Top-Down Proteomics for Rapid Identification of Intact Microorganisms

Abstract

We apply MALDI-TOF/TOF mass spectrometry for the rapid and high-confidence identification of intact Bacillus spore species. In this method, fragment ion spectra of whole (undigested) protein biomarkers are obtained without the need for biomarker prefractionation, digestion, separation, and cleanup. Laser-induced dissociation (unimolecular decay) of higher mass (>5 kDa) precursor ions in the first TOF analyzer is followed by reacceleration and subsequent high-resolution mass analysis of the resulting sequence-specific fragments in a reflectron TOF analyzer. In-house-developed software compares an experimental MS/MS spectrum with in silico-generated tandem mass spectra from all protein sequences, contained in a proteome database, with masses within a preset range around the precursor ion mass. A p-value, the probability that the observed matches between experimental and in silico-generated fragments occur by chance, is computed and used to rank the database proteins to identify the most plausible precursor protein. By inference, the source microorganism is then identified on the basis of the identification of individual, unique protein biomarker(s). As an example, intact Bacillus atrophaeus and Bacillus cereus spores, either pure or in mixtures, were unambiguously identified by this method after fragmenting and identifying individual small, acid-soluble spore proteins that are specific for each species. Factors such as experimental mass accuracy and number of detected fragment ions, precursor ion charge state, and sequence-specific fragmentation have been evaluated with the objective of extending the approach to other microorganisms. MALDI-TOF/TOF-MS in a lab setting is an efficient tool for in situ confirmation/verification of initial microorganism identification.

Enhanced In-Source Fragmentation in MALDI-TOF-MS of Oligonucleotides Using 1,5-Diaminonapthalene

“…A standard peptide mixture was used for external mass/charge calibration in both polarities. For comparison with ISD fragments, negative ion LID TOF/ TOF spectra (i.e., LIFT mode [25]) of selected oligonucleotides were also acquired using standard instrument parameters [26]. Typically, from 250 to 1000 individual laser shots were averaged for each MS or MS/MS spectrum.…”

Shiga Toxin 2 Subtypes of Enterohemorrhagic E. coli O157:H- E32511 Analyzed by RT-qPCR and Top-Down Proteomics Using MALDI-TOF-TOF-MS

“…Lesser abundant fragments ions are the result of fragmentation on the C-terminal side of E residues (y 43 , y 55 , y 61 , b 57 , b 64 ) and occasionally lysine residues (y 65 ). The USDA top-down analysis software confirmed the top identification as the B-subunit of Stx2a (P-value=3.4×10 -13 and USDA score=54.17 [24,38]). Figure 4b is the MS/MS-PSD spectrum of the peak at m/z 7774 shown in Figure 3.…”

Top-down proteomics: applications, recent developments and perspectives

“…More recently, a top-down MS method for the rapid and high-confidence identification of intact Bacillus spore species was developed. In this method, fragment ion spectra of undigested [whole] protein biomarkers are obtained without the need for biomarker pre-fractionation, digestion, separation, and cleanup [159]. Several proteins from E. coli, including an over expressed one directly from a cell lysate, using their charge stripping approach on an ESI/ ion trap instrument were identified by McLuckey and co-workers [160].…”