Direct infusion electrospray ionization mass spectra of crude cell extracts for microbial characterizations: influence of solvent conditions on the detection of proteins.
Direct infusion electrospray ionization mass spectrometry (DIES-MS) of crude bacterial extracts is a rapid method that can be used to characterize microbial cells. Phospholipids, metabolites, and proteins can be detected rapidly with minimal sample preparation. However, several factors influence the detection of signals in such high-throughput analyses. We studied the influence of solvent conditions, including the organic content and pH of the solvent, on the extraction and subsequent detection of signals in DIES-MS, with a view to improving the detection of protein signals. Unfractionated cell extracts from three strains of the Gram-negative Escherichia coli (including one encoding a recombinant green fluorescence protein), and the Gram-positive Bacillus sphaericus and B. subtilis were investigated. Both pH and the organic content of the solvent were found to influence the spectral information as observed from principal component analysis of the spectral data. A polar solvent with higher organic content resulted in the extraction of phospholipids that overtly dominate the spectral information. Decreasing the organic content of the extraction solvent resulted in the improved detection of protein peaks. Altering the pH of the extraction solvent resulted in different protein profiles from the same bacterium, as observed after spectral deconvolution. In addition, the protein profiles were also different when using different organic solvents. Spectral deconvolution showed several protein peaks that had mass-based homology with those in protein databases for the (sequenced) organisms studied. These results suggest that a combination of solvent conditions can be used to generate protein profiles rapidly that when combined can provide additional valuable proteomic information.
Vaidyanathan
Seetharaman
S
School of Chemistry, The University of Manchester, P.O. Box 88, Sackville Street, Manchester M60 1QD, UK. s.vaidyanathan@manchester.ac.uk
O'Hagan
Steve
S
Goodacre
Royston
R
D001407:Bacillus / Q000737:chemistry / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000302:isolation & purification; D012997:Solvents / Q000737:chemistry / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization
D001426:Bacterial Proteins; D012997:Solvents