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Implementation of normalized retention time (iRT) for bottom-up proteomic analysis of the aminoglycoside phosphotransferase enzyme facilitating method distribution

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Abstract

Improvements in mass spectrometry technology to include instrument duty cycle, resolution, and sensitivity suggest mass spectrometry as a highly competitive alternative to conventional microbiological proteomic techniques. Targeted mass spectral analysis, sans prior empirical measurements, has begun to solely use the enormous amount of available genomic information for assay development. An in silico tryptic digestion of a suspected antibiotic-resistant enzyme using only its genomic information for assay development was achieved. Both MRM and full-scan MS2 independent data acquisitions were obtained for an antibiotic-resistance microbe not previously measured using mass spectrometry. In addition, computation methods to determine highest responding peptides in positive ion mode liquid chromatography-mass spectrometry (LC-MS) were evaluated. Employment of the relative retention time (iRT) concept using a homemade peptide standard set revealed facile method transfer between two fundamental different mass spectral platforms: an ultra-high-pressure liquid chromatography triple quadrupole-mass spectrometer (UHPLC-MS) and nano-liquid chromatography parallel reaction monitoring (nano-LC-PRM) hybrid quadrupole orbitrap Q-exactive mass spectrometer supporting easy dissemination and rapid method implementation between laboratories.

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Authors and Affiliations

  1. U.S. Department of Agriculture, Residue Chemistry and Predictive Microbiology Research Unit, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA, 19038, USA

    Johnny J. Perez

  2. U.S. Department of Agriculture, Molecular Characterization of Foodborne Pathogens Research Unit, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA, 19038, USA

    Chin-Yi Chen

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Correspondence to Johnny J. Perez.

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Published in the topical collection Young Investigators in (Bio-)Analytical Chemistry with guest editors Erin Baker, Kerstin Leopold, Francesco Ricci, and Wei Wang.

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Perez, J.J., Chen, CY. Implementation of normalized retention time (iRT) for bottom-up proteomic analysis of the aminoglycoside phosphotransferase enzyme facilitating method distribution. Anal Bioanal Chem 411, 4701–4708 (2019). https://doi.org/10.1007/s00216-018-1377-z

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  • DOI: https://doi.org/10.1007/s00216-018-1377-z

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