Analysis of the Acidic
Proteome with Negative Electron-Transfer
Dissociation Mass Spectrometry
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Abstract
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 <i>Saccharomyces cerevisiae</i> representing the most comprehensive
analysis of peptide anions by tandem mass spectrometry to date. The
same <i>S. cerevisiae</i> 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