Mohamed Saiel Saeed Alhamdani

Oncoproteomic profiling with antibody microarrays

The incidence of cancer and its associated mortality are increasing globally, indicating an urgent need to develop even more effective and sensitive sets of biomarkers that could help in early diagnosis and consequent intervention. Given that many cellular processes are carried out by proteins, cancer research has recently shifted toward an exploration of the full proteome for such discovery. Among the advanced methodologies that are being developed for analyzing the proteome, antibody microarrays have become a prominent tool for gathering the information required for a better understanding of disease biology, early detection, discrimination of tumors and monitoring of disease progression. Here, we review the technical aspects and challenges in the development and use of antibody microarray assays and examine recently reported applications in oncoproteomics.

Affinity-based microarrays for proteomic analysis of cancer tissues

Based on about a decade of technical developments in analysing the human proteome with antibody microarrays and experience in performing such analyses, now there are the means at hand for detailed and simultaneously global investigations of this kind. Many technical aspects have been dealt with of both the microarray format itself – such as overcoming kinetic and mass transport limitations and thus achieving accurate measurements – and ancillary processes – such as extraction procedures that provide good protein solubilisation, produce reproducible yields and preserve the native protein conformation as much as possible. The overall analysis process is robust and reproducible, highly sensitive down to the level of single‐molecule detection and permits an analysis of several parameters on many molecules at a time. While the study of body liquids is widely applied, analyses of tissue proteomes are still scarce. However, conditions do exist to perform the latter at a quality level that meets the standards for clinical applications. This review highlights methodological aspects relevant for a biomedically useful analysis of cellular samples and discusses the potential of such studies, in particular, in view of personalised medicine approaches.

Analysis conditions for proteomic profiling of mammalian tissue and cell extracts with antibody microarrays

Antibody microarrays are a developing tool for global proteomic profiling. A protocol was established that permits robust analyses of protein extracts from mammalian tissues and cells rather than body fluids. The factors optimized were buffer composition for surface blocking, blocking duration, protein handling and processing, labeling parameters like type of dye, molar ratio of label versus protein, and dye removal, as well as incubation parameters such as duration, temperature, buffer, and sample agitation.

Single-Step Procedure for the Isolation of Proteins at Near-Native Conditions from Mammalian Tissue for Proteomic Analysis on Antibody Microarrays

The process of extracting comprehensive proteome representations is a crucial step for many proteomic studies. While antibody microarrays are an evolving and promising methodology in proteomics, the issue of protein extraction from tissues for this kind of analysis has never been addressed. Here, we describe a single-step extraction buffer for the isolation of proteins from mammalian tissues under native conditions in an effective and reproducible manner. Protein was extracted from cell lines BxPC-3 and SU.86.86, rat organs (pancreas, liver, heart and lung) and human pancreatic cancer tissues using several buffer systems that contained individual nonionic or zwitterionic detergents in comparison to commercial extraction buffers. Also, detergent combinations were used that included at least one polymeric phenylethylene glycol, a long-chain amidosulfobetaine, cholate and a zwitterionic detergent. Extracts were analyzed for protein quantity and quality. The detergent cocktails exhibited superior extraction capacity. Additionally, they demonstrated a substantially higher recovery of membrane and compartmental proteins as well as much better preservation of protein functionality. Also, they did not interfere with subsequent analysis steps such as labeling. In Western blot and antibody microarray assays, they outperformed the other buffer systems, indicating that they should also be useful for other types of proteomic studies.

Prediction of recurrence of non muscle-invasive bladder cancer by means of a protein signature identified by antibody microarray analyses

About 70% of newly diagnosed cases of bladder cancer are low‐stage, low‐grade, non muscle‐invasive. Standard treatment is transurethral resection. About 60% of the tumors will recur, however, and in part progress to become invasive. Therefore, surveillance cystoscopy is performed after resection. However, in the USA and Europe alone, about 54 000 new patients per year undergo repeated cystoscopies over several years, who do not experience recurrence. Analysing in a pilot study resected tumors from patients with (n = 19) and without local recurrence (n = 6) after a period of 5 years by means of an antibody microarray that targeted 724 cancer‐related proteins, we identified 255 proteins with significantly differential abundance. Most are involved in the regulation and execution of apoptosis and cell proliferation. A multivariate classifier was constructed based on 20 proteins. It facilitates the prediction of recurrence with a sensitivity of 80% and a specificity of 100%. As a measure of overall accuracy, the area under the curve value was found to be 91%. After validation in additional sample cohorts with a similarly long follow‐up, such a signature could support decision making about the stringency of surveillance or even different treatment options.

Secretome profiling with antibody microarrays

Following the advances in human genome sequencing, attention has shifted in part toward the elucidation of the encoded biological functions. Since proteins are the driving forces behind very many biological activities, large-scale examinations of their expression variations, their functional roles and regulation have moved to the central stage. A significant fraction of the human proteome consists of secreted proteins. Exploring this set of molecules offers unique opportunities for understanding molecular interactions between cells and fosters biomarker discovery that could advance the detection and monitoring of diseases. Antibody microarrays are among the relatively new proteomic methodologies that may advance the field significantly because of their relative simplicity, robust performance and high sensitivity down to single-molecule detection. In addition, several aspects such as variations in amount, structure and activity can be assayed at a time. Antibody microarrays are therefore likely to improve the analytical capabilities in proteomics and consequently permit the production of even more informative and reliable data. This review looks at recent applications of this novel platform technology in secretome analysis and reflects on the future.

Robust protein profiling with complex antibody microarrays in a dual-colour mode

Antibody microarrays are a multiplexing technique for the analyses of hundreds of different analytes in parallel from small sample volumes of few microlitres only. With sensitivities in the picomolar to femtomolar range, they are gaining importance in proteomic analyses. These sensitivities can be obtained for complex protein samples without any pre-fractionation or signal amplification. Also, no expensive or elaborate protein depletion steps are needed. As with custom DNA-microarrays, the implementation of a dual-colour assay adds to assay robustness and reproducibility and was therefore a focus of our technical implementation. In order to perform antibody microarray experiments for large sets of samples and analytes in a robust manner, it was essential to optimise the experimental layout, the protein extraction, labelling and incubation as well as data processing steps. Here, we present our current protocol, which is used for the simultaneous analysis of the abundance of more than 800 proteins in plasma, urine, and tissue samples.

Clinical proteomics: Promises, challenges and limitations of affinity arrays

After the establishment of DNA/RNA sequencing as a means of clinical diagnosis, the analysis of the proteome is next in line. As a matter of fact, proteome‐based diagnostics is bound to be even more informative, since proteins are directly involved in the actual cellular processes that are responsible for disease. However, the structural variation and the biochemical differences between proteins, the much wider range in concentration and their spatial distribution as well as the fact that protein activity frequently relies on interaction increase the methodological complexity enormously, particularly if an accuracy and robustness is required that is sufficient for clinical utility. Here, we discuss the contribution that protein microarray formats could play towards proteome‐based diagnostics.

Identification of novel biomarkers in chronic immune thrombocytopenia (ITP) by microarray-based serum protein profiling.

The pathological mechanisms underlying the development of immune thrombocytopenia (ITP) are unclear and its diagnosis remains a process of exclusion. Currently, there are no known specific biomarkers for ITP to support differential diagnosis and treatment decisions. Profiling of serum proteins may be valuable for identifying such biomarkers. Sera from 46 patients with primary chronic ITP and 34 healthy blood donors were analysed using a microarray of 755 antibodies. We identified 161 differentially expressed proteins. In addition to oncoproteins and tumour‐suppressor proteins, including apoptosis regulator BCL2, breast cancer type 1 susceptibility protein (BRCA1), Fanconi anaemia complementation group C (FANCC) and vascular endothelial growth factor A (VEGFA), we detected six anti‐nuclear autoantibodies in a subset of ITP patients: anti‐PCNA, anti‐SmD, anti‐Ro/SSA60, anti‐Ro/SSA52, anti‐La/SSB and anti‐RNPC antibodies. This finding may provide a rational explanation for the association of ITP with malignancies and other autoimmune diseases. While RUNX1mRNA expression in the peripheral blood mononuclear cells (PBMC) of patients was significantly downregulated, an accumulation of RUNX1 protein was observed in the platelets of ITP patients. This may indicate dysregulation of RUNX1 expression in PBMC and megakaryocytes and may lead to an imbalanced immune response and impaired thrombopoiesis. In conclusion, we provide novel insights into the pathogenic mechanisms of ITP that warrant further exploration.

Proteome Variations in Pancreatic Stellate Cells upon Stimulation with Proinflammatory Factors

Background: Stroma formation in pancreatic cancer is controlled via the activation of pancreatic stellate cells. Results: Pancreatic stellate cells demonstrate different proteomic and functional attributes following specific stimuli. Conclusion: The proteomic feature of pancreatic stellate cells shows TNF-α as a main contributor to activation. Significance: The shift of proteomes in activated cells may help in understanding the processes of stromal formation and finding new drug targets. Pancreatic stellate cells are key mediators in chronic pancreatitis and play a central role in the development of pancreatic fibrosis, stromal formation, and progression of pancreatic cancer. This study was aimed at investigating molecular changes at the level of the proteome that are associated with the activation of pancreatic stellate cells by proinflammatory factors, namely TNF-α, FGF2, IL6, and chemokine (C-C motif) ligand 4 (CCL4). They were added individually to cells growing in serum-free medium next to controls in medium supplemented with serum, thus containing a mixture of them all, or in serum-free medium alone. Variations were detected by means of a microarray of 810 antibodies targeting relevant proteins. All tested factors triggered increased proliferation and migration. Further analysis showed that TNF-α is the prime factor responsible for the activation of pancreatic stellate cells. CCL4 is associated with cellular neovascularization, whereas FGF2 and IL6 induction led to better cellular survival and decreased apoptotic activity of the stellate cells. The identified direct effects of individual cytokines on human pancreatic stellate cells provide new insights about their contribution to pancreatic cancer promotion.