Abstract
We report the use of neutron-encoded (NeuCode) stable isotope labeling of amino acids in cell culture for the purpose of C-terminal product ion annotation. Two NeuCode labeling isotopologues of lysine, (13)C6(15)N2 and (2)H8, which differ by 36 mDa, were metabolically embedded in a sample proteome, and the resultant labeled proteins were combined, digested, and analyzed via liquid chromatography and mass spectrometry. With MS/MS scan resolving powers of ~50,000 or higher, product ions containing the C terminus (i.e. lysine) appear as a doublet spaced by exactly 36 mDa, whereas N-terminal fragments exist as a single m/z peak. Through 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%) based on 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 relative to searching through MS/MS only. We use this tool to simplify spectra prior to database searching, to sort unmatched tandem mass spectra for spectral richness, for correlation of co-fragmented ions to their parent precursor, and for de novo sequence identification.
Highlights
The ability to make de novo sequence identifications directly from tandem mass spectra has long been a holy grail of the proteomic community
Metabolic incorporation of amino acids is an efficient method of introducing distinctive labels that eliminates in vitro labeling, but this method requires that the sample be amenable to cell culture [19, 20]
Under high resolving powers, this doublet is resolved. We first developed this NeuCode concept in the context of metabolic labeling, akin to stable isotope labeling with amino acids in cell culture (SILAC), except that instead of the precursor partners being separated by 4 to 8 Da, they are separated by only 6 to 40 mDa
Summary
The ability to make de novo sequence identifications directly from tandem mass spectra has long been a holy grail of the proteomic community. A set of identified spectra from 13,832 unique peptides (Ͼ7,400 per precursor charge 2–3) was produced to train PepNovoϩ so it could learn features such as the relative peak height ranks of b/y-ions and the probability of noise at each mass interval. These spectra contained 15,503 y-type product ion peaks as detected by the search algorithm.
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