Characterization of Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis-Separated M. Agalactiae Membrane Antigens by Mass Spectrometry

Mycoplasma membrane proteins are generally designated according to their apparent molecular weight measured by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). However, several results about mycoplasma membrane antigens are in conflict because some doubts are emerging about the accuracy of the method utilized to identify the antigens. The aim of this work was to characterize proteins separated after (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, have been excised from gel and their whole molecular weight and proteolytic patterns determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). The peptide pattern obtained using trypsin digestion allowed us to identify LipA, P48, P59, P80 and P40. Some other proteins showed analogies to the proteins of Mycoplasma genitalium or Mycoplasma pneumoniae, the only completely sequenced Mycoplasmas. There wasn't even a close correspondence between the apparent molecular weight from SDS-PAGE (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 tandem mass spectrometry (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.