on 10-04-201310:28 AM - edited on 10-15-202111:37 AM by AnalyteGuru
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.