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

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 MS 2 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. Graphical Abstract