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Development of narrow immobilized pH gradients covering one pH unit for human seminal plasma proteomic analysis
Author(s) -
StaritaGeribaldi Mireille,
Roux Françoise,
Garin Jérôme,
Chevallier Daniel,
Fénichel Patrick,
Pointis Georges
Publication year - 2003
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200300493
Subject(s) - spots , mass spectrometry , chromatography , chemistry , proteome , immobilized ph gradient , tandem mass spectrometry , matrix assisted laser desorption/ionization , proteomics , microbiology and biotechnology , analytical chemistry (journal) , biology , desorption , biochemistry , isoelectric focusing , enzyme , organic chemistry , adsorption , gene
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.