A Novel Peptidomic Approach to Strain Typing of Clinical Acinetobacter baumannii Isolates Using Mass Spectrometry

Abstract

Background Acinetobacter baumannii is a common nosocomial pathogen and strain-typing methods play important roles in hospital outbreak investigations and epidemiologic surveillance. We describe a method for identifying strain-specific peptide markers based on LC-MS/MS profiling of digested peptides. This method classified a test set of A. baumannii isolates collected from a hospital outbreak with discriminatory performance exceeding that of MALDI-TOF-MS. Methods Following the construction of a species “pan-peptidome” by in silico translation and digestion of whole genome sequences, a hypothetical set of genome-specific peptides for an isolate was constructed from the disjoint set of the pan-peptidome and the isolate’s calculated peptidome. The genome-specific peptidome guided selection of highly expressed genome-specific peptides from LC-MS/MS experimental profiles as potential peptide markers. The species specificity of each experimentally identified genome-specific peptide was confirmed through a Unipept lowest common ancestor analysis. Results Fifteen A. baumannii isolates were analyzed to derive a set of genome- and species-specific peptides that could be used as peptide markers. Identified peptides were cross-checked with protein BLAST against a set of 22 A. baumannii whole genome sequences. A subset of these peptide markers was confirmed to be present in the actual peptide profiles generated by multiple reaction monitoring and targeted LC-MS/MS. The experimentally identified peptides separated these isolates into six strains that agreed with multilocus sequence typing analysis performed on the same isolates. Conclusions This approach may be generalizable to other bacterial species, and the peptides may be useful for rapid MS strain tracking of isolates with broad application to infectious disease diagnosis.

Identification of the OXA-48 Carbapenemase Family by Use of Tryptic Peptides and Liquid Chromatography-Tandem Mass Spectrometry

“…The application of genoproteomics to identify unique marker peptides has been recently advanced as a novel means to identify bacterial strains, species, and resistance elements (19,(34)(35)(36). We describe the development and accuracy assessment of an LC-MS/MS assay for detecting OXA-48 family carbapenemases in bacterial isolates based on the detection of tryptic peptides.…”

“…Second, LC-MS/MS allows direct detection of proteins, verifying expression, as opposed to PCR, which confirms the presence of a gene but not protein expression. Third, MRM allows for the possibility of high-level multiplexing in which a large number of diagnostic peptides may be combined together into a single diagnostic assay including species-level identification (35,36).…”

Deciphering Multifactorial Resistance Phenotypes in Acinetobacter baumannii by Genomics and Targeted Label-free Proteomics

“…Quantitative mass spectrometry (MS) appears particularly attractive to achieve this goal (14). MS technologies are increasingly used to characterize A. baumannii at the protein level using both untargeted (4, [15][16] or targeted approaches (17). Untargeted MS has been extensively used in proteomics study for a long time since it allows stochastic identification of hundreds to thousands of proteins with a coarse quantitation.…”

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

“…Mass analysis was carried out in data-dependent analysis mode, where MS1 scans at 60,000 mass resolution were carried out with the full MS range from m/z 375 to 1,500 and 10 higher-energy collisional dissociation (HCD) MS2 scans at 30,000 resolution were sequentially carried out using an Orbitrap system. LC-MS/MS data were searched against a custom FASTA database composed of Escherichia coli protein sequences (4,212 sequences downloaded from https://www.uniprot.org/ in July 2016) and 15 sequences of NDM variants by the use of Proteome Discoverer 1.4 (Thermo Fisher Scientific) and Scaffold 4 (Proteome Software Inc., Portland, OR) as previously described (19,21). Additional total proteomic analysis for repeat extractions from new subcultures of samples L092 and L099 was performed as detailed in the supplemental material.…”