Simultaneous multiple substrate tag detection with ESI-ion trap MS for in vivo bacterial enzyme activity profiling.
A bacterial identification method in which multiple enzyme activities are measured simultaneously and in vivo with electrospray ionization-mass spectrometry (ESI-MS) is described. Whole-cell bacteria are immobilized onto a filter support and incubated with a mixture of substrates. Each substrate is chosen to measure a specific enzyme activity of a targeted bacterium and to produce a tag of unique molecular weight. After a predetermined incubation time, the solution is filtered, and the supernatant consisting of a mixture of released tags and unhydrolyzed substrates is directly analyzed, without chromatographic separation, by ESI-MS. Bacteria remain viable on the filter for further analyses. The method was tested by measuring the aminopeptidase activity of the bacteria Escherichia coli, Bacillus subtilis, Bacillus cereus, and Pseudomonas aeruginosa. The resulting aminopeptidase enzyme profiles allowed the differentiation between the four bacteria tested. The method is rapid, since a multiplex advantage is realized when assaying for multiple enzymes, and it is amenable to automation via a flow injection analysis setup.
fbasile@mines.edu
Franco
F
Basile
Imma
I
Ferrer
Edward T
ET
Furlong
Kent J
KJ
Voorhees
D000626:Aminopeptidases / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D013379:Substrate Specificity
D000626:Aminopeptidases