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.

A systematic assessment of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) application for rapid identification of pathogenic microbes that affect food crops: delivered and future deliverables

“…67–70 Further, Demarco and Burnham, 71 used diafiltration-assisted MALDI MS analysis to identify uropathogens namely Escherichia coli , Klebsiella pneumoniae , and Proteus mirabilis , after it was desalted, fractionated, and concentrated, the urine samples were used to MALDI MS. Recently, Doncevic et al , 72 had developed a tandem mass based MALDI MS method, and the custom program package for the identification of pathogens using peptide tandem mass library contained with 20 917 bacterial species which helped to identify the organisms without the need for bacterial culturing, from urine samples.…”

Deep learning-driven fragment ion series classification enables highly precise and sensitive de novo peptide sequencing

“…This prevents proteomics from being efficiently used in applications where the peptide sequences are not known a priori. This concerns neoepitope identification 9 , antibody sequencing 10 , pathogen surveillance 11 , microbial community studies 12 , and paleontology 13 . Therefore, efficient de novo peptide sequencing algorithms, which aim to identify peptides directly from spectra without relying on any database, are highly needed.…”

Rapid detection of Candida albicans in urine by an Electrochemical Impedance Spectroscopy (EIS)-based biosensor

“…However, each of these methods has its own set of advantages and limitations, including factors like availability, cost, laboratory expertise, and the clinical context in which the test is being performed. 12,13…”

“…However, each of these methods has its own set of advantages and limitations, including factors like availability, cost, laboratory expertise, and the clinical context in which the test is being performed. 12,13 Biosensors with enhanced sensitivity, selectivity, and easeof-use would be crucial for improving the diagnosis and management of candiduria. In fact, a variety of biosensors have been proposed for the detection of Candida, offering different advantages and performance characteristics.…”