Abstract
Matrix-assisted laser desorption with concomitant ionization, in combination with a linear time-of-flight mass spectrometer, was used to analyze underivatized and hard-to-solubilize surface layer proteins and glycoproteins by depositing them on top of a microcrystalline layer of the matrix alpha-cyano-4-hydroxycinnamic acid. Use of this special sample preparation technique allowed the first successful desorption-ionization of intact surface layer proteins and accurate determination of their molecular weights by mass spectrometry. The molecular mass of the monomeric subunit of the major surface layer protein isolated from Clostridium thermosaccharolyticum E207-71 was determined to be 75,621 +/- 81 Da. The obtainable mass accuracy of the technique is conservatively considered to be within +/- 0.2%. This result deviates from that given by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by approximately 7.4 kDa because this method is strongly affected and biased by the three-dimensional structure of this type of surface protein. With the apparent advantages of unsurpassed mass accuracy, low dependence on the physicochemical properties of the surface layer proteins, and high sensitivity, it can be concluded that a linear time-of-flight instrument combined with UV matrix-assisted laser desorption with concomitant ionization is better suited for molecular weight determination than is gel electrophoresis.
Highlights
Crystalline bacterial cell surface layers (S-layers) have been identified as the outermost cell envelope of many eubacteria and archaeobacteria
In the ongoing systematic survey of S-layer proteins and glycoproteins, several thermophilic strains of Clostridium thermosaccharolyticum obtained from the collection of the Austrian Sugar Research Institute (Tulln, Austria) were screened [19]
Even much higher molecular weights have been suggested for S-layer subunits on the basis of mass distribution data obtained by high-resolution electron microscopy [3]
Summary
Crystalline bacterial cell surface layers (S-layers) have been identified as the outermost cell envelope of many eubacteria and archaeobacteria (for reviews, see references 8, 15, 23, and 26). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been the method of choice for this task and for the monomeric subunits, molecular masses in the range of approximately 30 to 220 kDa were determined [23]. Even much higher molecular weights have been suggested for S-layer subunits on the basis of mass distribution data obtained by high-resolution electron microscopy [3].
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