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 #332773
Research Project: Molecular Identification and Characterization of Bacterial and Viral Pathogens Associated with Foods

Location: Produce Safety and Microbiology Research

Title: Complementary b/y fragment ion pairs from post-source decay of metastable YahO for calibration of MALDI-TOF-TOF-MS/MS

Author
item Fagerquist, Clifton - Keith
item Zaragoza, William

Submitted to: International Journal of Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/31/2017
Publication Date: 2/13/2017
Citation: Fagerquist, C.K., Zaragoza, W.J. 2017. Complementary b/y fragment ion pairs from post-source decay of metastable YahO for calibration of MALDI-TOF-TOF-MS/MS. International Journal of Mass Spectrometry. 415:29-37.

Interpretive Summary: Mass calibrants are critical to obtaining the highest mass accuracy obtainable for mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments. A new protein mass calibrant was developed for MS/MS top-down proteomic identification of protein biomarkers and toxins from unfractionated bacterial cell lysates using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) mass spectrometry. The calibrant is the YahO protein from Escherichia coli O157:H7 strain EDL933 whose gene (yahO) was cloned into Escherichia coli strain K-12 and over-expressed. The YahO calibrant was evaluated for MS/MS analysis of the B-subunit of Shiga toxin 2 from Escherichia coli O157:H7 strain EDL933 and found to be an effective calibrant for analysis for either co-location or separate (but adjacent) spot locations of analyte and calibrant. In addition, the fragmentation efficiency of metastable YahO suggests that it would be a useful and effective calibrant for other MS/MS platforms

Technical Abstract: Complementary b/y fragment ion pairs from post-source decay (PSD) of metastable YahO protein ion were evaluated for use in the calibration of MALDI-TOF-TOF for tandem mass spectrometry (MS/MS). The yahO gene from pathogenic Escherichia coli O157:H7 strain EDL933 was cloned into a pBAD18 plasmid vector and transferred to a yahO gene knockout (delta yahO) of non-pathogenic Escherichia coli strain K-12. YahO protein was over-expressed by overnight culturing of the K-12 mutant on Luria-Bertani agar (LBA) supplemented with arabinose. MS/MS-PSD of the YahO protein ion results in polypeptide backbone cleavage on the C-terminal side of seven aspartic acid (D) residues of the mature protein sequence resulting in a series of prominent b/y fragment ion pairs. Interestingly, we observe an unusual oscillatory pattern in the fragmentation efficiency of cleavage sites across the linear polypeptide chain of YahO. The instrument was calibrated for MS/MS-PSD using at least six of twelve of the most intense b- and y-type fragment ions of YahO. Calibration was tested on the disulfide bond-reduced B-subunit of Shiga toxin 2 from Escherichia coli O157:H7 strain EDL933 cultured overnight on LBA supplemented with ciprofloxacin. Two non-simultaneous calibrant/analyte acquisition scenarios were evaluated: 1. YahO and B-subunit on separate (but adjacent) spot locations; 2. YahO and the B-subunit co-located on the same spot. We observed no significant difference in mass accuracy for co-location vs. adjacent spot analysis in spite of a significant drop in ion intensity of both calibrant and analyte for co-location analysis.