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Reputable Mentor II
Reputable Mentor II
Richards AL, Vincent CE, Guthals A, Rose CM, Westphall MS, Bandeira N, Coon JJ.
Mol Cell Proteomics. 2013 Sep 16.
We report the use of neutron-encoded stable isotope labeling of amino acids in cell culture (NeuCode SILAC) for the purpose of C-terminal product ion annotation. Two NeuCode SILAC isotopologues of lysine, 13C615N2 and 2H8, which differ by 36 mDa are metabolically embedded into a sample proteome and the resultant labeled proteins are combined, digested, and analyzed by liquid chromatography and mass spectrometry. With MS/MS scan resolutions of ~ 50,000 or higher, product ions containing the C-terminus (i.e., lysine) appear as a doublet spaced by exactly 36 mDa while N-terminal fragments exist as a single m/z peak. With theory and experiment we demonstrate that over 90% of all y-type product ions have detectable doublets. We report on an algorithm that can extract these neutron signatures with high sensitivity and specificity. In other words, of 15,503 y-type product ion peaks, the y-type ion identification algorithm correctly identified 14,552 (93.2%) by detection of the NeuCode doublet. 6.8% were misclassified, i.e., other ion types that were assigned as y-type products. Searching NeuCode labeled yeast with PepNovo+ resulted in a 34% increase in correct de novo identifications compared with searching using MS/MS only. We use this tool to simplify spectra prior to database searching, sort unmatched tandem mass spectra for spectral richness, for correlation of co-fragmented ions to their parent precursor, and for de novo sequence identification.
University of Wisconsin-Madison, United States
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