparation, chromatographic separation, and MS technologies at multi-levels, for accurate and reliable characterization of protein deamidation in both simple and complex biological samples, yielding important new insight on how deamidation together with isomerization and racemization occurs. These technological progresses will lead to a better understanding of how deamidation contributes to the pathology of aging and other degenerative diseases and the development of biopharmaceutical drugs.
... Different detergents, digestion buffers, enzyme-tosubstrate ratio and digestion time were tested using a similar procedure to that previously described. 16 For details, see Table S1 (supporting information). ...
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Rationale: The high specificity of trypsin is a prerequisite for accurate protein identification and quantification in shotgun proteomics. It is of great importance to minimize nonspecific enzymatic cleavages during proteomic sample preparation. Methods: In this study, protein extraction and trypsin digestion conditions were extensively evaluated using the less complex E.coli lysates to improve the sensitivity of detecting low-abundance nonspecific peptides by liquid chromatography tandem mass spectrometry (LC/MS/MS). Results: Trypsin digestion buffers and digestion time were proven to have a significant effect on nonspecific cleavages. The triethylammonium bicarbonate buffer induces significantly lower nonspecific cleavages than the other two buffers, but a freshly prepared urea solution does not induce more than sodium dodecyl sulfate (SDS). Since prolonged trypsin digestion resulted in a considerable number of nonspecific cleavages, an optimized 2h protocol was developed with 45.2% less semi-specific tryptic peptides but 18.5% more unmodified peptides identified than with the commonly used 16h protocol. Conclusions: The significant reduction of nonspecific cleavages and artificial modifications improves the accuracy of protein quantification and the identification of low-abundance proteins, and it is especially useful for studying protein posttranslational modifications. The proposed 2h trypsin digestion protocol can potentially be a replacement for the traditional 16h one.
... Instead, to take advantage of the unique features of ERLIC, studies generally integrated RPLC with ERLC in a 2D-LC mode, in which ERLIC was either used in the first dimension for sample fractionation, or used in the second dimension to further analyze the purified fraction from RPLC. 16,[19][20][21] Recently, a long-length ERLIC (LERLIC) material packed capillary column was used for targeted glutamine deamidation analysis in proteomic study, but with a relatively high run time (1200 min) to achieve satisfactory separation. 22 This long run time could potentially introduce method artifacts and dilute the loaded sample, resulting in lower sensitivity. ...
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Electrostatic repulsion hydrophilic interaction chromatography (ERLIC) coupled with mass spectrometry (MS) is a technique that is increasingly being used as a trapping/enrichment tool for glycopeptides/phosphorylated peptides or sample fractionation in proteomics research. Here, we describe a novel ERLIC-MS/MS-based peptide mapping method that was successfully used for the characterization of denosumab, in particular the analysis of sequence coverage, terminal peptides, methionine oxidation, asparagine deamidation and glycopeptides. Compared to reversed phase liquid chromatography (RPLC)-MS/MS methods, ERLIC demonstrated unique advantages in the retention of small peptides, resulting in 100% sequence coverage for both the light and heavy chains. It also demonstrated superior performance in the separation and characterization of asparagine deamidated peptides, which is known to be challenging by RPLC-MS/MS. The developed method can be used alone for peptide mapping-based characterization of monoclonal antibodies, or as an orthogonal method to complement the RPLC-MS/MS method. This study extends the applications of ERLIC from that of a trapping/fractioning column to biologic therapeutics characterization. The ERLIC-MS/MS method can enhance biologic therapeutics analysis with more reliability and confidence for bottom-up peptide mapping-based characterization.
... Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) is a novel mode chromatography designed to allow selective isolation of phosphopep tides from a tryptic digest. It is similar to HILIC except that the solid support has the same (or similar) charge as the molecules being separated, which causes electrostatic repulsion (Hao et al. 2010). By using a high organic buffer in the mobile phase, sol utes are retained on the column despite being electrostatically repulsed. ...
Chapter
Mass spectrometry imaging (MSI) and mass spectrometry (MS) profiling are two especially suitable methods for recording and visualizing microscopic molecular details. Mass spectrometry imaging of larger molecules, such as proteins and intact peptides, was demonstrated using a gentler imaging technique, termed matrix‐assisted laser/desorption ionization (MALDI). This chapter presents an overview of MSI and MS profiling instrumentation, sample preparation, and data analysis, with a focus on peptide and protein detection. It provides specific examples of MSI and MS profiling in biomarker discovery. Biomarkers are commonly considered to be indicators of a disease state or a therapeutic response. They have proven useful in many fields of research, including fundamental neuroscience, microbiology, and plant biology. Matrix‐assisted laser/desorption ionization‐MSI and MS profiling are valuable tools for characterizing chemical signaling in animal nervous systems, particularly for signaling peptides, which can be both detected and sequenced on tissue.
... Plasma samples were purified by cold (−20 °C) acetone precipitation and redissolved in a sodium deoxycholate ammonium acetate buffer for reduction, alkylation and trypsin in solution digestion overnight 38 . The tryptic peptides were then fractionated using the ERLIC chromatography method as previously described 39 . Each fraction of the plasma tryptic peptides and three pairs of synthetic targeted peptides were separated and analyzed on a Dionex Ultimate 3000 RSLC NanoLC system coupled to a Q-Exactive tandem mass spectrometer (Thermo Fisher, MA) LC-MS/ MS system as previously described 9 . ...
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Metabolic disorders in T2DM generate multiple sources of free radicals and oxidative stress that accelerate nonenzymatic degenerative protein modifications (DPMs) such as protein oxidation, disrupt redox signaling and physiological function, and remain a major risk factor for clinical diabetic vascular complications. In order to identify potential oxidative biomarkers in the blood plasma of patients with T2DM, we used LC-MS/MS-based proteomics to profile plasma samples from patients with T2DM and healthy controls. The results showed that human serum albumin (HSA) is damaged by irreversible cysteine trioxidation, which can be a potential oxidative stress biomarker for the early diagnosis of T2DM. The quantitative detection of site-specific thiol trioxidation is technically challenging; thus, we developed a sensitive and selective LC-MS/MS workflow that has been used to discover and quantify three unique thiol-trioxidized HSA peptides, ALVLIAFAQYLQQC(SO3H)PFEDHVK (m/z 1241.13), YIC(SO3H)ENQDSISSK (m/z 717.80) and RPC(SO3H)FSALEVDETYVPK (m/z 951.45), in 16 individual samples of healthy controls (n = 8) and individuals with diabetes (n = 8). Targeted quantitative analysis using multiple reaction monitoring mass spectrometry revealed impairment of the peptides with m/z 1241.13, m/z 717.80 and m/z 951.45, with significance (P < 0.02, P < 0.002 and P < 0.03), in individuals with diabetes. The results demonstrated that a set of three HSA thiol-trioxidized peptides, which are irreversibly oxidatively damaged in HSA in the plasma of patients with T2DM, can be important indicators and potential biomarkers of oxidative stress in T2DM.
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Gene expression contributes to phenotypic traits and human disease. To date, comparatively less is known about regulators of protein abundance, which is also under genetic control and likely influences clinical phenotypes. However, identifying and quantifying allele-specific protein abundance by bottom-up proteomics is challenging since single nucleotide variants (SNVs) that alter protein sequence are not considered in standard human protein databases. To address this, we developed the GenPro software and used it to create personalized protein databases (PPDs) to identify single amino acid variants (SAAVs) at the protein level from whole exome sequencing. In silico assessment of PPDs generated by GenPro revealed only a 1% increase in tryptic search space compared to a direct translation of all human transcripts and an equivalent search space compared to the UniProtKB reference database. To identify a large unbiased number of SAAV peptides, we performed high-resolution mass spectrometry-based proteomics for two human post-mortem brain samples and searched the collected MS/MS spectra against their respective PPD. We found an average of ~117,000 unique peptides mapping to ~9,300 protein groups for each sample and of these 977 were unique variant peptides. We found that over 400 reference and SAAV peptide pairs were, on average, equally abundant in human brain by label-free ion intensity measurements and confirmed the absolute levels of three reference and SAAV peptide pairs using heavy labeled peptides standards coupled with parallel reaction monitoring (PRM). Our results highlight the utility of integrating genomic and proteomic sequencing data to identify sample-specific SAAV peptides and support the hypothesis that most alleles are equally expressed in human brain.
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Fungal biotechnology has been well established in food and healthcare sector, and now being explored for lignocellulosic biorefinery due to their great potential to produce a wide array of extracellular enzymes for nutrient recycling. Due to global warming, environmental pollution, green house gases emission and depleting fossil fuel, fungal enzymes for lignocellulosic biomass refinery become a major focus for utilizing renewal bioresources. Proteomic technologies tender better biological understanding and exposition of cellular mechanism of cell or microbes under particular physiological condition and are very useful in characterizing fungal secretome. Hence, in addition to traditional colorimetric enzyme assay, mass-spectrometry-based quantification methods for profiling lignocellulolytic enzymes have gained increasing popularity over the past five years. Majority of these methods include two dimensional gel electrophoresis coupled to mass spectrometry, differential stable isotope labeling and label free quantitation. Therefore, in this review, we reviewed more commonly used different proteomic techniques for profiling fungal secretome with a major focus on two dimensional gel electrophoresis, liquid chromatography-based quantitative mass spectrometry for global protein identification and quantification. We also discussed weaknesses and strengths of these methodologies for comprehensive identification and quantification of extracellular proteome.
Article
Purpose: To increase the proteome coverage of human atherosclerotic plaques and identify low-abundance proteins that may have important functions during the development and progression of atherosclerosis. Experimental design: Thirty-eight human carotid atherosclerotic plaques were pooled into two samples and analyzed in triplicate using offline multidimensional LC-MS/MS. The collected fractions of trypsin-digested peptides from Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) were analyzed by LC-MS/MS on a Q Exactive (Thermo Fisher, MA, USA). Results: A total of 4702 proteins were identified from atherosclerotic plaques at a false discovery rate (FDR) of 1%, of which 3846 were identified with at least 2 unique peptides. Many pathways related to the development and progression of atherosclerosis were identified, such as atherosclerosis signaling, toll receptor signaling pathway and inhibition of matrix metalloproteases. Many low-abundance proteins with important functions in atherosclerosis that were previously unidentifiable using mass spectrometry based proteomics methods, such as TGF-β, interleukins and other growth factors, were identified confidently from plaques. Conclusions and clinical relevance: This study has substantially increased the coverage of the atherosclerotic plaque proteome which represents a leap forward in understanding of plaque composition, development and progression. The identification of many low-abundance proteins may also facilitate biomarker discovery.
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Chemical proteomic strategies for simultaneous bioimaging and target identification of non-covalent bioactive compounds in live mammalian cells are currently not available, due to a lack of photo-affinity labels that are minimally modified from their parental compounds, yet chemically tractable using copper-free bioorthogonal chemistries. We have developed novel minimalist linkers containing both an alkyl diazirine and a cyclopropene. We have shown chemical probes made from such linkers could be used for simultaneous in situ imaging and covalent labeling of endogenous BRD-4 (an important epigenetic protein) via a raipd, copper-free, tetrazine-cyclopropene ligation reaction (k2 > 6 M-1 s-1). The key features of our cyclopropenes, with their unique C-1 linkage to BRD-4-targeting moiety, are their tunable reactivity and solubility, relative stability and synthetic accessibility (achievable in a single step from probes containing terminal alkynes).
Article
Deamidation is a spontaneous degenerative protein modification (DPM) that disrupts the structure and function of both endogenous proteins and various therapeutic agents. While deamidation has long been recognized as a critical event in human aging and multiple degenerative diseases, research progress in this field has been restricted by the technical challenges associated with studying this DPM in complex biological samples. Asparagine (Asn) deamidation generates L-aspartic acid (L-Asp), D-aspartic acid (D-Asp), L-isoaspartic acid (L-isoAsp) or D-isoaspartic acid (D-isoAsp) residues at the same position of Asn in the affected protein, but each of these amino acids displays similar hydrophobicity and cannot be effectively separated by reverse phase liquid chromatography. The Asp and isoAsp isoforms are also difficult to resolve using mass spectrometry since they have the same mass and fragmentation pattern in MS/MS. Moreover, the ¹³C peaks of the amidated peptide are often misassigned as monoisotopic peaks of the corresponding deamidated peptides in protein database searches. Furthermore, typical protein isolation and proteomic sample preparation methods induce artificial deamidation that cannot be distinguished from the physiological forms. To better understand the role of deamidation in biological aging and degenerative pathologies, new technologies are now being developed to address these analytical challenges, including mixed mode electrostatic-interaction modified hydrophilic interaction liquid chromatography (emHILIC). When coupled to high resolution, high accuracy tandem mass spectrometry this technology enables unprecedented, proteome-wide study of the ‘deamidome’ of complex samples. The current article therefore reviews recent advances in sample preparation methods, emHILIC-MS/MS technology, and MS instrumentation / data processing approaches to achieving accurate and reliable characterization of protein deamidation in complex biological and clinical samples.
Article
A meter-scale monolithic silica capillary column modified with urea-functional groups for hydrophilic interaction liquid chromatography (HILIC) was developed for highly efficient separation of biological compounds. We prepared a ureidopropylsilylated monolithic silica capillary column with a minimum plate height of 12 μm for nucleosides and a permeability of 2.1 × 10(-13) m(2), which are comparable with the parameters of monolithic silica-C18 capillary columns. Over 300,000 theoretical plates were experimentally obtained in HILIC with a 4 m-long column at 8 MPa; this is the best result yet reported for HILIC. A 2 m-long ureidopropylsilylated monolithic silica capillary column was utilized to develop a HILIC mode LC-MS system for proteomics applications. Using tryptic peptides from human HeLa cell lysate proteins, we identified the comparable numbers of peptides and proteins in HILIC with those in reversed-phase liquid chromatography (RPLC) using a C18-modified monolithic silica column when shallow gradients were applied. In addition, approximately 5-fold increase in the peak response on average was observed in HILIC for commonly identified tryptic peptides due to the high acetonitrile concentration in the HILIC mobile phase. Since HILIC mode LC-MS shows orthogonal selectivity to RPLC mode LC-MS, it is useful as a complementary tool to increase proteome coverage in proteomics studies.
Article
We have developed a fully automatable two-dimensional liquid chromatography platform for shotgun proteomics analyses based on the online coupling of hydrophilic interaction liquid chromatography (HILIC) – using a nonionic type of TSKgel Amide 80 at either pH 6.8 (neutral) or 2.7 (acidic) – with conventional low-pH reversed-phase chromatography. Online coupling of the neutral-pH HILIC and reversed phase chromatography systems outperformed the acidic HILIC–reversed phase chromatography combination, resulting in 18.4% (1914 versus 1617 nonredundant proteins) and 41.6% (12,989 versus 9172 unique peptides) increases in the number of identified peptides and proteins from duplicate analyses of Rat pheochromocytoma lysates. Armed with this optimized HILIC–reversed phase liquid chromatography platform, we identified 2554 nonredundant proteins from duplicate analyses of a Saccharomyces cerevisiae lysate, with the detected protein abundances spanning from approximately 41 to 106 copies per cell, which contained up to approximately 2092 different validated protein species with a dynamic range of concentrations of up to approximately 104. This present study establishes a fully automated platform as a promising methodology to enable online coupling of different hydrophilic HILIC and reversed phase chromatography systems, thereby expanding the repertoire of multidimensional liquid chromatography for shotgun proteomics.
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Unlabelled: Vascular dementia (VaD) is a leading cause of dementia in the elderly together with Alzheimer's disease with limited treatment options. Poor understanding of the pathophysiology underlying VaD is hindering the development of new therapies. Hence, to unravel its underlying molecular pathology, an iTRAQ-2D-LC-MS/MS strategy was used for quantitative analysis of pooled lysates from Brodmann area 21 of pathologically confirmed cases of VaD and matched non-neurological controls. A total of 144 differentially expressed proteins out of 2281 confidently identified proteins (false discovery rate=0.3%) were shortlisted for bioinformatics analysis. Western blot analysis of selected proteins using samples from individual patients (n=10 per group) showed statistically significant increases in the abundance of SOD1 and NCAM and reduced ATP5A in VaD. This suggested a state of hypometabolism and vascular insufficiency along with an inflammatory condition during VaD. Elevation of SOD1 and increasing trend for iron-storage proteins (FTL, FTH1) may be indicative of an oxidative imbalance that is accompanied by an aberrant iron metabolism. The synaptic proteins did not exhibit a generalized decrease in abundance (e.g. syntaxin) in the VaD subjects. This reported proteome offers a reference data set for future basic or translational studies on VaD. Biological significance: Our study is the first quantitative clinical proteomic study where iTRAQ-2D-LC-MS/MS strategy has been used to identify the differential proteome in the VaD cortex by comparing VaD and matched control subjects. We generate testable hypothesis about the involvement of various proteins in the vascular and parenchymal events during the evolution of VaD that finally leads to malfunction and demise of brain cells. This study also establishes quantitative proteomics as a complementary approach and viable alternative to existing neurochemical, electron microscopic and neuroimaging techniques that are traditionally being used to understand the molecular pathology of VaD. Our study could inspire fellow researchers to initiate similar retrospective studies targeting various ethnicities, age-groups or sub-types of VaD using brain samples available from brain banks across the world. Meta-analysis of these studies in the future may be able to shortlist candidate proteins or pathways for rationale exploration of therapeutic targets or biomarkers for VaD.
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Protein phosphorylation is an important post-translational modification (PTM) involved in embryonic development, adult homeostasis and diseases. Over the last decade several advances have been made in liquid chromatography tandem mass spectrometry (LC-MS/MS)-based technologies to identify thousands of phosphorylation sites. However, in-depth phosphoproteomics often require off-line enrichment and fractionation techniques. In this study we provide a detailed analysis of the physico-chemical characteristics of phosphopeptides, which have been fractionated by off-line high pH chromatography (HpH) before subsequent titanium dioxide (TiO2) enrichment and LC-MS/MS analysis. Our results demonstrate that HpH is superior to standard strong-cation exchange (SCX) fractionation in total number of phosphopeptides detected when analyzing the same number of fractions by identical LC-MS/MS gradients. From 14 HpH fractions we routinely identified over 30,000 unique phosphopeptide variants, which is more than twice the number compared to SCX fractionation. HpH chromatography displayed an exceptional ability to fractionate singly phosphorylated peptides with minor benefits for doubly phosphorylated peptides over SCX. Further optimizations in the pooling and concatenation strategy increased the total number of multi-phosphorylated peptides detected after HpH fractionation. In conclusion, we provide a basic framework and resource for performing in-depth phosphoproteome studies utilizing off-line basic reversed-phased fractionation. Raw data is available at ProteomeXchange: PXD001404.
Article
This review summarizes the recent advances in the analysis of amino acids, peptides and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a 2D-setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident, due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, of histones and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, larger pore size…) is still too limited. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Deamidation of asparagines and glutamines occurs spontaneously in proteins and results in protein degradation. Deamidation of asparaginyl residues in proteins produces a mixture of asparaginyl, n-aspartyl, and isoaspartyl residues, which has been linked to the pathology of some neurodegenerative diseases. However, accurate proteomic analysis of deamidation is challenging since it occurs quickly during conventional proteomic sample preparation, and the co-elution of the two resulting isomeric deamidated peptides in reversed-phase liquid chromatography (RPLC) compromises their identification and quantification using RPLC-MS/MS. To overcome these difficulties, a novel sample preparation protocol to minimize artificial deamidation has been developed alongside an offline RP-ERLIC-MS/MS (reversed-phase chromatography fractionation followed by electrostatic repulsion-hydrophilic interaction chromatography coupled with MS/MS) strategy to separate and quantify the three deamidation products from the same peptide on a proteome-wide scale. These protocols are detailed in this unit. © 2014 by John Wiley & Sons, Inc.
Article
Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) and first-dimensional fractionation is widely used for reducing sample complexity in large-scale proteomic profiling experiments. However, the limited number of proteins identified and the relatively long running time are a barrier to the successful application of this approach. In this study, off-line high pH reversed-phase fractionation (RPF) was combined with nano-LC–MS/MS in order to develop an improved method for global proteomic profiling of different cell lines. In the first dimensional reverse phase HPLC separation, 300 μg of digested cell protein was separated into 78 fractions under high pH conditions and condensed into 26 fractions for the second nano-LC–MS/MS analysis at low pH. The chromatographic conditions for the first and second steps were optimized, and the accuracy and reproducibility of protein quantification were investigated with an average Pearson correlation coefficient of 0.94. The method was then applied in the identification of proteins in six common cell lines (DMS, MFM, HepG2, U2OS, 293T and yeast), which resulted in identification of 7300–8500 and 8956 proteins in heavy/light labeled and label-free cell samples, respectively, in 1.5 days. The performance of the developed method was compared with isoelectric focusing (IEF)-nano-LC–MS/MS and the previously reported method; and off-line high pH RPF-nano-LC–MS/MS proved advantageous in terms of the number of proteins identified and the analytical time needed to achieve a successful global proteomic profiling outcome. The RPF-nano-LC–MS/MS method identified more proteins from low abundance (150 μg) samples with an average sequence coverage for each cell line of 23.4–35.1%. RPF-nano-LC–MS/MS may therefore be an efficient alternative tool for achieving improved proteomic coverage of multiple cell lines.
Article
Deamidation of glutamine (Gln) residues is a spontaneous or enzymatic process with significant implications in aging and human pathology. Although some methods are available to identify the γ/α-glutamyl products of deamidation, none of these methods allows the characterization of this post-translational modification (PTM) from complex biological samples by shotgun proteomics. Here we present LERLIC-MS/MS, a chromatographic strategy that uses a long (50 cm) anion-exchange capillary column operating in the electrostatic repulsion-hydrophilic interaction mode (ERLIC) and coupled directly to MS/MS for proteome analysis in a single injection. Profiling of soluble extracts of brain tissues by LERLIC-MS/MS distinguished for the first time γ/α-glutamyl isomers of deamidation, encountering a 1.7 γ/α-glutamyl ratio for most Gln deamidation products. A detailed analysis of any deviation from that observed ratio allowed the identification of transglutaminase-mediated γ-glutamyl isomers as intermediate products of transamidation. Furthermore, LERLIC-MS/MS was able to simultaneously separate Gln and asparagine (Asn) deamidation products even for those peptides showing multiple deamidated proteoforms. The characterization of Asn deamidated residues by LERLIC-MS/MS also uncovered novel PIMT (Protein L-isoaspartyl methyltransferase) substrate proteins in human brain tissues that deviated from the expected 3:1 isoAsp/Asp ratio. Taken together, our results demonstrate that LERLIC-MS/MS can be used to perform an in-depth study of protein deamidation on a global proteome scale. This new strategy should help to elucidate the biological implications of deamidation in aging and disease conditions.
Chapter
To achieve a more comprehensive analysis of protein phosphorylation, a wide variety of larger scale strategies were developed, all of which incorporated some form of enrichment prior to mass spectrometry analysis. Several strategies have been developed that enrich specifically for phosphopeptides based on an affinity for phosphate. These methods entail different molecular mechanisms, and thus are susceptible to different background problems. Improvements of the technique have been focused on achieving more efficient binding of phosphopeptides to the metal, more efficient elution from the column, and a reduction of nonphosphopeptide background. Many researchers over the years have optimized the pH, buffers, and other conditions to improve the recovery of peptides using Fe⁺³ immobilized metal ion affinity chromatography (IMAC). Large‐scale phosphoproteomics studies have been enabled by the combination of enrichment, shotgun proteomic methods and large‐scale data analysis methods. Advances in mass spectrometers continued to improve the analysis of phosphorylation in biological systems.
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Objective— Mutations affecting contractile-related proteins in the ECM (extracellular matrix), microfibrils, or vascular smooth muscle cells can predispose the aorta to aneurysms. We reported previously that the LRP1 (low-density lipoprotein receptor–related protein 1) maintains vessel wall integrity, and smLRP1 −/− mice exhibited aortic dilatation. The current study focused on defining the mechanisms by which LRP1 regulates vessel wall function and integrity. Approach and Results— Isometric contraction assays demonstrated that vasoreactivity of LRP1-deficient aortic rings was significantly attenuated when stimulated with vasoconstrictors, including phenylephrine, thromboxane receptor agonist U-46619, increased potassium, and L-type Ca ²⁺ channel ligand FPL-64176. Quantitative proteomics revealed proteins involved in actin polymerization and contraction were significantly downregulated in aortas of smLRP1 −/− mice. However, studies with calyculin A indicated that although aortic muscle from smLRP1 −/− mice can contract in response to calyculin A, a role for LRP1 in regulating the contractile machinery is not revealed. Furthermore, intracellular calcium imaging experiments identified defects in calcium release in response to a RyR (ryanodine receptor) agonist in smLRP1 −/− aortic rings and cultured vascular smooth muscle cells. Conclusions— These results identify a critical role for LRP1 in modulating vascular smooth muscle cell contraction by regulating calcium signaling events that potentially protect against aneurysm development.
Article
Characterization nd studies of proteome are challenging because biological samples are complex, with a wide dynamic range of abundance. At present the proteins are identified by digestion into peptides, with subsequent identification of the peptides by mass spectrometry (MS). MS is a powerful technique for the purpose, but it cannot identify every peptide in such complex mixtures simultaneously. For accurate analysis and quantification it is important to separate the peptides first by chromatography into fractions of a size that MS can handle. With these less complex fractions, the probability is increased of identifying peptides of low abundance that would otherwise experience ion suppression effects due to the presence of peptides of high abundance. Enrichment for peptides with certain post-translational modifications helps to increase their detection rates as well. Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) is a mixed-mode chromatographic technique which combines the use of electrostatic repulsion and hydrophilic interaction. This review provides an overview of ERLIC and its various proteomics applications. ERLIC has been demonstrated to have good orthogonality to reverse phase liquid chromatography (RPLC), making it useful as a first dimension in multidimensional liquid chromatography (MDLC) and fractionation of digests in general. Peptides elute in order of their isoelectric points and polarity. ERLIC has also been successfully utilized for the enrichment for phosphopeptides and glycopeptides, facilitating their identification. In addition, it is promising for the study of peptide deamidation. ERLIC performs comparably well or better than established methods for these various applications, and serves as a viable and efficient workflow alternative.
Article
Phosphorylation is an important post-translational modification that is involved in regulating many signaling pathways. Of particular interest are the growth factor mediated Ras and phosphoinositide 3-kinase (PI3K) signaling pathways which, if misregulated, can contribute to the progression of cancer. Phosphoproteomic methods have been developed to study regulation of signaling pathways; however, due to the low stoichiometry of phosphorylation, understanding these pathways is still a challenge. In this study, we have developed a multi-dimensional method incorporating electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with tandem IMAC/TiO2 enrichment for subsequent phosphopeptide identification by LC/MS/MS. We applied this method to PDGF-stimulated NIH 3T3 cells to provide over 11,000 unique phosphopeptide identifications. Upon motif analysis, IMAC was found to enrich for basophilic kinase substrates while the subsequent TiO2 step enriched for acidophilic kinase substrates, suggesting that both enrichment methods are necessary to capture the full complement of kinase substrates. Biological functions that were over-represented at each PDGF stimulation time point, together with the phosphorylation dynamics of several phosphopeptides containing known kinase phosphorylation sites, illustrate the feasibility of this approach in quantitative phosphoproteomic studies. Copyright © 2015 Elsevier B.V. All rights reserved.
Chapter
As advanced proteomic technologies have been developed, huge amounts of proteomic data have been generated from a broad spectrum of patient tissue and body fluid samples collected under various disease conditions. From these data, proteomic biomarkers that can serve as indicators of disease states have been searched for numerous diseases. To discover reliable protein biomarkers, high-throughput proteomic platforms have been developed, including two-dimensional gel electrophoresis, mass spectrometry (MS)-based proteomic analysis, and protein–antibody microarrays. Of these platforms, liquid chromatography–tandem MS (LC–MS/MS) analysis has been widely used for discovery of protein biomarkers due to its high sensitivity in analytical capability and large dynamic ranges of detection. Before LC–MS/MS analysis, exhaustive fractionations of complex protein samples are often employed to improve the depth of proteome profiles during the discovery phase of protein biomarkers. Moreover, LC–MS/MS analysis provides the high-throughput capability sufficient for verification of protein biomarker candidates selected from the discovery phase in large cohorts of clinical samples before their clinical use. Here, we summarize recent advances in MS-based proteomic methods applied for the discovery of protein biomarkers.
Chapter
This chapter discusses the use of hydrophilic interaction chromatography (HILIC) for analysis of biochemical compounds. Some compounds such as amino acids, carbohydrates, nucleobases, nucleosides, oligonucleotides, peptides, phospholipids, and proteins are of interest as components in pharmaceutical or food products. The chapter focuses on application of HILIC to separations of these types of compounds in problems of biochemical and related interests. Many recent glycan analysis applications have employed zwitterionic- HILIC (ZIC-HILIC) or neutral amide stationary phases. Other types of HILIC columns have been used for certain applications, but it seems that the field is moving toward the former two column types as first-line choices. Fluorescence detection of derivatized glycans or mass spectrometric detection is clearly the mode of choice, with mass spectrometry (MS) providing superior data for structural characterization. Amide or zwitterionic phases have also become popular for other biomolecules such as nucleobases, nucleosides, nucleotides, oligonucleotides, amino acids, and peptides.
Article
Mass spectrometry (MS)-based proteomic approaches have largely facilitated our systemic understanding of cellular processes and biological functions. Cutoffs in protein expression fold changes (FC) are often arbitrarily determined in MS-based quantification with no demonstrable determination of small magnitude changes in protein expression. Therefore, many biological insights may remain veiled due to high FC cutoffs. Herein we employ the intestinal epithelial cell (IEC) line Caco-2 as a model system to demonstrate the dynamicity of tandem-mass-tag (TMT) labeling over a range of 5 - 40% changes in protein abundance, with the variance controls of ±5% FC for around 95% of TMT ratios when sampling nine to twelve biological replicates. We further applied this procedure to examine the temporal proteome of Caco-2 cells upon exposure to human whey proteins (WP). Pathway assessments predict subtle effects due to WP in moderating xenobiotic metabolism, promoting proliferation and various other cellular functions in differentiating enterocyte-like Caco-2 cells. This demonstration of a sensitive MS approach may open up new perspectives in the system-wide exploration of elusive or transient biological effects by facilitating scrutiny of narrow windows of proteome abundance changes. Furthermore, we anticipate this study will encourage more investigations of WP on infant gastrointestinal tract development.
Article
Background and Objective: Unwanted pregnancy is a common health problem worldwide. Emergency contraception (EC) is the best effective approach for solving this problem. For reorganizing and correcting misunderstandings about the use of these methods, the present study was carried out on Zanjan Islamic Azad university students' in order to assess their attitudes towards emergency contraception. Materials and Methods: The attitude of 1019 students towards EC was investigated using a self administered questionnaire including questions about demographic data and their attitude status. The data analyzed using appropriate software. Results: About one third of the students (35.2%) had positive attitude, over half of them (64.1%) had impartial and less than one percent (0.7%) had negative attitude towards emergency contraception. Over half of them tended to use it and about one third believed that EC was very effective. 36% wished to obtain it from private health sectors and 35% cited that they did not do anything for the reason of modesty and shyness. Over one third of the subjects believed that using these methods is confronted with their religion. Conclusion: The most important obstacles for obtaining these devices were modesty, shyness and religious reasons which created a negative attitude. Consulting sessions can solve many of these barriers and will support a positive attitude.
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Hydrophilic interaction chromatography (HILIC) was presented as a general-purpose mode of chromatography in 1990.(1) It is now second only to reversed-phase (RP) chromatography as a method for analysis by HPLC. Studies of the mechanism generally confirm that it involves partitioning of analytes between the mostly organic mobile phase and a stagnant layer of water of hydration associated with the hydrophilic stationary phase. If the stationary phase has an electrostatic charge, then mixed-mode effects from this will be superimposed upon the hydrophilic interaction. A particularly useful combination involves the use of an ion-exchange column with the same charge as most of the analytes and a high concentration of organic solvent (HILIC conditions). In the absence of the organic solvent, the resulting electrostatic repulsion would normally cause exclusion of the analytes from the pore volume, leading to elution prior to the dead volume of the column. However, as hydrophilic interaction is independent of electrostatic effects, with sufficient organic solvent the solutes are retained despite the electrostatic repulsion. This combination is termed electrostatic repulsion-hydrophilic interaction chromatography -abbreviated as ERLIC?
Article
Reduction and alkylation are essential steps in shotgun proteomic sample preparation, but over-alkylation can occur on peptide N-terminus and amino acid residues other than cysteine, which adversely affects protein identification and quantification. To date, different sample preparation protocols are used in different laboratories, but a systematic comparison of the published protocols has not been done yet in the aspect of over-alkylation. Here, we comprehensively evaluated various protocols using different denaturants, different digestion buffers and different concentrations of reduction and alkylation reagents. The suggested protocols from the manufacturers of RapiGest and ProteaseMAX induce the highest degree of over-alkylation compared with others. Carbamidomethylation at lysine increases over 4 times while using triethylammonium bicarbonate buffer compared with that using others, and it should be minimized for improving the labeling efficiency of stable isotope labeling reagents that label lysine residues. The use of digestion buffers at pH 6 reduces over-alkylation, but greatly introduces some other artificial modifications. Excessive iodoacetamide was proved to be the major cause of over-alkylation during trypsin digestion. Therefore, over-alkylation can be minimized by using low concentration of dithiothreitol and iodoacetamide for reduction and alkylation or quenching the excessive iodoacetamide before digestion.
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Introduction Glycosylations range among the most common posttranslational modifications with an estimated 50% of all proteins supposed to be glycosylated. These modifications are required for essential cellular processes including cell–cell recognition, protein structure and activity, e.g., of surface receptors, as well as subcellular localization of proteins. Beside the elucidation of the carbohydrate structures, the annotation of glycosylation sites is of primary interest as a basis for subsequent functional characterization. Although mass spectrometry is the method of choice for large-scale analysis of glycosylation sites, it requires initial enrichment of glycopeptides prior mass spectrometric detection in most cases. Materials and Methods In this paper, we present a novel approach for glycopeptide enrichment by electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Glycopeptides were separated from the bulk of non-modified peptides and gradually eluted from the stationary phase with potential for isoform resolution. Applied to human platelets, 125 glycosylation sites on 66 proteins were identified including major platelet glycoproteins responsible for cellular function. Conclusion These sites add a major contribution to the now more than 250 glycosylation sites annotated for platelets, which enable the clinically relevant design of quantification assays for platelet glycoproteins.
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Characterization of glyco- and phosphoproteins as well as their modification sites poses many challenges, the greatest being loss of their signals during mass spectrometric detection due to substoichiometric amounts and the ion suppression effect caused by peptides of high abundance. We report here an optimized protocol using electrostatic repulsion hydrophilic interaction chromatography for the simultaneous enrichment of glyco- and phosphopeptides from mouse brain membrane protein digest. With this protocol, we successfully identified 544 unique glycoproteins and 922 glycosylation sites, which were significantly higher than those from the commonly used hydrazide chemistry method (192 glycoproteins and 345 glycosylation sites). Moreover, a total of 383 phosphoproteins and 915 phosphorylation sites were recovered from the sample, suggesting that this protocol has the potential to enrich both glycopeptides and phosphopeptides simultaneously. Of the total 995 glycosylation sites identified from both methods, 96% were considered new as they were either annotated as putative or not documented in the newly released Swiss-Prot database. Thus, this study could be of significant value in complementing the current glycoprotein database and provides a unique opportunity to study the complex interaction of two different post-translational modifications in health and disease without being affected by interexperimental variations.
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Mass spectrometry (MS) is the most comprehensive and versatile tool in large-scale proteomics. In this review, we dissect the overall framework of the MS experiment into its key components. We discuss the fundamentals of proteomic analyses as well as recent developments in the areas of separation methods, instrumentation, and overall experimental design. We highlight both the inherent strengths and limitations of protein MS and offer a rough guide for selecting an experimental design based on the goals of the analysis. We emphasize the versatility of the Orbitrap, a novel mass analyzer that features high resolution (up to 150,000), high mass accuracy (2-5 ppm), a mass-to-charge range of 6000, and a dynamic range greater than 10(3). High mass accuracy of the Orbitrap expands the arsenal of the data acquisition and analysis approaches compared with a low-resolution instrument. We discuss various chromatographic techniques, including multidimensional separation and ultra-performance liquid chromatography. Multidimensional protein identification technology (MudPIT) involves a continuum sample preparation, orthogonal separations, and MS and software solutions. We discuss several aspects of MudPIT applications to quantitative phosphoproteomics. MudPIT application to large-scale analysis of phosphoproteins includes (a) a fractionation procedure for motif-specific enrichment of phosphopeptides, (b) development of informatics tools for interrogation and validation of shotgun phosphopeptide data, and (c) in-depth data analysis for simultaneous determination of protein expression and phosphorylation levels, analog to western blot measurements. We illustrate MudPIT application to quantitative phosphoproteomics of the beta adrenergic pathway. We discuss several biological discoveries made via mass spectrometry pipelines with a focus on cell signaling proteomics.
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Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
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Liquid chromatography and tandem mass spectrometry (LC-MS/MS) has become the preferred method for conducting large-scale surveys of proteomes. Automated interpretation of tandem mass spectrometry (MS/MS) spectra can be problematic, however, for a variety of reasons. As most sequence search engines return results even for 'unmatchable' spectra, proteome researchers must devise ways to distinguish correct from incorrect peptide identifications. The target-decoy search strategy represents a straightforward and effective way to manage this effort. Despite the apparent simplicity of this method, some controversy surrounds its successful application. Here we clarify our preferred methodology by addressing four issues based on observed decoy hit frequencies: (i) the major assumptions made with this database search strategy are reasonable; (ii) concatenated target-decoy database searches are preferable to separate target and decoy database searches; (iii) the theoretical error associated with target-decoy false positive (FP) rate measurements can be estimated; and (iv) alternate methods for constructing decoy databases are similarly effective once certain considerations are taken into account.
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DAF-2, an insulin receptor-like protein, regulates metabolism, development, and aging in Caenorhabditis elegans. In a quantitative proteomic study, we identified 86 proteins that were more or less abundant in long-lived daf-2 mutant worms than in wild-type worms. Genetic studies on a subset of these proteins indicated that they act in one or more processes regulated by DAF-2, including entry into the dauer developmental stage and aging. In particular, we discovered a compensatory mechanism activated in response to reduced DAF-2 signaling, which involves the protein phosphatase calcineurin.
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We describe the practical implementation of a new RP (pH 10 - pH 2) 2D HPLC-ESI/MS scheme for large-scale bottom-up analysis in proteomics. When compared to the common SCX-RP approach, it provides a higher separation efficiency in the first dimension and increases the number of identified peptides/proteins. We also employed the methodology of our sequence-specific retention calculator (SSRCalc) and developed peptide retention prediction algorithms for both LC dimensions. A diverse set of approximately 10,000 tryptic peptides from the soluble protein fraction of whole NK-type cells gave retention time versus hydrophobicity correlations, with R (2) values of 0.95 for pH 10 and 0.945 for pH 2 (formic acid) separation modes. The superior separation efficiency and the ability to use retention prediction to filter out false-positive MS/MS identifications gives promise that this approach will be a method of choice for large-scale proteomics analyses in the future. Finally, the "semi-orthogonal" separation selectivity permits the concatenation of fractions in the first dimension of separation before the final LC-ESI MS step, effectively cutting the analysis time in half, while resulting in a minimal reduction in protein identification.
Article
A computer program that progressively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence has been devised. For this purpose, a hydropathy scale has been composed wherein the hydrophilic and hydrophobic properties of each of the 20 amino acid side-chains is taken into consideration. The scale is based on an amalgam of experimental observations derived from the literature. The program uses a moving-segment approach that continuously determines the average hydropathy within a segment of predetermined length as it advances through the sequence. The consecutive scores are plotted from the amino to the carboxy terminus. At the same time, a midpoint line is printed that corresponds to the grand average of the hydropathy of the amino acid compositions found in most of the sequenced proteins. In the case of soluble, globular proteins there is a remarkable correspondence between the interior portions of their sequence and the regions appearing on the hydrophobic side of the midpoint line, as well as the exterior portions and the regions on the hydrophilic side. The correlation was demonstrated by comparisons between the plotted values and known structures determined by crystallography. In the case of membrane-bound proteins, the portions of their sequences that are located within the lipid bilayer are also clearly delineated by large uninterrupted areas on the hydrophobic side of the midpoint line. As such, the membrane-spanning segments of these proteins can be identified by this procedure. Although the method is not unique and embodies principles that have long been appreciated, its simplicity and its graphic nature make it a very useful tool for the evaluation of protein structures.
Article
A compelling need exists for the development of technologies that facilitate and accelerate the discovery of novel protein biomarkers with therapeutic and diagnostic potential. Comparisons among shotgun proteome technologies, including capillary isotachophoresis (CITP)-based multidimensional separations and multidimensional LC system, are therefore performed in this study regarding their abilities to address the challenges of protein complexity and relative abundance inherent in glioblastoma multiforme-derived cancer stem cells. Comparisons are conducted using a single processed protein digest with equal sample loading, identical second-dimension separation (RPLC) and MS conditions, and consistent search parameters and cutoff established by the target-decoy determined false-discovery rate. Besides achieving superior overall proteome performance in total peptide, distinct peptide, and distinct protein identifications; analytical reproducibility of the CITP proteome platform coupled with the spectral counting approach are determined by a Pearson R(2) value of 0.98 and a CV of 15% across all proteins quantified. In contrast, extensive fraction overlapping in strong cation exchange greatly limits the ability of multidimensional LC separations for mining deeper into the tissue proteome as evidenced by the poor coverage in various protein functional categories and key protein pathways. The CITP proteomic technology, equipped with selective analyte enrichment and ultrahigh resolving power, is expected to serve as a critical component in the overall toolset required for biomarker discovery via shotgun proteomic analysis of tissue specimens.
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The analysis and quantitation of membrane proteins have proved challenging for proteomics. Although several approaches have been introduced to complement gel-based analysis of intact proteins, the literature is rather limited in comparing major emerging approaches. Peptide fractionation using IEF (OFFGel), strong cation exchange HPLC using a pH gradient (SCX-pG), and RP HPLC at high pH, have been shown to increase peptide and protein identification over classic MudPIT approaches. This article compares these three approaches for first-dimensional separation of peptides using a detergent phase (Triton X-114) enriched membrane fraction from mouse cortical brain tissue. Results indicate that RP at high pH (pH 10) was superior for the identification of more peptides and proteins in comparison to the OFFGel or the SCX-pG approaches. In addition, gene ontology analysis (GOMiner) revealed that RP at high pH (pH 10) successfully identified an increased number of proteins with "membrane" ontology, further confirming its suitability for membrane protein analysis, in comparison to SCX-pG and OFFGel techniques.
Article
High-resolution peptide separation is pivotal for successful shotgun proteomics. The need for capable techniques propels invention and improvement of ever more sophisticated approaches. Recently, Agilent Technologies has introduced the OFFGEL fractionator, which conducts peptide separation by isoelectric focusing in an off-gel setup. This platform has been shown to accomplish high resolution of peptides for diverse sample types, yielding valuable advantages over comparable separation techniques. In this study, we deliver the first comparison of the newly emerging OFFGEL approach to the well-established on-line MudPIT platform. Samples from a membrane-enriched fraction isolated from murine C2C12 cells were subjected to replicate analysis by OFFGEL (12 fractions, pH 3-10) followed by RP-LC-MS/MS or 12-step on-line MudPIT. OFFGEL analyses yielded 1398 proteins (identified by 10,269 peptides), while 1428 proteins (11,078 peptides) were detected with the MudPIT approach. Thus, our data shows that both platforms produce highly comparable results in terms of protein/peptide identifications and reproducibility for the sample type analyzed. We achieve more accurate peptide focusing after OFFGEL fractionation with 88% of all peptides binned to a single fraction, as compared to 61% of peptides detected in only one step in MudPIT analyses. Our study suggests that both platforms are equally capable of high quality peptide separation of a sample with medium complexity, rendering them comparably valuable for comprehensive proteomic analyses.
Article
The high complexity and large dynamic range of blood plasma proteins currently prohibit the sensitive and high-throughput profiling of disease and control plasma proteome sample sets large enough to reliably detect disease indicating differences. To circumvent these technological limitations we describe here a new two-stage strategy for the mass spectrometry (MS) assisted discovery, verification and validation of disease biomarkers. In an initial discovery phase N-linked glycoproteins with distinguishable expression patterns in primary normal and diseased tissue are detected and identified. In the second step the proteins identified in the initial phase are subjected to targeted MS analysis in plasma samples, using the highly sensitive and specific selected reaction monitoring (SRM) technology. Since glycosylated proteins, such as those secreted or shed from the cell surface are likely to reside and persist in blood, the two-stage strategy is focused on the quantification of tissue derived glycoproteins in plasma. The focus on the N-glycoproteome not only reduces the complexity of the analytes, but also targets an information-rich subproteome which is relevant for remote sensing of diseases in the plasma. The N-glycoprotein based biomarker discovery and validation workflow reviewed here allows for the robust identification of protein candidate panels that can finally be selectively monitored in the blood plasma at high sensitivity in a reliable, non-invasive and quantitative fashion.
Article
The repeatability of peptide identifications in shotgun proteome analyses employing strong cation-exchange-xion-pair RP HPLC hyphenated to ESI MS/MS was compared to an alternative scheme, comprising high-pH RP chromatography combined with low-pH ion-pair RP chromatography. Equivalent results were obtained with both methods in proteome analysis of Corynebacterium glutamicum. From a total number of 1350 to 1850 peptides identified in triplicate analyses of five consecutive fractions chosen from the first-dimension separation, 41-45% of the peptides were identified three times, whereas 16-22 and 37-39% of the peptides were identified only twice or once, respectively. A comparison of the repeatability of peptide identifications from complex samples upon 1- or 2-D chromatographic separation revealed that an additional separation dimension decreases the repeatability by approximately 25%.
Article
Multidimensional liquid-based separation techniques are described for maximizing the resolution of the enormous number of peptides generated upon tryptic digestion of proteomes, and hence, reduce the spatial and temporal complexity of the sample to a level that allows successful mass spectrometric analysis. This review complements the previous contribution on unidimensional high performance liquid chromatography (HPLC). Both chromatography and electrophoresis will be discussed albeit with reversed-phase HPLC (RPLC) as the final separation dimension prior to MS analysis.
Article
1-D and 2-D LC methods were utilized for proteome analysis of undepleted human serum. Separation of peptides in 2-D LC was performed either with strong cation exchange (SCX)-RP chromatography or with an RP-RP 2-D LC approach. Peptides were identified by MS/MS using a data-independent acquisition approach. A peptide retention prediction model was used to highlight the potential false-positive peptide identifications. When applying selected data filtration, we identified 52 proteins based on 316 peptides in serum in 1-D LC setup. One hundred and eighty-four proteins/1036 peptides and 142 proteins/905 peptides were identified in RP-RP and SCX-RP 2-D LC, respectively. The performance of both 2-D LC methods for proteomic analysis is critically compared.
Article
We describe a detailed and widely applicable method for comprehensive proteomic profiling of the fission yeast Schizosaccharomyces pombe by 2-dimensional high performance liquid chromatography-electrospray ionization-tandem mass spectrometry that demonstrates high sensitivity and robust operation. Steps ranging from the preparation of total proteins, digestion of proteins to peptides, and separation of peptides by two-dimensional (1. strong cation exchange and 2. reversed-phase) high performance liquid chromatography followed by tandem mass spectrometry and data processing have been optimized for our instrumentation platform. Using this technology, we identify ca. 3400 proteins per sample and have identified an estimated 4600 proteins in vegetative cells (equal to ca. 90% of the predicted S. pombe proteome) at a false discovery rate of 0.02. Considering the fact that approximately 500 genes are strongly induced during sexual differentiation, and sexual differentiation was not included in our experiments, the proteomic profiling technique affords what should be virtually complete coverage of the vegetative S. pombe proteome. In addition, these methods are widely applicable, having been used for proteomic profiling of several other organisms.
Article
Human ductal saliva contributes over a thousand unique proteins to whole oral fluids. The mechanism by which most of these proteins are secreted by salivary glands remains to be determined. The present study used a mass spectrometry-based, shotgun proteomics approach to explore the possibility that a subset of the proteins found in saliva are derived from exosomes, membrane-bound vesicles of endosomal origin within multivesicular endosomes. Using MudPIT (multidimensional protein identification technology) mass spectrometry, we catalogued 491 proteins in the exosome fraction of human parotid saliva. Many of these proteins were previously observed in ductal saliva from parotid glands (265 proteins). Furthermore, 72 of the proteins in parotid exosomes overlap with those previously identified as urinary exosome proteins, proteins which are also frequently associated with exosomes from other tissues and cell types. Gene Ontology (GO) and KEGG pathway analyses found that cytosolic proteins comprise the largest category of proteins in parotid exosomes (43%), involved in such processes as phosphatidylinositol signaling system, calcium signaling pathway, inositol metabolism, protein export, and signal transduction, among others; whereas the integral plasma membrane proteins and associated/peripheral plasma membrane proteins (26%) were associated with extracellular matrix-receptor interaction, epithelial cell signaling, T-cell and B-cell receptor signaling, cytokine receptor interaction, and antigen processing and presentation, among other biological functions. In addition, these putative saliva exosomal proteins were linked to specific diseases (e.g., neurodegenerative disorders, prion disease, cancers, type I and II diabetes). Consequently, parotid glands secrete exosomes that reflect the metabolic and functional status of the gland and may also carry informative protein markers useful in the diagnosis and treatment of systemic diseases.
Article
Complex protein mixtures have traditionally been separated by 2-DE. Görg introduced IPGs as the first dimension of protein separation. In recent years, MS-based proteomics has increasingly become the method of choice for identifying and quantifying large number of proteins. In that technology, to decrease analyte complexity, proteins are often separated by 1-D SDS-gel electrophoresis before online MS analysis. Here, we investigate a recently introduced device for peptide separation with IPGs (Agilent OFFGEL). Loading capacity for optimal peptide focusing is below 100 microg and--similar to 2-D gels--IEF is more efficient in the acidic than the basic pH region. The 24-well fractionation format resulted in about 40% additional peptide identifications but less than 20% additional protein identifications than the 12-well format. Compared to in-gel digestion, peptide IEF consistently identified a third more proteins with equal number of fractions. Low protein starting amounts (10 microg) still resulted in deep proteome coverage. Advantages of the in-gel format include better reliability and robustness. Considering its superior performance, diminished sample and work-up requirements, peptide IEF will become a method of choice for sample preparation in proteomics.
Article
Shotgun proteome analysis platforms based on multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a powerful means to discover biomarker candidates in tissue specimens. Analysis platforms must balance sensitivity for peptide detection, reproducibility of detected peptide inventories and analytical throughput for protein amounts commonly present in tissue biospecimens (< 100 microg), such that platform stability is sufficient to detect modest changes in complex proteomes. We compared shotgun proteomics platforms by analyzing tryptic digests of whole cell and tissue proteomes using strong cation exchange (SCX) and isoelectric focusing (IEF) separations of peptides prior to LC-MS/MS analysis on a LTQ-Orbitrap hybrid instrument. IEF separations provided superior reproducibility and resolution for peptide fractionation from samples corresponding to both large (100 microg) and small (10 microg) protein inputs. SCX generated more peptide and protein identifications than did IEF with small (10 microg) samples, whereas the two platforms yielded similar numbers of identifications with large (100 microg) samples. In nine replicate analyses of tryptic peptides from 50 microg colon adenocarcinoma protein, overlap in protein detection by the two platforms was 77% of all proteins detected by both methods combined. IEF more quickly approached maximal detection, with 90% of IEF-detectable medium abundance proteins (those detected with a total of 3-4 peptides) detected within three replicate analyses. In contrast, the SCX platform required six replicates to detect 90% of SCX-detectable medium abundance proteins. High reproducibility and efficient resolution of IEF peptide separations make the IEF platform superior to the SCX platform for biomarker discovery via shotgun proteomic analyses of tissue specimens.
Article
Coupling of multiplex isobaric tags for relative and absolute quantitation (iTRAQ) to a sensitive linear ion trap (LTQ) mass spectrometer (MS) is a challenging, but highly promising approach for quantitative high-throughput proteomic profiling. Integration of the advantages of pulsed-Q dissociation (PQD) and collision-activated dissociation (CAD) fragmentation methods into a PQD-CAD hybrid mode, together with PQD optimization and data manipulation with a bioinformatics algorithm, resulted in a robust, sensitive and accurate iTRAQ quantitative proteomic workflow. The workflow was superior to the default PQD setting when profiling the proteome of a gastric cancer cell line, SNU5. Taken together, we established an optimized PQD-CAD hybrid workflow in LTQ-MS for iTRAQ quantitative proteomic profiling that may have wide applications in biological and biomedical research.
Article
Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) has been introduced recently for phosphopeptide enrichment. Here we compared ERLIC with the well-established SCX-IMAC for identifying phosphopeptides in EGF-treated A431 cells. The ERLIC approach detected a higher number of phosphopeptides (17 311) than SCX-IMAC (4850), but it only detected 926 unique phosphopeptides compared to 1315 in SCX-IMAC. Only 12% unique phosphopeptides were common to both approaches, suggesting that more comprehensive phosphoproteomes could be generated by complementing SCX-IMAC with ERLIC.
Article
Two modes of separation coupled with MS enable researchers to study complicated biological structures.
Article
Proteomics has progressed radically in the last 5 years and is now on par with most genomic technologies in throughput and comprehensiveness. Analyzing peptide mixtures by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS) has emerged as the main technology for in-depth proteome analysis whereas two-dimensional gel electrophoresis, low-resolution MALDI, and protein arrays are playing niche roles. MS-based proteomics is rapidly becoming quantitative through both label-free and stable isotope labeling technologies. The latest generation of mass spectrometers combines extremely high resolving power, mass accuracy, and very high sequencing speed in routine proteomic applications. Peptide fragmentation is mostly performed in low-resolution but very sensitive and fast linear ion traps. However, alternative fragmentation methods and high-resolution fragment analysis are becoming much more practical. Recent advances in computational proteomics are removing the data analysis bottleneck. Thus, in a few specialized laboratories, "precision proteomics" can now identify and quantify almost all fragmented peptide peaks. Huge challenges and opportunities remain in technology development for proteomics; thus, this is not "the beginning of the end" but surely "the end of the beginning."
Article
The complexity and diversity of biological samples in proteomics require intensive fractionation ahead of mass spectrometry identification. This work developed a chromatographic method called virtual three-dimensional chromatography to fractionate complex protein mixtures. By alternate elution with different pHs and salt concentrations, we implemented pH and salt steps by turns on a single strong cation exchange column to fully exploit its chromatographic ability. Given standard proteins that were not resolved solely by pH or salt gradient elution could be successfully separated using this combined mode. With a reversed phase column tandem connected behind, we further fractionated as well as desalted proteins as the third dimension. This present strategy could readily be adapted with respect to special complexity of biological samples. Crude plasma without depleting high abundance proteins were fractionated by this three-dimensional mode and then analyzed by reversed phase liquid chromatography coupled with LTQ mass spectrometry. In total, 1933 protein groups with wide dynamic ranges were identified from a single experiment. Some characteristics that correlated to the behavior of proteins on strong cation exchange columns are also discussed.
Article
Heart diseases resulting in heart failure are among the leading causes of morbidity and mortality in the Western world and can result from either systemic disease (e.g., hypertensive heart disease, ischemic heart disease) or specific heart muscle disease (e.g., dilated cardiomyopathy/DCM). Subproteome analysis of such disease subsets affords a reduction in sample complexity, potentially revealing biomarkers of cardiac failure that would otherwise remain undiscovered in proteome wide studies. Label-free nanoscale LC-MS has been applied in this study to validate a Triton X-114-based phase enrichment method for cardiac membrane proteins. Annotation of the subcellular location combined with GRAVY score analysis indicates a clear separation between soluble and membrane-bound proteins with an enrichment of over 62% for this protein subset. LC-MS allowed confident identification and annotation of hydrophobic proteins in this control sample pilot study and demonstrates the power of the proposed technique to extract integral membrane-bound proteins. This approach should be applicable to a wider scale study of disease-associated changes in the cardiac membrane subproteome.
Article
Two-dimensional (2D) fractionation is a commonly used tool to increase dynamic range and proteome coverage for bottom-up, shotgun proteomics. However, there are few reports comparing the relative separation efficiencies of 2D methodologies using low-microgram sample quantities. In order to systematically evaluate 2D separation techniques, we fractionated microgram quantities of E. coli protein extract by seven different methods. The first dimension of separation was performed with either reversed-phase high-pressure liquid chromatography (RP-HPLC), gel electrophoresis (SDS-PAGE), or strong cation exchange (SCX-HPLC). The second dimension consisted of a standard reversed-phase capillary HPLC coupled to an electrospray ionization quadrupole time-of-flight mass spectrometer for tandem mass spectrometric analysis. The overall performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The protein-level RP-HPLC and the high-pH RP-HPLC peptide-level separations performed the best, identifying 281 and 266 proteins, respectively. The online pH variance SCX and the SDS-PAGE returned modest performances with 178 and 139 proteins identified, respectively. The offline SCX had the worst performance with 81 proteins identified. We also examined various chromatographic factors that contribute to separation efficiency, including resolving power, orthogonality, and sample loss.
Article
This review represents a summary of the development and application of a novel mixed-mode HPLC approach to the separation and analysis of peptides and proteins termed hydrophilic interaction/cation-exchange chromatography (HILIC/CEX). This approach combines the most advantageous aspects of two widely different separation mechanisms, i.e. a separation based on hydrophilicity/hydrophobicity differences between polypeptides overlaid on a separation based on net charge. Applications described include HILIC/CEX separations of cyclic peptides, alpha-helical peptides, random coil peptides and modified or deletion products of synthetic peptides. In addition, the excellent resolving ability of HILIC/CEX for modified histone proteins is described. This approach is shown to represent an excellent complement to RP chromatography (RPC), as well as being a potent analytical tool in its own right.
Article
When a hydrophilic chromatography column is eluted with a hydrophobic (mostly organic) mobile phase, retention increases with hydrophilicity of solutes. The term hydrophilic-interaction chromatography is proposed for this variant of normal-phase chromatography. This mode of chromatography is of general utility. Mixtures of proteins, peptides, amino acids, oligonucleotides, and carbohydrates are all resolved, with selectivity complementary to those of other modes. Typically, the order of elution is the opposite of that obtained with reversed-phase chromatography. A hydrophilic, neutral packing was developed for use in high-performance hydrophilic-interaction chromatography. Hydrophilic-interaction chromatography is particularly promising for such troublesome solutes as histones, membrane proteins, and phosphorylated amino acids and peptides. Hydrophilic-interaction chromatography fractionations resemble those obtained through partitioning mechanisms. The chromatography of DNA, in particular, resembles the partitioning observed with aqueous two-phase systems based on polyethylene glycol and dextran solutions.
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Article
Highly complex protein mixtures can be directly analyzed after proteolysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this paper, we have utilized the combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS. One milligram of whole yeast protein was proteolyzed and separated by SCX chromatography (2.1 mm i.d.) with fraction collection every minute during an 80-min elution. Eighty fractions were reduced in volume and then re-injected via an autosampler in an automated fashion using a vented-column (100 microm i.d.) approach for RP-LC-MS/MS analysis. More than 162,000 MS/MS spectra were collected with 26,815 matched to yeast peptides (7,537 unique peptides). A total of 1,504 yeast proteins were unambiguously identified in this single analysis. We present a comparison of this experiment with a previously published yeast proteome analysis by Yates and colleagues (Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-7). In addition, we report an in-depth analysis of the false-positive rates associated with peptide identification using the Sequest algorithm and a reversed yeast protein database. New criteria are proposed to decrease false-positives to less than 1% and to greatly reduce the need for manual interpretation while permitting more proteins to be identified.
Article
Recently, we have developed a high-resolution two-dimensional separation strategy for the analysis of complex peptide mixtures. This methodology employs isoelectric focusing of peptides on immobilized pH gradient (IPG) gels in the first dimension, followed by reversed-phase chromatography in the second dimension, and subsequent tandem mass spectrometry analysis. The traditional approach to this mixture problem employs strong-cation-exchange (SCX) chromatography in the first dimension. Here, we present a direct comparison of these two first-dimensional techniques using complex protein samples derived from the testis of Rattus norvegicus. It was found that the use of immobilized pH gradients (narrow range pH 3.5-4.5) for peptide separation in the first dimension yielded 13% more protein identifications than the optimized off-line SCX approach (employing the entire pI range of the sample). In addition, the IPG technique allows for a much more efficient use on mass spectrometer analysis time. Separation of a tryptic digest derived from a rat testis sample on a narrow range pH gradient (over the 3.5-4.5 pH range) yielded 7626 and 2750 peptides and proteins, respectively. Peptide and protein identification was performed with high confidence using SEQUEST in combination with a data filtering program employing pI and statistical based functions to remove false-positives from the data.
Article
A novel integrated multidimensional liquid chromatography (IMDL) method is demonstrated for the separation of peptide mixtures by two-dimensional HPLC coupled with ion trap mass spectrometry. The method uses an integrated column, containing both strong cation exchange and reversed-phase sections for two-dimensional liquid chromatography. The peptide mixture was fractionated by a pH step using a series of pH buffers, followed by reversed-phase chromatography. Since no salt was used during separation, the integrated multidimensional liquid chromatography can be directly connected to mass spectrometry for peptide analysis. The pH buffers were injected from an autosampler, and the entire process can be carried out on a one-dimensional liquid chromatography system. In a single analysis, the IMDL system, coupled with linear ion trap mass spectrometry, identified more than 2000 proteins in mouse liver. The peptides were eluted according to their pI distribution. The resolution of the pH fractionation is approximately 0.5 pH unit. The method has low overlapping across pH fractions, good resolution of peptide mixture, and good correlation of peptide pIs with pH steps. This method provides a technique for large-scale protein identification using existing one-dimensional HPLC systems.
Article
Two-dimensional liquid chromatography is often used to reduce the proteomic sample complexity prior to tandem mass spectrometry analysis. The 2D-LC performance depends on the peak capacity in both chromatographic dimensions, and separation orthogonality. The peak capacity and selectivity of many LC modes for peptides is not well known, and mathematical characterization for orthogonality is underdeveloped. Consequently, it is difficult to estimate the performance of 2D-LC for peptide separation. The goal of this paper was to investigate a selectivity of common LC modes and to identify the 2D-LC systems with a useful orthogonality. A geometric approach for orthogonality description was developed and applied for estimation of a practical peak 2D-LC capacity. Selected LC modes including various RP, SCX, SEC, and HILIC were combined in 2D-LC setups. SCX-RP, HILIC-RP, and RP-RP 2D systems were found to provide suitable orthogonality. The RP-RP system (employing significantly different pH in both RP separation dimensions) had the highest practical peak capacity of 2D-LC systems investigated.
Article
Shotgun proteomics, where a tryptic digest of a complex proteome sample is directly analyzed by either single dimensional or multidimensional liquid chromatography tandem mass spectrometry, has gained acceptance in the proteomics community at large and is widely used in core facilities. Here we review the development in our laboratory of an alternative first-dimension separation technique for shotgun proteomics, immobilized pH gradient isoelectric focusing (IPG-IEF). The key advantages of the technology over other multidimensional separation formats (simplicity, high resolution, and high sensitivity) are discussed. The concept of using peptide pI to filter large shotgun proteomics datasets generated by the IPG-IEF technique to minimize false positives and negatives is also introduced. Finally, an account of the comparison of the technique with the established gold standard for multidimensional separation of peptides, strong cation exchange chromatography, is presented, along with the prospects for the use of peptide pI along with accurate mass measurement for the identificat