Characterization of surface layer proteins from Clostridium difficile by liquid chromatography electrospray ionization mass spectrometry

Surface layers (S-layers) are regularly ordered protein subunits found as t he outermost cell envelope component of many bacteria. Most S-layers are co mposed of a single protein or glycoprotein species with a molecular weight varying between 40 and 200 kDa, Clostridium difficile is the most common ca use of antibiotic associated diarrhea (AAD) and pseudomembranous colitis (P MC) in humans. Detection of the Slayer in some C. difficile strains, and pr eliminary characterization of two glycoproteins, P36 and P47, involved in t he composition of the S-layer of one of these strains (C. difficile C253), led us to investigate the most appropriate conditions for purification and chemical characterization of these proteins, This work describes the result s obtained when liquid chromatograpy (LC) coupled to mass spectrometry (MS) using electrospray ionization was applied to the analysis of C. difficile S-layer proteins (SLPs). In this way the molecular weights of the two SLP c omponents, P36 and P47, were detected to be 34258 +/- 2 and 39545 +/- 3 Da, respectively, These data deviate from sodium dodecyl sulfate-polyacrylamid e gel electrophoresis (SDS-PAGE) results by 1.85 and 7.5 kDa, To confirm th e LC-MS results, an alternative molecular weight analysis was performed: th e two S-layer proteins were isolated by semipreparative high performance li quid chromatography (HPLC), concentrated, and analyzed by matrix-assisted l aser desorption/ionization time-of-flight (MALDI-TOF). The two SLP subunits were digested with protease V8, and the peptide maps were determined by LC -MS using a C-18 column. Finally, preliminary results about peptide glycosy lation were obtained, Copyright (C) 1999 John Wiley & Sons, Ltd.