| dc.description.abstract | Protein phosphorylation is a reversible modification of cellular proteins that regulate protein function, cellular localization and formation of protein complexes. It is involved in almost all cellular processes and deregulation of protein phosphorylation has been implicated in the development of many diseases. Currently, mass spectrometry (MS) is the method of choice for studying cellular phosphorylation events, as the state-of-art MS techniques are able to analyze thousands of phosphopeptides present in complex biological samples in one experiment, in a rapid and highly sensitive fashion. However, phosphorylation analysis with MS is challenged by the substoichiometric levels of phosphorylated peptides, suppression of phosphopeptide ionization inside mass spectrometers and the loss of the phosphate group during peptide ion fragmentation in tandem mass spectrometry (MS/MS). With this respect, various developments in sample preparation strategies, instrumentation, quantitative methodologies and data analysis tools have been achieved to enhance MS-based phosphorylation analysis. Selective isolation and enrichment of phosphorylated proteins/peptides in analytical samples can be achieved prior to MS analysis by techniques such as, immobilized metal affinity chromatography (IMAC) using metal ions, metal oxide chromatography (MOAC) using metal oxides, other chromatographic methods such strong cation exchange (SCX) chromatography or the use of specific antibodies directed against specific phosphorylated residues in immunoprecipitation, while, combining different enrichment techniques are frequently employed to enhance the efficiency of enrichment and the subsequent phosphorylation analysis. Several MS approaches were developed to facilitate the analysis of phosphopeptides which can be difficult due the loss of the phosphate group. These include multi-stage MS with additional activation steps (e.g. MS3), mass spectrometers with high-energy implementations and special scan modes and the employment of ion fragmentation techniques that avoid the phosphate group loss, such as electron-based dissociation techniques. In this literature review, the principles and applications of the state-of-art MS methodologies, current challenges and future directions in phosphoproteomics are described. In addition, some of the advances made in chemical biology tools for studying protein phosphorylation are discussed. | |
