Efficient identification of phosphorylation by mass spectrometric phosphopeptide fingerprinting.
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).
Woo
Eileen M
EM
The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.
Fenyo
David
D
Kwok
Benjamin H
BH
Funabiki
Hironori
H
Chait
Brian T
BT
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
D010748:Phosphopeptides; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases