Phosphorylated peptides chromatography

Another means of moving beyond pure protein preparations to high-throughput characterization of proteomes is to enrich for phosphopeptides from complex mixtures by metal affinity chromatography (Andersson and Porath, 1986). Using this method, protein mixtures are proteolyzed to create peptides and phosphorylated peptides are enriched by metal affinity chromatography and subsequently identified by mass spectrometry. This method is limited, however, because in many cases phosphopeptides absorb poorly or nonphosphorylated peptides absorb nonspecifically to the metal affinity resins (Ahn and Resing, 2001). 

A more traditional but still successful method for the detection of a protein phosphorylation is by radioactive labeling with 35P. The labeled protein is digested, the peptides are separated by high-performance liquid chromatography, and the phosphorylated peptides are detected in specific fractions via their radioactivity. The fraction with the phosphorylated peptides can be further analyzed by mass spectrometry (Figeys et al., 1999). 

Ding J., Burkhart W., and Kassel D.B. (1994), Identification of phosphorylated peptides from complex mixtures using negative-ion orifice-potential stepping and capillary liquid chromatography/electrospray ionization mass spectrometry, Rapid Commun. Mass Spectrom. 8 94—98. 

Fractionation of proteins by strong cation exchange (SCX) chromatography, followed by IMAC enrichment of phosphopeptides from SCX fractions, led to a comprehensive identification of phosphoproteins of PSD isolated from mouse brain using LC-MS/MS (Trinidad et al. 2006). In this study, phosphorylation site(s) were mapped to 287 proteins from a total of 1,264 unique proteins identified. This translates into a 23% phosphorylation rate, comparable to an expected 33% rate in the general proteome (Johnson et al. 2005). The 287 phosphoproteins were derived from a total of 998 unique phosphorylated peptides, and the phosphorylations were mapped to 723 unique sites. Most of these occurred on serines, to a lesser extent on threonines, and only minimally on tyrosines (Figure 5A). 

Mass spectrometry of phosphopeptides has become a powerful tool for phosphorylation site identification. However, proteolytic digests examined by MS are often likely to fail to detect phosphopeptides because the ionization of phosphorylated peptides in positive ion mode MS is generally less efficient compared with the ionization of their nonphosphorylated counterparts resulting in ion suppression effects. A further problem is that phosphopeptides may not be retained by RP chromatography because they are too small and/or hydrophilic to bind to the CIS stationary phase. Therefore, capillary electrophoresis coupled to MS is a powerful method to enhance the detection of phosphoproteins and phosphopeptides due to their efficient separation by CE. 

Nishio,T., Ayano, E., Suzuki, Y, Kanazawa, H. and Okano,T. (2011). Separation of phosphorylated peptides utilizing dual pH- and temperature-responsive chromatography. Journal of Chromatography A, 1218,2079-2084. 

Tholey, A., Toll, H., Huber, C.G. (2005). Separation and detection of phosphorylated and nonphosophorylated peptides in hquid chromatography—mass spectrometry using monolithic columns and acidic or alkaline mobile phases. Anal. Chem. 77, 4618 1625. 

Phosphorylation on serine, threonine, and tyrosine residues is an extremely important modulator of protein function. Phosphorylation can be analyzed by mass spectrometry with enrichment of compounds of interest using immobilized metal affinity chromatography and chemical tagging techniques, detection of phosphopep-tides using mass mapping and precursor ion scans, localization of phosphorylation sites by peptide sequencing, and quantitation of phosphorylation by the introduction of mass tags (McLachlin and Chait 2001). 

Collins, M.O. and Yu, L. et al. (2005b) Robust enrichment of phosphorylated species in complex mixtures by sequential protein and peptide metal-affinity chromatography and analysis by tandem mass spectrometry. Sci. STKE 2005,16. 

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