Characterization of sodium dodecylsulfate polyacrylamide gel electrophoresis-separated M. agalactiae membrane antigens by mass spectrometry

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Eur. J. Mass Spectrom. DOI: 10.1255/ejms.621

Characterization of sodium dodecylsulfate polyacrylamide gel electrophoresis-separated M. agalactiae membrane antigens by mass spectrometry

Franco Carta
Porto Conte Ricerche, 07041 Alghero (SS), Italy
Salvatore Crobu
Consorzio Proteomics 07100 Sassari (SS), Italy
Franco Turrini
Dipartimento di Genetica, Sezione di Biochimica, University of Turin, 10126 Turin, Italy

ABSTRACT:

Mycoplasma membrane proteins are generally designated according their apparent molecular weight measured by SDS-PAGE. Several results about mycoplasma membrane antigens are conflicting because some doubts are emerging about the accuracy of the method utilised to identify the antigens. Aim of this work, was to characterise proteins separated after sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)-mass spectrometry to allow an uncontroversial designation of the antigens. Fifteen proteins with molecular weights ranging from 15,000 to 80,000 Da had been excised from gel and their whole molecular weight and proteolytic pattern had been determined using MALDI-TOF. The peptide pattern obtained using trypsin digestion allowed us to identify LipA, P48, P59, P80 and P40. Some other proteins showed analogies to proteins of Mycoplasma genitalium or Mycoplasma pneumoniae the only Mycoplasmas completely sequenced. There wasn't a close correspondence between the SDS-PAGE apparent molecular weight (generally used to name the proteins), the gene derived calculated mass and the molecular weight of whole proteins measured by MALDI-TOF. Only micro sequence data obtained by MS/MS allowed us to identify LipC, described as one of the most important Mycoplasma agalactiae antigens. This protein was found in correspondence with the 50 kDa region, instead of the 25 kDa region, confirming a phenomenon that we previously described.

Keywords: mycoplasma agalactiae, SDS-PAGE, 2D-PAGE, MALDI-ToF, tandem MS, in-gel digestion, database search, peptide mass fingerprint, membrane antigens, protein identification

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