title: Neutron-encoded signatures enable product ion annotation from tandem mass spectra.
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.
authors.affiliation: Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706;
authors.forename: Alicia L
authors.initials: AL
authors.lastname: Richards
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Vincent
authors.forename: Adrian
authors.initials: A
authors.lastname: Guthals
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Rose
authors.forename: Michael S
authors.initials: MS
authors.lastname: Westphall
authors.forename: Nuno
authors.initials: N
authors.lastname: Bandeira
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D015870:Gene Expression; D007477:Ions; D007553:Isotope Labeling / Q000379:methods*; D008239:Lysine / Q000378:metabolism; D058977:Molecular Sequence Annotation / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009502:Neutrons; D020543:Proteome / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D007477:Ions; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D008239:Lysine
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