Advances in Proteomic Technologies and Its Contribution to the Field of Cancer

Figure 4

MRM-MS-based assay workflows (± immunoaffinity enrichment of proteins or peptides). SISCAPA workflow using proteolytic peptides as surrogates for their respective proteins, as illustrated in the top panel of the schematic, is a sensitive approach to measure protein concentrations using immunoaffinity enrichment of surrogate peptides prior to MRM-MS. To achieve quantitation of the targeted protein(s), they are digested to component peptides using an enzyme such as trypsin. A stable isotope standard (SIS, blue asterisk) is added to the sample at a known concentration for quantitative analysis. The selected peptides are then enriched using anti-peptide antibodies immobilized on a solid support. Following washing and elution from the anti-peptide antibody, the amount of surrogate peptide is measured relative to the stable isotope standard using targeted mass spectrometry. Alternatively, an assay can start with immunoaffinity enrichment of intact target proteins from biospecimens using an internal stable isotope-labeled protein standard (red asterisk, such as PSAQ approach) and an antibody, as illustrated in the bottom panel, followed by proteolysis and final quantitation of the target.