Identification of Bacterial Proteins Observed in MALDI TOF Mass Spectra from Whole Cells

Abstract

Characteristic ions in the MALDI TOF mass spectra from bacterial cells have been associated with four known proteins. The proteins, observed both from cells and in filtered cellular suspensions, were isolated by HPLC and identified on the basis of their mass spectra and their partial amino acid sequence, determined using the Edman method (10-15 residues). The acid resistance proteins HdeA and HdeB give rise to ions near m/z 9735 and 9060 in MALDI TOF mass spectra from cells and from extracts of both Escherichia coli 1090 and Shigella flexneri PHS-1059. However, the proteins associated with proteolytic cleavage by the peptidase Lep, rather than the precursor proteins, were observed, both using cells and from cellular extracts. A cold-shock protein, CspA, was associated with the ion near m/z 7643 from Pseudomonas aeruginosa. Similarly, a cold-acclimation protein, CapB, was identified as the source of the ion near m/z 7684 in P. putida. This last protein was homologous with a known CapB from P. fragi. While these experiments involved the detection of known or homologous proteins from typical bacteria, this same approach could also be applied to the detection of unique proteins or biomarker proteins associated with other bacteria of public health significance.

Escherichia coli aconitases and oxidative stress: post-transcriptional regulation of sodA expression

“…Its expression is enhanced in stationary phase and repressed by H-NS (Yoshida et al, 1993 ;Holland et al, 1999) and the pattern of expression observed here suggests that AcnA (the stationary-phase enzyme) has a positive effect on HdeB synthesis, consistent with the view that Acn proteins assist in co-ordinating a stress response. Finally, the RpsA contents are lowered in all of the acn mutants (Table 2).…”

MALDI mass spectrometry analysis of high molecular weight proteins from whole bacterial cells: Pretreatment of samples with surfactants

“…A majority of the early research published on whole cell bacterial MALDI analysis described protein profiles up to 30 kDa [2,3]. The signals in this range were attributed to cellular proteins or cell wall associated proteins [8,9].Numerous studies were reported to expand the upper mass range for bacterial proteins obtained from whole cell MALDI-TOF-MS analysis [7,9]. The influence of matrix, solvent, and spotting techniques on the mass range and the quality of the MALDI spectra were reported.…”

Applications of MALDI‐TOF Mass Spectrometry in Clinical Diagnostic Microbiology

“…Several research groups have demonstrated that biomolecules desorbed from whole unfractionated cells and detected above 4 kDa are intact proteins (Arnold et al, 1999;Dai et al, 1999;Holland et al, 1999;Ryzhov & Fenselau, 2001). Most of the biomarkers detected in MALDI-TOF spectra of intact bacterial cells have a molecular mass below 15 kDa.…”

“…Similar to ribosomal proteins, these protein families are highly abundant, basic, and of medium hydrophobicity. Holland et al (1999) potentially identified the acid-resistant precursor proteins HdeA and HdeB observed in the MALDI analysis of both intact E. coli and Shigella flexneri. The ion at m/z 7643 in the spectra from Pseudomonas aeruginosa was mapped to the cold-shock protein CspA and similarly the ion at m/z 7684 observed in Pseudomonas putida was identified as the cold-acclimation protein CapB (Fenselau & Demirev, 2001).…”