Liquid Chromatography and Mass Spectrometry (LC-MS) Based Strategies for Quantitative Phosphoproteomics

Abstract

Reversible protein phosphorylation is among the most widespread and important protein modifications in nature, regulating a vast number of key cellular signaling pathways, and deregulation of phosphorylation-mediated processes is often implicated in many human diseases including cancers. Large-scale qualitative and quantitative analysis of protein phosphorylation is thus essential for understanding these important cellular signaling pathways and their underlying mechanisms. This challenging task has been enabled by exciting progress in both effective phosphopeptide enrichment strategies and powerful liquid chromatography coupled to with mass spectrometry (LC-MS). This review will detail some of the most recent developments in LC-MS based phosphoproteomics with an emphasis on quantitative analysis of protein phosphorylation or quantitative phosphoproteomics. We discuss a variety of LC-MS based in vivo and in vitro stableisotope labeling and label-free methods for global quantitative phosphoproteomics, and briefly introduce the progress in targeted quantitative phosphoproteomics while highlighting the application of some up-to-date popular techniques with corresponding representative workflows. For each strategy, the benefits and drawbacks are compared and highlighted in order to aid scientists in tailoring the most appropriate strategy under various circumstances for the global or targeted analysis of protein phosphorylation pertinent to any biological or biomedical questions in their own research.

Newly Developed Poly(Allyl Glycidyl Ether/Divinyl Benzene) Polymer for Phosphopeptides Enrichment and Desalting of Biofluids

“…The phosphorylated peptides and proteins have been of great significance as they are ready to lend a hand in understanding the disease status and act as biomarkers in biomedical research. 6,7 The mass spectrometric detection of the phosphorylated peptides has always been tricky because of the ionization suppression caused by its low abundance. 8,9 To overcome this problem, the enrichment of phosphopeptides prior to MS analysis has become a necessity to identify the peptides involved in different diagnostic works.…”

“…Thus functionalization of the polymer helps in selective binding of the molecules of interest such as phosphopeptides and phosphoproteins. The phosphorylated peptides and proteins have been of great significance as they are ready to lend a hand in understanding the disease status and act as biomarkers in biomedical research. , The mass spectrometric detection of the phosphorylated peptides has always been tricky because of the ionization suppression caused by its low abundance. , To overcome this problem, the enrichment of phosphopeptides prior to MS analysis has become a necessity to identify the peptides involved in different diagnostic works. − Among various techniques, immobilized metal ion affinity chromatography (IMAC) with different metal ions such as Fe 3+ , Ga 3+ , Zr 4+ , and Ti 4+ is predominant for the enrichment before analysis by MALDI-MS. − Immobilized metal ions capture the phosphopeptides through negatively charged phosphate groups of peptides. , …”