Development of narrow immobilized pH gradients covering one pH unit for human seminal plasma proteomic analysis.
Micropreparatively loaded two-dimensional (2-D) electrophoresis gels were used to identify human seminal plasma polypeptides by using narrow immobilized pH gradients (IPGs) covering one pH unit as first dimension. This investigation was restricted to IPG 4.5-5.5 and 5-6 because of the main spot distribution in the acidic part of the 2-D map performed with IPG 3-10, a zone presumed to be rich in spermatogenic markers. Both highly expressed and minor spots of the 2-D map were analyzed by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectroscopy analysis. Identification was obtained by a combination of mass spectrometry and database searching. Identified proteins were of different origin from the male genital tract and some proteolysis was observed. They appeared as either isolated molecules or isoforms. At the analytical level, narrow IPGs allowed a two-fold increase in the number of spots, improved resolution of minor spots particularly in surrounding of highly expressed spots and sensitivity level. Some of these faint spots were differently expressed in azoospermic patients as compared to normospermic men. Therefore, zooming-in on the proteome of human seminal plasma allowed more accurate differential expression analysis of impaired spermatogenesis associated markers.
Starita-Geribaldi
Mireille
M
INSERM EMI 00-09, Groupe de Recherche en Endocrinologie et Reproduction, IFR 50, UFR de Médecine, 28 avenue de Valombrose, 06107 Nice cedex 2, France. mgeribal@unice.fr
Roux
Françoise
F
Garin
Jérôme
J
Chevallier
Daniel
D
Fénichel
Patrick
P
Pointis
Georges
G
D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020543:Proteome; D012661:Semen / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D020543:Proteome