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

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 (Δ 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.