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

Ming-Quan      Guo; Bill      X. Huang

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.

Keywords: Liquid chromatography, Mass spectrometry, Phosphoproteomics, Stable isotope labeling, Label-free quantification, Protein phosphorylation, Absolute quantification, Limit of detection, Fourier transform-Ion Cyclotron Resonance