Identification of marker proteins for Bacillus anthracis using MALDI-TOF MS and ion trap MS/MS after direct extraction or electrophoretic separation.

Direct extraction of bacterial vegetative cells or spores followed by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI TOF MS) has become popular for bacterial identification, since it is simple to perform and mass spectra are readily interpreted. However, only high-abundance proteins that are of low mass and ionize readily are observed. In the case of B. anthracis spores, small acid-soluble spore proteins (SASPs) have been the most widely studied. Additional information can be obtained using tandem mass spectrometry (MS-MS) to confirm the identity of proteins by sequencing. This is most readily accomplished using ion trap (IT) MS-MS. However, enzymatic digestion of these proteins is needed to generate peptides that are within the mass range of the ion trap. The use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), or other forms of electrophoresis, allows one to focus on specific proteins of interest (e.g. the high mass exosporium glycoproteins BcIA and BcIB) that provide additional species- and strain-specific discrimination.

Identification of a second collagen-like glycoprotein produced by Bacillus anthracis and demonstration of associated spore-specific sugars.

Certain carbohydrates (rhamnose, 3-O-methyl rhamnose, and galactosamine) have been demonstrated to be present in Bacillus anthracis spores but absent in vegetative cells. Others have demonstrated that these spore-specific sugars are constituents of the glycoprotein BclA. In the current work, spore extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A second collagen-like glycoprotein, BclB, was identified in B. anthracis. The protein moiety of this glycoprotein was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and the carbohydrate components by gas chromatography-mass spectrometry and tandem mass spectrometry. Spore-specific sugars were also demonstrated to be components of BclB.

Strategies and data analysis techniques for lipid and phospholipid chemistry elucidation by intact cell MALDI-FTMS.

Ions attributed to lipids and phospholipids are directly observed by desorption from whole bacteria using intact cell (IC) matrix-assisted laser desorption-ionization (MALDI) Fourier transform mass spectrometry (FTMS). Saccharomyces cerevisiae are grown in rich media broth, concentrated, and applied directly to the MALDI surface without lysis or chemical treatment. FTMS of MALDI ions gives excellent signal to noise ratios with typical resolving powers of 90,000 and mass precision better than 0.002 Da. Use of accurate mass measurements and a simple set of rules allow assignment of major peaks into one of twelve expected lipid classes. Subsequently, fractional mass versus whole number mass plots are employed to enhance visual interpretation of the high-resolution data and to facilitate detection of related ions such as those representing homologous series or different degrees of unsaturation. This approach, coupled with rules based on bacterial biochemistry, is used to classify ions with m/z up to about 1000. Major spectral peaks in the range m/z 200-1000 are assigned as lipids and phospholipids. In this study, it is assumed that biologically-derived ions with m/z values lower than 1000 are lipids. This is not unreasonable in view of the facts that molecular weights of lipids are almost always less than 1000 Da, that the copy numbers for lipids in a cell are higher than those for any single protein or other component, and that lipids are generally collections of distinct homologous partners, unlike proteins or other cell components. This paper presents a new rapid lipid-profiling method based on IC MALDI-FTMS.

Use of double-depleted 13C and 15N culture media for analysis of whole cell bacteria by MALDI time-of-flight and Fourier transform mass spectrometry.

In the present paper, results demonstrating the significant advantages of matrix-assisted laser desorption/ionization (MALDI) analysis of whole cell samples of bacteria grown on double isotopically-depleted (13C and 15N) media are presented. It is shown that several advantages accrue for MALDI with a 9.4 T Fourier transform mass spectrometer (FTMS). Of particular note, for analysis of whole cells, sample preparation is simple and chemical interference is reduced. Moreover, ion coalescence problems are minimized, and data-base identification of proteins facilitated. Furthermore, high resolution mass spectra obtained from such whole cells show significant improvement in apparent mass resolving power and mass measurement accuracy, whether time-of-flight or FTMS MALDI is used. As a consequence, it becomes possible to detect subtle details in the chemistry of the organism, such as the presence of both post-translationally modified and unmodified versions of the same proteins. This approach is also adaptable to direct assay of over-expressed proteins from Escherichia coli cultures and should facilitate studies aimed at the detection of medically important cellular biomarker proteins.

Investigation of MALDI-TOF and FT-MS techniques for analysis of Escherichia coli whole cells.

Recently, it has been demonstrated that bacteria can be characterized using whole cells and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). However, identification of specific bacterial proteins usually requires analysis of cellular fractions or purified extracts. Here, the first application of Fourier transform mass spectrometry (FTMS) to analysis of bacterial proteins directly from whole cells is reported. It is shown that accurate mass MALDI-FTMS can be used to characterize specific ribosomal proteins directly from Escherichia coli cells. High-accuracy mass measurements and high-resolution isotope profile data confirm posttranslational modifications proposed previously on the basis of low-resolution mass measurements. Seven ribosomal proteins from E. coli whole cells were observed with errors of less than 27 ppm. This was accomplished directly from whole cells without fractionation, concentration, or overt overexpression of characteristic cellular proteins. MALDI-FTMS also provided information regarding E. coli lipids in the low-mass region. Although ions with m/z values below 1000 have been observed by FTMS of whole cells, this represents the first report of detection of ions in the 5000 to 10,000 m/z range by MALDI-FTMS using whole cells.