The current state of the art of quantitative phosphoproteomics and its applications to diabetes research

Abstract

Protein phosphorylation is a fundamental regulatory mechanism in many cellular processes and aberrant perturbation of phosphorylation has been implicated in various human diseases. Kinases and their cognate inhibitors have been considered as hotspots for drug development. Therefore, the emerging tools, which enable a system-wide quantitative profiling of phosphoproteome, would offer a powerful impetus in unveiling novel signaling pathways, drug targets and/or biomarkers for diseases of interest. This review highlights recent advances in phosphoproteomics, the current state of the art of the technologies and the challenges and future perspectives of this research area. Finally, some exemplary applications of phosphoproteomics in diabetes research are underscored.

Keywords: LC-MS/MS; Phosphoproteomics; diabetes; phosphopeptide enrichment; phosphorylation; quantification; targeted proteomics.

Figure 1. The overall workflows of MS-based global quantitative phosphoproteomics

Both label-free and stable isotope labeling based quantitative approaches can be applied. For labeling approach, SILAC can be performed at cell culture level and iTRAQ/TMT at peptide level. The peptides can either be subjected to LC fractionation prior to enrichment or directly subjected to phosphopeptide enrichment. LC-fractionation can also be applied after phosphopeptide enrichment.

Figure 2. Overview of targeted approaches for phosphoproteomics

(A) Multiplex immunoassays. In planar array-based assay, or ELISA, Specific primary phospho-antibodies are anchored to a carbon electrode plate (Antibody-1, and -2), which recognize phosphoproteins of interest. With the application of a fluorescent-tagged secondary antibody, the fluorescence signal measured corresponds to the expression level of that particular phosphoprotein in the sample. In the microbead-based array, primary antibodies are conjugated to the microspheres, and each microsphere batch is distinctly barcoded with different fluorophores mixed at a specific ratio (red, and yellow beads). (B) Protein microarray (or protein chip).In the forward-phase protein microarrays (FPPAs), phospho-antibodies are anchored on the chip to detect the phosphoproteins of interest; whereas in the reverse-phase protein microarrays (RPPAs), the clinical samples are directly spotted on the surface. With the sequential incubation of the cognate primary and secondary antibodies, phosphoprotein of interest could then be detected. (C) Targeted mass spectrometry. Heavy isotope-labeled synthetic peptides are typically spiked into protein digest as internal standards prior to IMAC/MOAC enrichment. The enriched phosphopeptides are subjected to SRM/PRM based targeted quantification, and the levels of phosphorylation are quantified based on light-to-heavy isotopic ratios.