Article

Recent applications of protein chips in biomarker discovery

October 2006
Molecular & Cellular Proteomics 5(10):S84-S84

Authors:

Dolores Cahill

University College Dublin

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Article

Expression Microarray Proteomics and the Search for Cancer Biomarkers

November 2006 · Current Genomics

Farid E Ahmed

Protein microarrays allow the simultaneous determination of a variety of parameters in parallel from minute amounts of sample, which allows for a high throughput analysis of translated gene functions. This article provides a wide and updated coverage of topics such as the complexity of this technology as compared to the gene expression technology, classification of protein microarrays, surface ... [Show full abstract] chemistry, detection methods, capture agents, experimental design, data analysis, standards, databases and interactions. The role of protein microarrays for the identification of cancer biomarkers in tissue and serum has also been elucidated. There are currently no guidelines on how to standardize these assays for biomarker discovery. Issues of sensitivity, specificity, difficulty in maintaining the native state of the protein upon surface immobilization, and limitations of the current arrays represent challenges that need to be overcome, and should be understood by those who are employing this parallel multiplexed technology in order to be able to achieve high throughput applications and minimize the occurrence of false positive and negative results.

Article

Clinical Proteomics and Biomarker Discovery

July 2004 · Annals of the New York Academy of Sciences

Early detection of disease generally provides much-improved outcomes by a definitive medical procedure or through lifestyle modification along with specific medical management strategies. For serum biomarkers, which are central to the diagnosis of many diseases, to become truly useful sentinels of pathogenesis, their sensitivity and specificity in both early detection and recurrence monitoring ... [Show full abstract] must be improved. Currently, the detection and monitoring of disease markers is based on solitary proteins, and this approach is not always reliable. New classes of biomarkers derived from mass spectroscopy analysis of the low molecular weight proteome have shown improved abilities in the early detection of disease and hence in patient risk stratification and outcome. The development of a modular platform technology with sufficient flexibility and design abstractions allowing for concurrent experimentation, test, and refinement will help speed the progress of mass spectroscopy-derived proteomic pattern-based diagnostics from the scientific laboratory to the medical clinic. For acceptance by scientists, physicians, and regulatory personnel, new bioinformatic tools are essential system components for data management, analysis, and intuitive display of these new and complex data. Clinically engineered mass spectroscopy systems are essential for the further development and validation of multiplexed biomarkers that have shown tremendous promise for the early detection of disease.

Article

Clinical Proteomics for Cancer Biomarker Discovery and Therapeutic Targeting

September 2002 · Technology in Cancer Research & Treatment

As we emerge into the post-genome era, proteomics finds itself as the driving force field as we translate the nucleic acid information archive into understanding how the cell actually works and how disease processes operate. Even so, the traditionally held view of proteomics as simply cataloging and developing lists of the cellular protein repertoire of a cell are now changing, especially in the ... [Show full abstract] sub-discipline of clinical proteomics. The most relevant information archive to clinical applications and drug development involves the elucidation of the information flow of the cell; the "software" of protein pathway networks and circuitry. The deranged circuitry of the cell as the drug target itself as well as the effect of the drug on not just the target, but also the entire network, is what we now are striving towards. Clinical proteomics, as a new and most exciting sub-discipline of proteomics, involves the bench-to-bedside clinical application of proteomic tools. Unlike the genome, there are potentially thousands of proteomes: each cell type has its own unique proteome. Moreover, each cell type can alter its proteome depending on the unique tissue microenvironment in which it resides, giving rise to multiple permutations of a single proteome. Since there is no polymerase chain reaction equivalent to proteomics- identifying and discovering the "wiring diagram" of a human diseased cell in a biopsy specimen remains a daunting challenge. New micro-proteomic technologies are being and still need to be developed to drill down into the proteomes of clinically relevant material. Cancer, as a model disease, provides a fertile environment to study the application of proteomics at the bedside. The promise of clinical proteomics and the new technologies that are developed is that we will detect cancer earlier through discovery of biomarkers, we will discover the next generation of targets and imaging biomarkers, and we can then apply this knowledge to patient-tailored therapy.

Article

Full-text available

Protein Microarrays as Tools for Functional Proteomics: Achievements, Promises and Challenges

December 2013 · Journal of Proteomics & Bioinformatics

Abstract The quest for a better understanding of organisms, and the human body in particular, at a comprehensive level has stimulated the development of techniques and processes that permit the analysis and assessment of biomedical information at high throughput. This has had substantial impact, not only by merely gathering knowledge but also by making scientists aware of the necessity to view ... [Show full abstract] organisms as complex biological systems rather than an assembly of individual biochemical pathways. Proteomics is still lagging behind genomics in this holistic analysis, however, because of the much higher degree of complexity that needs to be dealt with. Protein arrays, among other techniques, offer the prospect of advancing global protein analysis, similar to the impact of arrays in genomics. In basic research, protein arrays already contributed substantially, permitting the identification of many protein interactions and providing information on expression variations and structural changes, for example. Beside the fact of high throughput, arrays require relatively small reaction volumes, which is critical in view of the lack of means for in vitro protein amplification and beneficial for good assay sensitivity. Applications comprise many facets, such as the search for drug targets, analysis of host-parasite interactions and, above all, biomarker discovery. Despite the achievements and promises of the technology, it is still far from being a standard approach and many technical developments are ongoing. In this review, we look at the state of protein array technology and discuss future perspectives.

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Thesis

Biomarker discovery and pathway activation profiling in breast cancer using protein microarrays

January 2013

Johanna Sonntag

Breast cancer, the most frequent cancer entity among women, is nowadays widely recognized as a heterogeneous disease in terms of histopathology as well as on the molecular level. Over the last few years, gene expression profiling studies have improved our understanding of the underlying molecular mechanisms associated with the very heterogeneous outcomes of breast cancer patients. The existence ... [Show full abstract] of intrinsic molecular subtypes, which are linked to unique biological and prognostic features, was repeatedly demonstrated and points to the need of tailored therapy options. However on the functional level, breast cancer is not only a genomic but mainly a proteomic disease and gene expression profiling might provide only limited insights. Following the hypothesis that intrinsic biologic features of breast tumors affect prognosis and also therapy response, the general aim of this thesis was to further explore breast cancer heterogeneity with protein microarrays on the functional proteomics level. Around 70 80% of all breast cancer patients belong to the luminal intrinsic molecular subtype, characterized as a surrogate marker by overexpression of hormone receptors. An improved classification of this subtype is crucial for therapy decision as part of the patients are at higher risk of recurrence requiring chemo-endocrine treatment, whereas the other part is at lower risk and does not benefit from chemotherapy. However, accurate definition of low and high risk hormone receptor-positive breast cancer has remained a challenge so far. Thus, the first objective of this thesis was the identification of a robust and quantitative protein biomarker signature to facilitate risk classification of hormone receptor-positive breast cancer. To approach this aim reverse phase protein arrays were used to screen across over 120 breast cancer relevant proteins and a novel bioinformatics workflow for biomarker hit selection was applied. Using this approach, a biomarker signature consisting of caveolin-1, NDKA, RPS6, and Ki-67, was identified as most promising to distinguish between low and high risk hormone receptor-positive breast cancer. Since genomic and transcriptomic profiling alone cannot sufficiently predict protein pathway activation, it is important to explore and define the heterogeneity of hormone receptor-positive breast cancer on the proteome level. Especially as protein signaling pathways present the direct targets of new classes of therapeutics. Thus, the second thesis objective addressed the question of whether hormone receptor-positive breast cancer can be further categorized according to similar signaling pathway activation patterns and whether these patterns reflect common molecular mechanisms. Therefore, comprehensive protein pathway activation profiles of breast cancer specimens were obtained using reverse phase protein arrays. To complement this analysis, a microspot immunoassay was developed, which enabled the simultaneous quantification of eight different growth factors in tumor lysate as well as blood plasma of matching patient samples. Four subgroups were identified, based on differential expression of 90 cancer-relevant signaling proteins. Each subgroup showed unique characteristics which were also related to established clinicopathological features as well as growth factor expression. One subgroup, for example, was characterized by high expression levels of almost all analyzed proteins. In addition, VEGF tumor lysate levels were significantly higher in this subgroup and an enrichment of poorly differentiated tumors was observed underlining the aggressive phenotype. In contrast, another subgroup was characterized by weak signaling activity. Interestingly, this subgroup was mostly associated with invasive lobular carcinoma, the second most common histologic type of breast cancer, reflecting a link between histopathology and underlying molecular mechanisms. In summary, the reverse phase protein array based pathway activation profiling of hormone receptor-positive breast cancer, presented in this thesis, provides a comprehensive snapshot of the heterogeneity of this subtype on the proteomic level. Insights obtained can serve as basis to refine the concept of clinically relevant subtypes towards an improved definition of patient-tailored therapy options.

Article

Full-text available

New technologies in cancer. Protein microarrays for biomarker discovery

March 2011 · Clinical and Translational Oncology

The emergence of proteomic techniques and methodological approaches to study disease has kindled the quest for new biomarkers. Thus, the use of protein microarrays has surged as a powerful tool for large-scale testing of biological samples. In this mini-review, we will discuss the application of protein microarray technologies that offer unique opportunities to find novel biomarkers.

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Article

Proteomic approaches to biomarker discovery in lung cancers by SELDI technology

November 2003 · Science in China Series C Life Sciences

The purpose of the present work is to identify protein profiles that could be used to discover specific biomarkers in serum and discriminate lung cancer. Thirty serum samples from patients with lung cancer (15 cases of primary brochogenic carcinoma, 9 cases of metastasis lung cancer and 6 cases of lung cancer after chemotherapy) and twelve from healthy individuals were analyzed by SELDI (Surfaced ... [Show full abstract] Enhanced Laser Desorption/Ionization) technology. Anion-exchange columns were used to fractionate the sera with 6 designated pH washing solutions. Two types of protein chip arrays, IMAC-Cu and WCX2, were employed. Protein chips were examined in PBSII ProteinChip Reader (Ciphergen Biosystems Inc.) and the resulting profiles between cancer and normal were analyzed with Biomarker Wizard System. In total, 15 potential lung cancer biomarkers, of which 6 were up-regulated and 9 were down-regulated, were discovered in the serum samples from patients with lung cancer. 5 of 15 these biomarkers were able to be detected on both WCX2 and IMAC-Cu protein chips. The sensitivities provided by the individual markers range from 44.8% to 93.1% and the specificities were 85.0%-94.4%. Our results suggest that serum is a capable resource for detection of lung cancer with specific biomarkers. Moreover, protein chip array system was shown to be a useful tool for identification, as well as detection of disease biomarkers in sera.

Article

Biomarker Discovery by Tissue Microdissectionand ProteinChip® Array Analysis

May 2003 · LaboratoriumsMedizin

In recent years, the search for new cancer biomarkers received a strong impulse from genomic and proteomic high throughput techniques. Biomarkers or biomarker patterns should enable scientists or medical staff to make a more reliable early diagnosis of certain human diseases, especially malignant tumors, and to make prediction of their progression, which could further contribute to a more ... [Show full abstract] differentiated, individually orientated tumor therapy. However, until now relevant markers have been established for only a few tumor diseases. One of the most promising proteomic tools for the detection of new cancer biomarkers is the ProteinChip® System technology. This mass spectrometry based system (SELDI; Surface Enhanced Laser Desorption and Ionization) appears to have a high potential for biomarker discovery and may serve in future as a clinical diagnostic assay platform. Herein, we will review the current scientific literature dealing with cancer biomarker discovery by users of the ProteinChip System. Special emphasis is given to the investigation of microdissected tumor material. Further, we will discuss bioinformatic tools, suitable for the localisation and assessment of potential biomarkers.

Article

Colorectal cancer proteomics, molecular characterization and biomarker discovery

February 2010 · PROTEOMICS - CLINICAL APPLICATIONS

Colorectal cancer (CRC) is a widespread disease, whose major genetic changes and mutations have been well characterized in the sporadic form. Much less is known at the protein and proteome level. Still, CRC has been the subject of multiple proteomic studies due to the urgent necessity of finding clinically relevant markers and to elucidate the molecular mechanisms underlying the progression of ... [Show full abstract] the disease. These proteomic approaches have been limited by different technical issues, mainly related with sensitivity and reproducibility. However, recent advances in proteomic techniques and MS systems have rekindled the quest for new biomarkers in CRC and an improved molecular characterization. In this review, we will discuss the application of different proteomic approaches to the identification of differentially expressed proteins in CRC. In particular, we will make a critical assessment about the use of 2-D DIGE, MS and protein microarray technologies, in their different formats, to identify up- or downregulated proteins and/or autoantibodies profiles that could be useful for CRC characterization and diagnosis. Despite a wide list of potential biomarkers, it is clear that more scientific efforts and technical advances are still needed to cover the range of low-abundant proteins, which may play a key role in CRC diagnostics and progression.

Article

Comparative protein profiling of serum and plasma using an antibody suspension bead array approach

February 2010 · Proteomics

In the pursuit towards a systematic analysis of human diseases, array-based approaches within antibody proteomics offer high-throughput strategies to discover protein biomarkers in serum and plasma. To investigate the influence of sample preparation on such discovery attempts, we report on a systematic effort to compare serum and plasma protein profiles determined with an antibody suspension bead ... [Show full abstract] array. The intensity levels were used to define protein profiles and no significant differences between serum and plasma were observed for 79% of the 174 antibodies (targeting 156 proteins). By excluding 36 antibodies giving rise to differential intensity levels, cluster analysis revealed donor-specific rather than preparation-dependent grouping. With a cohort from a clinically relevant medical condition, the metabolic syndrome, the influence of the sample type on a multiplexed biomarker discovery approach was further investigated. Independent comparisons of protein profiles in serum and plasma revealed an antibody targeting ADAMTSL-4, a protein that would qualify to be studied further in association with the condition. In general, the preparation type had an impact on the results of the applied antibody suspension bead array, and while the technical variability was equal, plasma offered a greater biological variability and allowed to give rise to more discoveries than serum.

Article

Evaluation of Biomarker Discovery Approaches to Detect Protein Biomarkers of Acute Renal Allograft R...

April 2005 · Journal of Proteome Research

Management of host responses to allografts by immunosuppressive therapy is the cornerstone of transplantation medicine, but it is still deficient in one important element: biomarkers that are readily accessible and predict the fate of the transplant early, specifically, and reliably. Using a Brown Norway (BN)-to-Lewis rat renal allograft model of kidney transplantation, this study aims at ... [Show full abstract] evaluating two proteomic approaches to discover biomarkers for acute rejection: SELDI-MS technology and 2D gel electrophoresis combined with mass spectrometry. Several novel potential serum biomarkers have been identified for follow up. Overall, the conclusion is that apparently at the serum protein level, dramatic changes only occur at a stage where kidney function is already severely affected. Multivariate analysis of serum profiles suggests that there is an ensemble of subtle changes, comprising a proteomic signature of acute rejection at an early stage, a more detailed evaluation of which might provide novel opportunities for the diagnosis of acute rejection. Profiling of the excreted proteins indicates that urine might even present the earliest signs of the rejection process.

Article

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ProteinChips: the essential tools for proteomic biomarker discovery and future clinical diagnostics

February 2005 · Clinical Chemistry and Laboratory Medicine

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Recent applications of protein chips in biomarker discovery

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