Relative quantification of peptide phosphorylation in a complex mixture using 18O labeling. Physiol Genomics 2007 Oct 22;31(2):357-63
Date
08/09/2007Pubmed ID
17684036Pubmed Central ID
PMC2771636DOI
10.1152/physiolgenomics.00096.2007Scopus ID
2-s2.0-35549008176 (requires institutional sign-in at Scopus site) 22 CitationsAbstract
We have developed a method to determine the degree of phosphorylation of a peptide in a complex mixture without enrichment or operation of the mass spectrometer in negative ion mode. Yeast lysate containing known amounts of synthetic peptides (VPQLEIVPNSAEERLHSMK and VPQLEIVPN[pS]AEERLHSMK) was labeled with (16)O and (18)O during hydrolysis. After treatment of one sample with a cocktail of phosphatases, the two samples were pooled. The intensity of the dephosphorylated peptide peaks was used to infer the degree of phosphorylation present before treatment. The linear dynamic range of this method is >10-fold before either of the peptide envelopes becomes indistinguishable from the surrounding noise. Since both the site of posttranslational modification and the proportion of the protein population that is modified are vital in protein function, the employment of this technique will provide a valuable tool for the analysis of the functional implications of protein phosphorylation.
Author List
Smith JR, Olivier M, Greene ASMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceChromatography, Liquid
Hydrolysis
Molecular Sequence Data
Oxygen Isotopes
Peptides
Phosphoric Monoester Hydrolases
Phosphorylation
Protein Processing, Post-Translational
Sensitivity and Specificity
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
