02930nas a2200337 4500008004100000245016200041210006900203260001300272300001200285490000600297520178400303653002402087653001202111653002902123653002102152653002102173653000902194653002202203653000902225653002802234653002002262653002002282653001302302653002302315653001602338100001902354700002202373700002002395700002302415856015402438 2009 eng d00aUse of differential isotopic labeling and mass spectrometry to analyze capacitation-associated changes in the phosphorylation status of mouse sperm proteins.0 aUse of differential isotopic labeling and mass spectrometry to a c2009 Mar a1431-400 v83 a
Mammalian 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.
10aAmino Acid Sequence10aAnimals10aChromatography, Affinity10aFourier Analysis10aIsotope Labeling10aMale10aMass Spectrometry10aMice10aMolecular Sequence Data10aPhosphopeptides10aPhosphorylation10aProteome10aSperm Capacitation10aSpermatozoa1 aPlatt, Mark, D1 aSalicioni, Ana, M1 aHunt, Donald, F1 aVisconti, Pablo, E uhttps://www.umass.edu/veterinary-animal-sciences/research-faculty/publications/use-of-differential-isotopic-labeling-and-mass-spectrometry-to-analyze