Proteomic Technologies Used in Cancer Proteomics

Proteomics is the study of the proteome—defined as the total set of proteins expressed in a given cell type at a given time [27]. Tremendous progress has been made in the past few years in generating large-scale cellular protein profiles, organelle composition, protein activity patterns, and datasets for protein-protein interactions.

The key technologies underpinning cancer proteomic studies have been and remain 2D-PAGE for protein separation and mass spectrometry (MS) for protein identification. 2D-PAGE has been used for decades to separate complex mixtures of proteins, but recent technological improvements (in particular, immobilized pH-gradients and gels meeting industrial standards) have greatly enhanced resolution, sensitivity, and reproducibility of this separation method. 2D-PAGE is capable of displaying 103-104 polypeptide chains, characterized by a single isoelectric point (pi) and M(r) value as coordinates. This technology provides separation of proteins according to their physicochemical characteristics independent of immunochemical methods typically used to separate proteins. Data output from 2D-PAGE is typically slow and analysis is limited to low-throughput means. These limitations prevent this technology from being used to rapidly screen large sample numbers, an important concept in biomarker discovery. Despite these considerations, 2D-PAGE has been effectively used as a discovery tool in numerous human cancers, both for expression and functional purposes.

4.2. Differential In-Gel Electrophoresis

Differential in-gel electrophoresis (DIGE) facilitates protein expression by labeling different populations of proteins with fluorescent dyes. Typically, paired samples from the normal and tumor region are labeled with Cy3 and Cy5. After analysis by differential analysis image software, protein spots that exhibit a significant difference in intensity are excised for in-gel tryptic digestion and MS analysis.

4.3. Isotope-Coded Affinity Tagging and Amino Acid-Coded Mass Tagging

The isotope-coded affinity tag (ICAT) technology enables the concurrent identification and comparative quantitative analysis of proteins present in biological fluids by coupling microcapillary chromatography with electro-spray ionization tandem MS. A two-step approach was developed to identify proteins whose abundance changes between samples. In the first step, a software program for the automated quantification of ICAT reagent labeled peptides analyzed by microcapillary electrospray ionization TOF-MS determines those peptides that differ in abundance. In the second step, peptides are identified by tandem MS using an electrospray quadrupole TOF-MS and sequence database searching [28]. Amino acid-coded mass tagging (AACT)-assisted MS is an ICAT-derived method. This technology utilizes a number of different heavy amino acids as internal markers that significantly increase the peptide sequence coverage for both quantitation and identification. Despite their promise, ICAT and AACT are still emerging technologies with limited application in cancer research.

4.4. Gel-Free Technologies

Over the recent years, proteomic technologies evolved rapidly and now offer new perspectives in discovery of new cancer biomarkers based on the detection of low molecular weight proteins or peptides by MS. Two main peptidomic approaches are currently under investigation. These include pattern recognition and single/oligo biomarker detection.

4.4.1. Pattern Recognition

Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF)-MS provides a mean to rapidly assess complex protein mixtures in body fluids and, to a lesser degree, in tissues. Combined with pattern recognition algorithms, SELDI technology generates highly accurate diagnostic information. This system appeared to have potential in biomarker discovery and, to a lesser degree, as a clinical diagnostic assay platform [29], especially in human cancer. Despite its initial enthusiastic appeal, substantial skepticism developed for SELDI due to questionable reproducibility and lack of definitive protein characterization. In the meantime, it was possible to identify the peaks of interest by various techniques, that is by gel electrophoresis, in-gel trypsin digestion, and tandem MS. Identities of proteins could then be confirmed by conventional technologies like ELISA and immunodepletion assays.

4.4.2. Single/Oligo Biomarker Detection

The MALDI time-of-flight mass spectrometer (MALDI-TOF-MS) is a powerful analytical tool for comprehensive profiling of peptides. This sensitive instrument provides the ability to simultaneously detect minor peptide changes in complex biological mixtures. Usually, a few micrograms of sample are sufficient to create a detailed mass spectrum. The resolution and specificity of this analysis is further increased by coupling MS with liquid chromatography (LC) or other high-resolution technologies. For example, biological samples separated by means of high-performance LC (HPLC) are individually analyzed with MALDI-MS. The result of this peptide profiling process is the generation of a two-dimensional (2D) dataset with fraction number and mass-to-charge ratio (m/z) as coordinates. With proper methodological care, peptide abundance is represented by the mass spectra signal intensity. This can be visualized in a 2D intensity map, using a color intensity code. Specialized software tools are often used to data mine via statistical analysis in order to obtain the most significant experimental changes [30].

4.5. Targeted Glycoproteomics

In human cancer, surface gylcoproteins have been shown to play a key role in immune response generation and metastasis (reviewed, for example, in [31]). Thus, there is an opportunity for direct targeting of glycoproteins in comparative proteomics analysis. This approach will provide analysis of oligosaccharides released from glycoproteins as well as the recovery and identification of proteins with aberrant glycosylation. Technological advances, in particular application of capillary electrophoresis in combination with MS and tandem MS, is expected to facilitate biomedical glycoscreening projects [32].

4.6. Reverse-Phase Protein Arrays-Based Studies

Reverse-phase protein arrays offer a robust new method of quantitatively assessing expression levels and the activation status of a panel of proteins. For this purpose, the lysate of protein(s) of interest is arrayed without selection via a capture molecule. This array can then be queried with an antibody or ligand probe, or an unknown biological component. Since an individual test sample is immobilized in each array spot, this array can be composed of a variety of different patient samples. Each array is incubated with one detection protein or antibody, and a single end point is measured across the arrayed cohort and can be directly compared across multiple samples. Replicates can be reproducibly printed at a given sitting, increasing quality control over a series of queried arrays (reviewed in [33]).

4.7. Antibody-Based Proteomic Studies

The significance of antibody microarrays in cancer research has been reviewed [34, 35]. Several applications of this technology have been reported, including protein analysis in serum, resected frozen tumors, cell lines, and on membranes of blood cells. Antibody microarray experimental formats can be categorized into two categories: direct labeling and dual antibody sandwich assays. Each format has its distinct advantages and disadvantages. In the direct labeling method, the covalent labeling of all proteins in a complex mixture provides a means for detecting bound proteins after incubation on an antibody microarray. If proteins are labeled with a tag, such as biotin, the signal from bound proteins can be amplified. In the sandwich assay, proteins captured on an antibody microarray are detected by a cocktail of specific detection antibodies.

4.8. Combinatorial Beads

Another technique that could facilitate biomarker discovery in the serum of cancer patients consists in the use of combinatorial beads to reduce the dynamic range of proteins. This technique consists in a library of combinatorial ligands coupled to small beads. This library comprises hexameric ligands composed of amino acids, resulting in millions different structures. When these beads are impregnated with complex proteomes (e.g., human body fluids) of widely differing protein compositions, they are able to significantly reduce the concentration differences and thus greatly enhance the possibility of identifying species of low abundance [36].

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