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

Authors: Perez Jr, Johnny; Chen, Chinyi

Submitted to: Analytical and Bioanalytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/13/2018
Publication Date: 10/25/2018
Citation: Perez Jr, J.J., Chen, C. 2018. Implementation of normalized retention time (IRT) for bottom-up proteomic analysis of the aminoglycoside phosphotransferase enzyme facilitating method distribution. Analytical and Bioanalytical Chemistry. 411:4701-4708. https://doi.org/10.1007/s00216-018-1377-z.
DOI: https://doi.org/10.1007/s00216-018-1377-z

Interpretive Summary: Mass spectrometry is in the beginning stages of becoming a truly viable alternative to conventional microbial analysis methods. Analysis of all the proteins synthesized by an organism coined the “proteome” is where mass spectrometry has no equal in terms of speed, cost and interpretable results. Here, we use open-source software packages to predict how an unknown targeted antibiotic resistant enzyme will digest in the presence of trypsin, a common protease, and how each potential newly generated tryptic peptide will ionize in positive ion mode electrospray ionization. Full characterization and confirmation of each tryptic peptide was also performed by fragmenting each peptide followed with observing all fragments using mass spectrometry. Finally, we demonstrated the ability to generate a home-made standard peptide set using myoglobin, a common mass spectral calibrant, to map where each suspected peptide will elute using the peptide set as benchmarks for facile method implementation between highly diverse mass spectrometry technologies and laboratories.

Technical 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, have 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) was evaluated. Employment of the relative retention time concept (iRT) using a home-made peptide standard set revealed facile method transfer between two fundamental different mass spectral platforms: a ultrahigh-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.