Use of differential isotopic labeling and mass spectrometry to analyze capacitation-associated changes in the phosphorylation status of mouse sperm proteins.

TitleUse of differential isotopic labeling and mass spectrometry to analyze capacitation-associated changes in the phosphorylation status of mouse sperm proteins.
Publication TypeJournal Article
Year of Publication2009
AuthorsPlatt MD, Salicioni AM, Hunt DF, Visconti PE
JournalJournal of proteome research
Volume8
Issue3
Pagination1431-40
Date Published2009 Mar
ISSN1535-3893
KeywordsAmino Acid Sequence, Animals, Chromatography, Affinity, Fourier Analysis, Isotope Labeling, Male, Mass Spectrometry, Mice, Molecular Sequence Data, Phosphopeptides, Phosphorylation, Proteome, Sperm Capacitation, Spermatozoa
AbstractMammalian sperm need to reside in the female reproductive tract for a finite period of time before acquiring fertilizing competence. The biochemical changes associated with this process are collectively known as “capacitation”. With the use of the mouse as an experimental model, we have previously demonstrated that capacitation is associated with a cAMP-dependent increase in protein tyrosine phosphorylation. However, little is known about the identity and function of the protein targets of this phosphorylation cascade. In the present work, we have used differential isotopic labeling coupled with immobilized metal affinity chromatography (IMAC)-based phosphopeptide enrichment and analysis on a hybrid linear ion trap/FT-ICR mass spectrometer to measure the changes in protein phosphorylation resulting from the capacitation process. As no kinase activators and/or phosphatase inhibitors were used in the preparation of the sperm samples, phosphorylated residues identified in this study represent in vivo sites of phosphorylation. Also, in contrast to other methods which rely on the incorporation of isotopically labeled amino acids at the protein level (e.g., SILAC), the present technique is based on the Fisher esterification of protein digests, allowing for the comparison of phosphorylation status in the absence of protein synthesis. This approach resulted in the identification of 55 unique, in vivo sites of phosphorylation and permitted the relative extent of phosphorylation, as a consequence of capacitation, to be calculated for 42 different phosphopeptides. This work represents the first effort to determine which specific protein phosphorylation sites change their phosphorylation status in vivo as a result of the mammalian capacitation process.
DOI10.1021/pr800796j
Alternate JournalJ. Proteome Res.
PubMed ID19186949