title: Efficient identification of phosphorylation by mass spectrometric phosphopeptide fingerprinting.
abstract: We describe a rapid and efficient method for the identification of phosphopeptides, which we term mass spectrometric (MS) phosphopeptide fingerprinting. The method involves quantitative comparison of proteolytic peptides from native versus completely dephosphorylated proteins. Dephosphorylation of serine, threonine, and tyrosine residues is achieved by in-gel treatment of the separated proteins with hydrogen fluoride (HF). This chemical dephosphorylation results in enrichment of those unmodified peptides that correspond to previously phosphorylated peptides. Quantitative comparison of the signal-to-noise ratios of peaks in the treated versus untreated samples are used to identify phosphopeptides, which can be confirmed and further studied by tandem mass spectrometry (MS/MS). We have applied this method to identify eight known phosphorylation sites of Xenopus Aurora A kinase, as well as several novel sites in the Xenopus chromosome passenger complex (CPC).
authors.affiliation: The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.
authors.forename: Eileen M
authors.initials: EM
authors.lastname: Woo
authors.forename: David
authors.initials: D
authors.lastname: Fenyo
authors.forename: Benjamin H
authors.initials: BH
authors.lastname: Kwok
authors.forename: Hironori
authors.initials: H
authors.lastname: Funabiki
authors.forename: Brian T
authors.initials: BT
authors.lastname: Chait
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D064127:Aurora Kinases; D002875:Chromosomes / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D008970:Molecular Weight; D010449:Peptide Mapping / Q000379:methods*; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D010748:Phosphopeptides; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases