Figure 1

Approach for peptide quantification using ¹⁵N mass spectra. (a) The method uses accurate mass and charge measurements to search a ¹⁴N-unlabeled database and a ¹⁵N database of intact peptide masses and nitrogen counts obtained by in silico digestion of an organism's proteome. The number of nitrogens in each of the returned peptides is used to examine a limited number of mass differences designated by arrows. On finding an unambiguous ¹⁴N-¹⁵N pair, the method labels each member of the pair as originating from either the unlabeled or the ¹⁵N-labeled sample. (b) During peptide identification, a fragmentation spectrum (1) associated with a member of the pair is searched against an unlabeled database or ¹⁵N-labeled database of peptides based on the assigned label status. Note that only one member of the peptide pair needs to have an associated fragmentation spectrum. The monoisotopic masses of the peptides (2) and their nitrogen composition (3) are used to limit the search space of peptides scored against the spectrum. The intensity of each member of a ¹⁴N-¹⁵N pair is used to derive a peptide ratio. (c) Top, a population of incompletely labeled peptides can generate a complex isotope distribution pattern for ¹⁵N-labeled peptides. The peaks in purple, in order of decreasing m/z, correspond to peptides lacking one and two labeled nitrogens. Bottom, even in highly enriched samples, such peaks can be found in real mass spectra as illustrated for a +2 charge peptide, LGFFETVDTDTQR from E. coli protein yaeT (b0177). (d) For a range of charge states, monoisotopic XICs can be paired with an XIC with an additional ¹³C, but lack any XICs detected at a negative ¹³C shift (top). In contrast, ¹⁵N XICs will lack both XICs at negative and positive ¹³C shifts (bottom).