title: Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation.
abstract: Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.
authors.affiliation: Department of Chmeistry, University of Virginia, Charlottesville, Virginia 22908, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Ficarro
authors.forename: Olga
authors.initials: O
authors.lastname: Chertihin
authors.forename: V Anne
authors.initials: VA
authors.lastname: Westbrook
authors.forename: Forest
authors.initials: F
authors.lastname: White
authors.forename: Friederike
authors.initials: F
authors.lastname: Jayes
authors.forename: Petr
authors.initials: P
authors.lastname: Kalab
authors.forename: Jarrod A
authors.initials: JA
authors.lastname: Marto
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.forename: John C
authors.initials: JC
authors.lastname: Herr
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
authors.forename: Pablo E
authors.initials: PE
authors.lastname: Visconti
mesh_terms: D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D020543:Proteome; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D000074405:Valosin Containing Protein
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D010750:Phosphoproteins; D020543:Proteome; D014443:Tyrosine; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein