ACCURATE DETERMINATION OF THE MOLECULAR-WEIGHT OF THE MAJOR SURFACE-LAYER PROTEIN ISOLATED FROM CLOSTRIDIUM-THERMOSACCHAROLYTICUM BY TIME-OF-FLIGHT MASS-SPECTROMETRY

Matrix-assisted laser desorption with concomitant ionization, in combi nation with a linear time-of-flight mass spectrometer, was used to ana lyze underivatized and hard-to-solubilize surface layer proteins and g lycoproteins by depositing them on top of a microcrystalline layer of the matrix alpha-cyano-4-hydroxycinnamic acid. Use of this special sam ple preparation technique allowed the first successful desorption-ioni zation of intact surface layer proteins and accurate determination of their molecular weights by mass spectrometry. The molecular mass of th e monomeric subunit of the major surface layer protein isolated from C lostridium thermosaccharolyticum E207-71 was determined to be 75,621 /- 81 Da. The obtainable mass accuracy of the technique is conservativ ely considered to be within +/-0.2%. This result deviates from that gi ven by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by ap proximately 7.4 kDa because this method is strongly affected and biase d by the three-dimensional structure of this type of surface protein. With the apparent advantages of unsurpassed mass accuracy, low depende nce on the physicochemical properties of the surface layer proteins, a nd high sensitivity, it can be concluded that a linear time-of-flight instrument combined with UV matrix-assisted laser desorption with conc omitant ionization is better suited for molecular weight determination than is gel electrophoresis.