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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Biology of Human Pathogens Associated with Food

Location: Produce Safety and Microbiology Research

Title: A new calibrant for MALDI-TOF-TOF-PSD-MS/MS of non-digested proteins for top-down proteomic analysis

Authors
item Fagerquist, Clifton
item Sultan, Omar

Submitted to: Rapid Communications in Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 6, 2012
Publication Date: April 13, 2012
Repository URL: http://onlinelibrary.wiley.com/doi/10.1002/rcm.6220/abstract;jsessionid=74F6236AD2008DFFF45E68698F702FEE.d01t04
Citation: Fagerquist, C.K., Sultan, O. 2012. A new calibrant for MALDI-TOF-TOF-PSD-MS/MS of non-digested proteins for top-down proteomic analysis. Rapid Communications in Mass Spectrometry. 1241:1248.

Interpretive Summary: Accurate mass measurement is critical to obtaining the highest molecular identification possible using mass spectrometric (MS) instrumentation. Analysis of an unknown compound is always preceded by instrument calibration using known compounds, i.e. calibrants. With advances in MS instrumentation and their applications, it is important to use and/or develop calibrants that take full advantage of instrument mass accuracy. We have developed a new calibrant, disulfide-reduced/alkylated thioredoxin (AlkTrx), for post-source decay (PSD) tandem mass spectrometric analysis (MS/MS) of un-digested proteins from bacterial cell lysates analyzed by matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) mass spectrometry. Fragment ion mass accuracy was significantly improved using the AlkTrx calibrant compared to use of a lower molecular weight peptide calibrant. The increased mass accuracy with this new calibrant resulted in a significant improvement in top-down proteomic identification.

Technical Abstract: RATIONALE: Matrix-assisted laser desorption/ionization (MALDI) time-of-flight-time-of-flight (TOF-TOF) tandem mass spectrometry (MS/MS) has seen increasing use for post-source decay (PSD)-MS/MS analysis of non-digested protein ions for top-down proteomic identification. However, there is no commonly accepted calibrant for this purpose beyond the use of peptide calibrants whose fragment ions span a lower mass-to-charge (m/z) range. METHODS: We have used the PSD-generated fragment ions of disulfide-reduced/alkylated thioredoxin (AlkTrx) for TOF-TOF calibration in reflectron mode for the purpose of PSD-MS/MS analysis. The average m/z of AlkTrx fragment ions were used for calibration. The quality of the calibration was assessed from the observed fragment ion mass error of MS/MS of the YahO protein from an un-fractionated bacterial cell lysate of Escherichia coli O157:H7 as well as from MS/MS of bovine ubiquitin. The fragment ion mass error of these two analytes were also used to assess instrument calibration using the monoisotopic fragment ions of [Glu1]-fibrinopeptide B (GluFib). RESULTS: A general improvement in fragment ion mass accuracy was observed using the AlkTrx calibration compared to the GluFib calibration which resulted in a more significant top-down proteomic identification of these analyte proteins. CONCLUSIONS: Our results suggest that AlkTrx may be useful as a calibrant for MALDI-TOF-TOF-PSD-MS/MS of small and modest-sized protein ions. The uniform fragmentation efficiency of YahO across its sequence suggests that it may be useful as a post-calibration standard to assess PSD-MS/MS instrument performance as well as establishing appropriate top-down proteomic fragment ion tolerances.

Last Modified: 10/30/2014
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