Identification of LifA Protein Fragments

Excised SDS-PAGE gel-bands of rLifA^C1480A were incubated in 50 mM ammonium bicarbonate containing porcine trypsin (Promega) in a trypsin:lymphostatin ratio of ~1:30, overnight at 32 °C. Peptides were identified by MALDI mass spectroscopy on an ultrafleXtreme™ mass spectrometer (Bruker) using an α-cyano-4-hydroxycinnamic acid matrix. A peptide mass map was generated from spectral data using Compass DataAnalysis 4.4 software (Bruker). Peptide masses were searched against the National Center for Biotechnology Information database of non-identical protein sequences (NCBInr) with the MASCOT search engine,⁵⁰ (link) mass tolerance of 10 ppm (Matrix Science). Peptide masses were also compared to the sequence of full-length LifA from EPEC E2348/69 (Protein Prospector software).12 (link), 13 (link), 51 The instrument was calibrated prior to each data acquisition and an internal calibration performed on the digest products of porcine trypsin.

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Bease A.G., Blackburn E.A., Chintoan-Uta C., Webb S., Cassady-Cain R.L, & Stevens M.P. (2021). Activity of Lymphostatin, A Lymphocyte Inhibitory Virulence Factor of Pathogenic Escherichia coli, is Dependent on a Cysteine Protease Motif. Journal of Molecular Biology, 433(19), 167200.

Publication 2021

porcine trypsin ultrafleXtreme™ mass spectrometer Compass DataAnalysis 4.4 software

Ammonium bicarbonate Epec Hydroxycinnamic acid Maldi Mass spectroscopy Peptide Porcine Promega Protein Sds page Tolerance Trypsin

Corresponding Organization : University of Edinburgh

Other organizations : Roslin Institute

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Variable analysis

independent variables

Incubation of excised SDS-PAGE gel-bands of rLifA^C1480A in 50 mM ammonium bicarbonate containing porcine trypsin

dependent variables

Peptides identified by MALDI mass spectroscopy

control variables

Trypsin:lymphostatin ratio of ~1:30
Overnight incubation at 32 °C
Use of α-cyano-4-hydroxycinnamic acid matrix for MALDI
Peptide mass tolerance of 10 ppm for database search
Calibration of the instrument prior to each data acquisition
Internal calibration performed on the digest products of porcine trypsin

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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