Detection and identification of a protein biomarker in antibiotic‐resistant Escherichia coli using intact protein LC offline MALDI‐MS and MS/MS
Authors:
Open Access
- 12 November 2019
- journal article
- research article
- Published by Oxford University Press (OUP) in Journal of Applied Microbiology
- Vol. 128 (3) , 697-709
Abstract
Aims The identification and differentiation of antibiotic‐resistant bacteria by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) profiling remains a challenge due to the difficulty in detecting unique protein biomarkers associated with this trait. To expand the detectable proteome in antibiotic‐resistant bacteria, we describe a method implementing offline LC protein separation/fractionation prior to MALDI‐ToF‐MS and top‐down MALDI‐ToF/ToF‐MS (tandem MS or MS/MS) for the analysis of several antibiotic‐resistant Escherichia coli isolates. Methods and Results Coupling offline LC with MALDI‐ToF‐MS increased the number of detected protein signals in the typically analyzed mass regions (m/z 3000–20 000) by a factor of 13. Using the developed LC‐MALDI‐ToF‐MS protocol in conjunction with supervised principal components analysis, we detected a protein biomarker at m/z 9355 which correlated to β‐lactam resistance among the E. coli bacteria tested. Implementing a top‐down MALDI‐ToF/ToF‐MS approach, the prefractionated protein biomarker was inferred as a DNA‐binding HU protein, likely translated from the blaCMY‐2 gene (encoding AmpC‐type β‐lactamase) in the incompatibility plasmid complex A/C (IncA/C). Conclusions Our results demonstrate the utility of LC‐MALDI‐MS and MS/MS to extend the number of proteins detected and perform MALDI‐accessible protein biomarker discovery in microorganisms. Significance and Impact of the Study This outcome is significant since it expands the detectable bacterial proteome via MALDI‐ToF‐MS.Keywords
Funding Information
- National Institute of General Medical Sciences (P20GM103432)
- National Institute of Food and Agriculture (WYO‐511‐14)
- Directorate for Mathematical and Physical Sciences (NSF‐MRI 1429615)
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