Direct Identification of Urinary Tract Pathogens by MALDI-TOF/TOF Analysis and De Novo Peptide Sequencing

Abstract

For mass spectrometry-based diagnostics of microorganisms, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently routinely used to identify urinary tract pathogens. However, it requires a lengthy culture step for accurate pathogen identification, and is limited by a relatively small number of available species in peptide spectral libraries (≤3329). Here, we propose a method for pathogen identification that overcomes the above limitations, and utilizes the MALDI-TOF/TOF MS instrument. Tandem mass spectra of the analyzed peptides were obtained by chemically activated fragmentation, which allowed mass spectrometry analysis in negative and positive ion modes. Peptide sequences were elucidated de novo, and aligned with the non-redundant National Center for Biotechnology Information Reference Sequence Database (NCBInr). For data analysis, we developed a custom program package that predicted peptide sequences from the negative and positive MS/MS spectra. The main advantage of this method over a conventional MALDI-TOF MS peptide analysis is identification in less than 24 h without a cultivation step. Compared to the limited identification with peptide spectra libraries, the NCBI database derived from genome sequencing currently contains 20,917 bacterial species, and is constantly expanding. This paper presents an accurate method that is used to identify pathogens grown on agar plates, and those isolated directly from urine samples, with high accuracy.

Keywords: de novo peptide sequencing; peptide identification software; tandem mass spectrometry; uropathogenic infection.

Figure 1

Pipeline for the identification of species by de novo peptide sequencing where (A) positive and negative MS/MS spectra were aligned, transformed in silico to y-ions, and used for the prediction of peptide sequence. (B) Elucidated peptide sequences were scored according to the highest intensities, and searched against NCBInr database by BLASTp.

Figure 2

Obtained results used to identify the Putative stress protein found in K. pneumoniae (accession number STS794191). (A) The mirror image of positive and negative MS/MS spectra, where the positive spectrum (green) represents y-ions prevalently, and the negative spectrum (orange) represents mostly b-ions of the analyzed peptide. (B) The denoted peptide sequence was determined as “MNKDEIGGNWKQFK” by Protein Acrobat, where red lines represent matched amino acids to each ion mass.

Figure 3

Identification from pure bacterial colonies by MALDI-TOF/TOF and de novo peptide sequencing. Results are plotted for E. coli, P. mirabilis, K. pneumoniae, and P. aeruginosa. The Y-axis indicates the probability of correct species identification, and the bars represent the five bacterial species with the highest number of matched peptides for each bacterium separately.