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Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology
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Rapid detection of colistin resistance protein MCR-1 by LC-MS/MS

Honghui Wang1, Yong Chen2, Jeffrey R Strich1

  • 11Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD USA.

Clinical Proteomics
|March 21, 2019
PubMed
Abstract

Insights

Rapidly detect the MCR-1 protein, a marker for colistin resistance, using liquid chromatography tandem mass spectrometry (LC-MS/MS). This assay achieves 100% sensitivity and specificity in under 90 minutes, aiding in the fight against multi-drug resistant Gram-negative organisms.

Area of Science:

  • Microbiology and Infectious Diseases
  • Analytical Chemistry
  • Proteomics

Background:

  • Colistin and polymyxin B are crucial antibiotics for treating severe infections caused by multidrug-resistant Gram-negative bacteria.
  • The emergence of plasmid-mediated colistin resistance via the MCR-1 gene presents a significant global health challenge.
  • Rapid identification of MCR-1 protein in clinical isolates is essential for effective treatment strategies.

Purpose of the Study:

  • To develop a rapid and accurate method for detecting the MCR-1 protein in bacterial isolates.
  • To identify unique MCR-1 peptides suitable for detection by liquid chromatography tandem mass spectrometry (LC-MS/MS).
  • To establish the feasibility of using LC-MS/MS for timely MCR-1 protein identification in clinical settings.

Main Methods:

  • A genoproteomic approach was employed to identify unique MCR-1 protein peptides.
  • Tryptic peptides of MCR-1 were characterized for ionization efficiency and detectability using triple quadrupole LC-MS.
  • A rapid multiple reaction monitoring LC-MS/MS assay was developed using three selected optimal peptides, validated on a 99-sample blinded test set.

Main Results:

  • The developed LC-MS/MS assay demonstrated 100% sensitivity and specificity in identifying MCR-1-containing isolates.
  • No false positive detections were observed across all tests conducted by three independent operators.
  • The assay provides results in under 90 minutes, with DTFPQLAK identified as the best-performing peptide.

Conclusions:

  • This study successfully demonstrates the feasibility of rapid MCR-1 protein detection in bacterial isolates using LC-MS/MS.
  • The developed assay offers a highly sensitive and specific tool for identifying colistin resistance mediated by MCR-1.
  • This rapid detection method can significantly aid in clinical decision-making for infections involving multidrug-resistant Gram-negative bacteria.

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