Current Proteomics - Volume 10, Issue 2, 2013

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating the spatial distribution of proteins and small molecules within biological specimen. This technique combines the potential of MALDI-ToF mass spectrometry with the ability to scan series of pixels across the surface of tissues, which generates multiplex space-correlated mass spectra. Numerical processing of the data allows visualization of specific molecular species and their correlation with histological image of the tissue. Hence, IMS is a multiplex untargeted analysis that enables characterization of tissue regions based on their endogenous biomolecular content. The major advantage of this technique is its potential to define tissue regions based on their molecular profiles independently of their histological and morphological characteristics given by traditional tools. IMS can detect potential cancer foci within histologically normal tissue, reveal intra-tumor heterogeneity or altered protein expression at tumor/normal tissue interface zones. IMS is an emerging technology that could fill gaps in the knowledge of spatial nature of a disease allowing better understanding of connections between structure and molecular processes.

Protein arrays represent a step forward in proteomic technologies, as they enable to address biological and functional questions by large-scale approaches (i.e., up to several thousand proteins may be monitored in a single experiment). Here we review recent achievements of protein arrays and their classification. We also summarize current and future applications of this technology to address the study of the human proteome including protein identification, biomarker discovery, screening of protein-protein/molecule interactions, detection of protein post-translational modifications and serum profiling. Advantages and limitations of protein arrays compared to other proteomic technologies are also discussed here. The use, advances and recent applications of fluorescent microspheres as a promising alternative to planar arrays are also summarized in this report. Finally, we propose future perspectives of protein arrays from a comprehensive standpoint that includes technological, biological, economical and practical aspects.

Posttranslational modifications (PTMs) are dynamic regulators of protein function, and play important roles in diseases such as cancer. PTM analysis can be challenging, the stoichiometry of PTMs is often low, and various combinatorial modifications are possible. Currently, two major techniques are used to detect and characterize PTMs, immunoassays and mass spectrometry. Immunoassays rely on antibodies for detection of the protein of interest, and are therefore limited to targeted analysis. Mass spectrometry, on the other hand, is capable of characterizing posttranslational modifications both in targeted or non-targeted methods. Recently, new immunoassays were introduced that improve current methods, but also appear particularly promising in the analysis of PTMs. Two of these new immunoassays, proximity ligation assay and nanoscale immunoassay, are discussed in this review. In contrast to immunoassays, mass spectrometry enables characterization of a priori unknown PTM sites. A bottom-up approach, in which proteins are digested into smaller peptides, is well suited for targeted assays as well as cataloging PTMs. A top-down approach, where intact proteins are measured, is challenging but allows mapping of combinatorial PTMs. Mass spectrometry and immunoassays are therefore complementary techniques in analysis of PTMs. Advances in these methods now enable extremely sensitive detection of PTMs from very little material (immunoassays), or can fully characterize combinatorial modifications on proteins in both targeted and non-targeted ways (mass spectrometry). Recent developments in these techniques discussed in this review will therefore likely play an important role in current and future PTM analysis, particularly in the field of cancer research.

Although proteomics is a very recently developed science, important advances have already occurred in this field. There is growing interest in the scientific community for using this technology as a high-throughput innovative strategy for biomarker discovery in the field of cancer research. However, certain challenges must be overcome before reaching this goal. The basis for discovering reliable biomarkers is rooted in technical and biological parameters. The main biological limit is outlining tumor heterogeneity, while methodological issues include appropriate study designs, the use of sensitive technologies for screening, and the employment of confident strategies for data validation. This review focuses on the major questions related to the strengths and limitations of the development of cancer biomarkers by proteomic- based studies.

A plenitude of reasons, including tumour heterogeneity, mechanisms of resistance to therapy, a complex pathogenesis, in which cancer stem cells are involved, contribute to the poor outcome of HNSCC patients after therapy. The roles of vital signalling proteins are constantly unravelled, and different types of inhibitors are examined yielding novel potential targeting strategies. However, there will be a long way to go, before drugs targeting these molecules will be available in routine clinical practice. In this review the focus will be on these novel targets, as well as inhibiting agents, which nowadays are combined to an increasing degree. Additionally, the role proteomics plays in modern drug research will be highlighted.

Transgelins, which have been studied by proteomic methods for more than ten years as tumor-associated proteins, are the members of calponin family, since calponin homology domains are present in their amino acid sequences and there are other typical structural features. According to the available information there is a specific tissue distribution of these proteins. Three closely related genes which coded transgelins are revealed in the human genome. Single nucleotide substitutions and the expression peculiarities of these genes can be the reasons for existence of several isoforms of three main transgelins. Different manifestations of biochemical polymorphism of transgelins are detected by now. Transgelin (or SM22-alpha) is well known as a biomarker of smooth muscle cell differentiation. It was revealed that transgelin acts as repressor of MMP9 gene, coding matrix metalloproteinase-9. At the same time the oppositely directed changes in the content of transgelin proteins were found in tissue samples of malignant tumors of the respiratory, digestive and urogenital systems, as well as in the cultivated tumor cell using proteomic technologies. Correspondingly, according to some authors transgelins can be potential biomarkers of malignant neoplasms, but in the opinion the others – transgelins are suppressors of tumor growth. In this review, we consider possible reasons for such differences and discuss the prospects of solution of the existing contradictions.

Clinical proteomics studies are extensively being conducted in recent years. We have outlined some views about proteomics progresses for clinical research, via the understanding the molecular mechanisms of carcinogenesis, tumor response to therapy and biomarker identification versus novel therapeutic target-discovery; as well as proteomerelated methods used in cancer research. Integrative concepts of development of novel therapeutic targets and the application of proteomics in drug development are also discussed. Several aspects are touched including the development of novel therapeutic targets, proteomics as a tool to discover biomarkers, discovery and validation of prognostic and predictive biomarkers, and biobanking versus proteomics. Finally, we envision the application of clinical proteomics for future personalized cancer therapies can set up a consensus that will be reached amongst the scientific community.