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

Abstract

Summary 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.

Mass spectrometry-based proteomics for system-level characterization of biological responses to engineered nanomaterials

“…With the advances of MS-based proteomics, global phosphoproteome profiling now allows quantifying > 10,000 site-specific phosphorylation events in one experiment. Various enrichment techniques such as immobilized metal affinity chromatography (or IMAC) have been developed to overcome the low-abundance nature of phosphorylation [60]. For instance, in one study using IMAC-enrichment of phosphopeptides, differential phosphorylation on 32 proteins were observed in human lung cells treated with CuO [61], and many proteins were involved in multiple signaling pathways such as lipid antigen presentation and telomerase signaling.…”

Advances in microscale separations towards nanoproteomics applications

“…However, the sensitivity challenge for most types of PTM analyses is much more significant compared to global proteome profiling due to sub-stoichiometric and low-abundance nature of PTMs. Using phosphoproteomics as an example, typically hundreds of micrograms of peptides are required even when the most advanced nanoLC-MS platforms were used [102,103]. Recently, online integration of TiO 2 enrichment with nanoLC-MS [104,105] or orthogonal 3D separations [64] have significantly improved the overall sensitivity of the phosphoproteomics workflow.…”

“…Recently, online integration of TiO 2 enrichment with nanoLC-MS [104,105] or orthogonal 3D separations [64] have significantly improved the overall sensitivity of the phosphoproteomics workflow. However, the current state-of-the-art of phosphoproteomics still falls far short of analyzing nanogram-scale samples [103]. …”

Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines

“…Quantitative analysis of redox PTMs with significant coverage (i.e., identifying >10,000 Cys sites), similar to those achieved with phosphoproteome, may soon become a reality. 135 The development of novel approaches to enable broad quantification of both relative changes and site occupancy of multiple types of redox PTMs will significantly enhance our understanding of the role of protein thiols in redox signaling and regulation. The simultaneous measurement of multiple redox PTMs and other PTMs, such as phosphorylation or acetylation by either global or targeted quantification methods, may reveal significant insight into cell signaling through “cross-talk” between different types of PTMs.…”