Rapid Identification of New Delhi Metallo-β-Lactamase (NDM) Using Tryptic Peptides and LC-MS/MS

Abstract

There is significant interest in the development of mass spectrometry (MS) methods for antimicrobial resistance protein detection, given the ability of these methods to confirm protein expression. In this work, we studied the performance of a liquid chromatography, tandem MS multiple-reaction monitoring (LC-MS/MS MRM) method for the direct detection of the New Delhi metallo-β-lactamase (NDM) carbapenemase in clinical isolates. Using a genoproteomic approach, we selected three unique peptides (SLGNLGDADTEHYAASAR, AFGAAFPK, and ASMIVMSHSAPDSR) specific to NDM that were efficiently ionized and spectrally well-defined. These three peptides were used to build an assay with turnaround time of 90 min. In a blind set, the assay detected 21/24 bla_NDM-containing isolates and 76/76 isolates with negative results, corresponding to a sensitivity value of 87.5% (95% confidence interval [CI], 67.6% to 97.3%) and a specificity value of 100% (95% CI, 95.3% to 100%). One of the missed identifications was determined by protein fractionation to be due to low (∼0.1 fm/μg) NDM protein expression (below the assay limit of detection). Parallel disk diffusion susceptibility testing demonstrated this isolate to be meropenem susceptible, consistent with low NDM expression. Total proteomic analysis of the other two missed identifications did not detect NDM peptides but detected other proteins expressed from the bla_NDM-containing plasmids, confirming that the plasmids were not lost. The measurement of relative NDM concentrations over the entire isolate test set demonstrated variability spanning 4 orders of magnitude, further confirming the remarkable range that may be seen in levels of NDM expression. This report highlights the sensitivity of LC-MS/MS to variations in NDM protein expression, with implications for how this technology may be used.

Keywords: New Delhi metallo-β-lactamase; mass spectrometry; multiple-reaction monitoring; tryptic peptide.

FIG 1

Workflow diagram of peptide selection for NDM detection by MRM LC-MS. Abbreviations used: ESP, enhanced signature peptide (predictor); DDA, data-dependent acquisition; BLASTp, protein blast; Unipept, Unipept Peptidome Analysis (Web tool).

FIG 2

Representative LC-MS chromatograms of three NDM peptides for two isolates used in 20-sample assay development. S6 is a bla_NDM-containing isolate and S7 was used as a negative control. (A) SLGNLGDADTEHYAASAR. (B) ASMIVMSHSAPDSR. (C) AFGAAFPK. rdotp/R-ratio values are shown for each peptide. For S7, the signals are shown as insertions to show the details.