Analysis of protein phosphorylation using mass spectrometry

Protein phosphorylation has been known to be a pivotal modification regulating many cellular activities and functions. Except for several conventional techniques, mass spectrometry-based strategies are increasingly considered as vital tools that can be utilized to characterize phosphorylated peptides or proteins. In this article, we summarized currently available mass spectrometry-based techniques for the analysis of phosphorylation. Due to the low abundance of phosphopeptides, enrichment steps such as specific antibodies, immobilized metal affinity chromatography, and specific tags are crucial for their use in detection. Since the non-specific binding of the enrichment techniques are constantly of major concerns, phosphatase treatment, neutral loss scan, or precursor ion scan enable the recognition of the phosphopeptide signals. In addition, quantitative methods including isotope labeling and mass tags are also discussed. Phosphoproteome analysis seems to provide elucidation of signaling networks and global decipherment of cell activities, which require powerful analytical methods for complete and routine identification of the phosphorylation event. Despite that numerous approaches have been exploited, comprehensive analysis of protein phosphorylation remains a challenging task. With the progressively more improvements of instruments and methodologies, we can foresee the implementation of a comprehensive approach for the analysis of phosphorylation states of proteins.