We describe the first implementation of negative electron-transfer dissociation (NETD) on a hybrid ion trap-orbitrap mass spectrometer and its application to high-throughput sequencing of peptide anions. NETD, coupled with high pH separations, negative electrospray ionization (ESI), and an NETD compatible version of OMSSA, is part of a complete workflow that includes the formation, interrogation, and sequencing of peptide anions. Together these interlocking pieces facilitated the identification of more than 2000 unique peptides from Saccharomyces cerevisiae representing the most comprehensive analysis of peptide anions by tandem mass spectrometry to date. The same S. cerevisiae samples were interrogated using traditional, positive modes of peptide LC-MS/MS analysis (e.g., acidic LC separations, positive ESI, and collision activated dissociation), and the resulting peptide identifications of the different workflows were compared. Due to a decreased flux of peptide anions and a tendency to produce lowly charged precursors, the NETD-based LC-MS/MS workflow was not as sensitive as the positive mode methods. However, the use of NETD readily permits access to underrepresented acidic portions of the proteome by identifying peptides that tend to have lower pI values. As such, NETD improves sequence coverage, filling out the acidic portions of proteins that are often overlooked by the other methods.

• PMID: 22335612
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Journal Article | Research Support, N.I.H., Extramural | Research Support, N.I.H., Intramural | Research Support, Non-U.S. Gov't

Authors

McAlister GC, Russell JD, Rumachik NG, Hebert AS, Syka JE, Geer LY, Westphall MS, Pagliarini DJ, Coon JJ

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