Investigation of MALDI-TOF and FT-MS Techniques for Analysis of Escherichia coli Whole Cells

Abstract

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.

High Accuracy Mass Measurement Approach in the Identification of Phospholipids in Lipid Extracts: 7 T Fourier-transform Mass Spectrometry and MS/MS Validation

“…However, considering that the mass tolerance of 21.38 ppm is known to be a required mass accuracy for the correct assignments of lipid species under the assumption of proton cationization, 10 ppm is not a loose criterion for accurate mass measurements. 16 On the other hand, when a multiple number of lipid candidates existed within a 10 ppm tolerance from the observed monoisotopic mass, only the candidate with the smallest delta-mass (Δm) was selected (Table 1-6). In the case where two closely-spaced peaks existed, both peaks were considered to find lipid candidates as long as these two peaks showed 'A' and 'A+1' isotopic distributions.…”

“…4,[10][11][12][13][14][15][16][17] Even with the disadvantage of relatively low sensitivity, the direct-infusion ESI-FTMS approach is very easy to implement and also provides a good method to compare the global lipid distributions of two total lipid extracts under inspection.…”

“…[15][16][17] Wilkins and coworkers developed a data analysis strategy that utilizes a mass defect plot (fractional mass versus whole number mass) for the identification of lipid species detected in the high-resolution MALDI FTMS spectra. 4,16,17 Using only accurate mass measurements and a simple set of rules related to mass defects, a majority of ion-peaks below m/z 1,000 could be distinguished into twelve lipid classes. The classified lipids were further identified using their in-house lipid database with more than 50,000 entries.…”

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

“…Using a ribosomal fraction (not whole cells) Arnold and Reilly [29] observed that several ribosomes seem to be present without the expected post-translational modifications, most notably the loss of the C-terminal methionine. Recently, we have confirmed the identity of several ribosomes and their modified forms by accurate mass, high resolution MALDI FTMS analysis of whole cell Escherichia coli [39]. Because the FTMS measurements were done with high accuracy, reliable assignments of the detected ions to ribosomes are possible even without separation from the other cellular proteins.…”

“…Monoisotopic ion species from five of the previously-identified ribosomal proteins [39] were used as calibrants. The broadband spectra were divided into two mass ranges (m/z 2000 -7000 and m/z 7000 -13000) and the calibrant peaks falling within the ranges used to separately calibrate those subspectral regions.…”