Sample preparation in matrix-assisted laser desorption/ionization mass spectrometry of whole bacterial cells and the detection of high mass (>20 kDa) proteins.
Three sample preparation strategies commonly employed in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS) of whole bacterial cells were investigated for the detection of high mass signals; these included the dried droplet, the seed-layer/two-layer, and the bottom-layer methods. Different sample preparation approaches favoured the detection of high- or low-mass proteins. The low-mass peaks were best detected using the bottom-layer method. By contrast, the dried droplet method using a solvent with higher water content, and hence effecting a slower crystallization process, gave the best results for the detection of high-mass signals. Signals up to m/z 158 000 could be detected with this methodology for Bacillus sphaericus. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the same extracts used for MALDI-TOFMS showed bands in the molecular weight range in which high-mass peaks were observed in MALDI-MS, suggesting that the high-mass signals are not polymeric adducts of low-mass protein monomers. In addition, one of the high molecular weight proteins (approximately 126 kDa) was putatively identified as an S-layer protein by an in-gel tryptic digest. The bacterial samples spotted on the target wells for MALDI-TOFMS, using the different sample preparation strategies, were examined under a scanning electron microscope and differences were observed between the different strategies, suggesting that the nature of the crystals and the distribution of the analytes amidst the crystals could influence the spectral pattern observed in MALDI-TOFMS of whole bacterial cells. Finally, evidence is presented to indicate that, although the determinants are intact cells, cell lysis occurs both before and during the MALDI process.
Vaidyanathan
Seetharaman
S
Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth, Ceredigion SY23 3DD, Wales, UK. ssv@aber.ac.uk
Winder
Catherine L
CL
Wade
Steve C
SC
Kell
Douglas B
DB
Goodacre
Royston
R
D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism*; D003373:Coumaric Acids / Q000032:analysis; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D006094:Gram-Positive Bacteria / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008970:Molecular Weight; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001426:Bacterial Proteins; D003373:Coumaric Acids; C073734:sinapinic acid