Skip to main content
ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #329693
Research Project: Molecular Identification and Characterization of Bacterial and Viral Pathogens Associated with Foods

Location: Produce Safety and Microbiology Research

Title: YahO protein as a calibrant for top-down proteomic identification of Shiga toxin using MALDI-TOF-TOF-MS/MS and post-source decay

Author
item Fagerquist, Clifton - Keith
item Zaragoza, William

Submitted to: Proceedings of the ASMS Conference on Mass Spectrometry and Allied Topics
Publication Type: Proceedings
Publication Acceptance Date: 6/5/2016
Publication Date: 7/29/2016
Citation: Fagerquist, C.K., Zaragoza, W.J. 2016. YahO protein as a calibrant for top-down proteomic identification of Shiga toxin using MALDI-TOF-TOF-MS/MS and post-source decay. Proceedings of the ASMS Conference on Mass Spectrometry and Allied Topics. 280941 MP 719.

Interpretive Summary:

Technical Abstract: Matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF-TOF) mass spectrometry is increasingly utilized for rapid top-down proteomic identification of proteins. This identification may involve analysis of either a pure protein or a protein mixture. For analysis of a pure protein, in-source decay (ISD) is often used, however ISD is not feasible for a protein mixture because of the difficulty in attributing fragment ions to a specific precursor ion. For analysis of a protein mixture, e.g. bacterial cell lysate, it is necessary to isolate a protein ion prior to its fragmentation by either collision-induced dissociation (CID) or post-source decay (PSD) such that fragment ions can be attributed to a specific precursor ion. We have developed and tested the use of a new calibrant for top-down proteomic identification using MALDI-TOF-TOF-MS/MS-PSD. The calibrant is the YahO protein from Escherichia coli O157:H7 strain EDL933. The yahO gene of Escherichia coli O157:H7 strain EDL933 was cloned into a pBAD18 plasmid vector and transferred to a yahO gene knockout of Escherichia coli strain K-12. YahO was strongly expressed by overnight culturing of the K-12 mutant strain on Luria-Bertani agar (LBA) supplemented with arabinose. Bacterial cells were harvested, bead-beat, centrifuged and the supernatant spotted onto the target of a MALDI-TOF-TOF mass spectrometer (4800, SCIEX). MS and MS/MS-PSD was performed. For MS/MS-PSD, the instrument was calibrated using at least six of twelve of the most intense b and y fragment ions of the mature YahO protein sequence (Mr = 7707.6 Da). Prominent fragment ions are the result of polypeptide backbone fragmentation on the C-terminal side of seven aspartic acid residues. Instrument calibration was tested on the disulfide bond-reduced B-subunit of Shiga toxin 2a (Stx2a) from Escherichia coli O157:H7 strain EDL933 that was cultured overnight on LBA supplemented with ciprofloxacin. Two non-simultaneous data acquisitions of calibrant and analyte were tested: 1. YahO and Stx2a B-subunit on separate (but adjacent) spot locations; 2. YahO and the B-subunit co-located on the same spot. MS/MS-PSD data was acquired for YahO, the instrument was then re-calibrated and then MS/MS-PSD data was acquired for the B-subunit. The timed-ion selector (TIS) allowed isolation of the precursor ion for each MS/MS experiment. We observed no significant difference in mass accuracy for co-location vs. adjacent spot analysis in spite of a significant decrease in both YahO and the B-subunit ion intensities for co-location spot analysis. This represents the first reported use of the YahO as a calibrant for mass spectrometry.