Chromatographic and mass spectrometric methods for the identification of phosphorylation sites in phosphoproteins.
The phosphorylation sites in a model phosphoprotein, alpha s1-casein from bovine milk, have been identified by tryptic peptide mapping (Gibson and Cohen, Methods Enzymol. vol. 193, p. 480 (1990)) employing reversed-phase high performance liquid chromatography (RPHPLC)/electrospray ionization mass spectrometry (ES-MS); by infusion tandem mass spectrometry (MS/MS) and LC/MS/MS in neutral loss mode of tryptic digests of alpha s1-casein, in which the characteristic neutral loss of phosphoric acid by phosphopeptides under collision-induced dissociation (CID) conditions is exploited to highlight phosphopeptides in a tryptic digest (Covey et al., in Methods in Protein Sequence Analysis, Jörnvall et al. (Eds), Birkhäuser Verlag, Basel 1991), and by a novel method, termed LC/CID-MS, in which phosphopeptides are located in mixtures of peptides by the generation and detection of phosphate-specific fragment ions during LC/ES-MS (Huddleston et al., J. Am. Soc. Mass Spectrom. vol. 4, p. 710 (1993)). An appraisal of the efficiency, sensitivity and practicality of each of these methods in the identification of phosphorylation sites in post-translationally modified proteins is given.
Hunter
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Mass Spectrometry Research Unit, University College of Swansea, UK.
Games
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D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D014357:Trypsin
D002364:Caseins; D010750:Phosphoproteins; D000469:Alkaline Phosphatase; D014357:Trypsin