Modification site localization scoring: strategies and performance

Abstract

Using enrichment strategies many research groups are routinely producing large data sets of post-translationally modified peptides for proteomic analysis using tandem mass spectrometry. Although search engines are relatively effective at identifying these peptides with a defined measure of reliability, their localization of site/s of modification is often arbitrary and unreliable. The field continues to be in need of a widely accepted metric for false localization rate that accurately describes the certainty of site localization in published data sets and allows for consistent measurement of differences in performance of emerging scoring algorithms. In this article are discussed the main strategies currently used by software for modification site localization and ways of assessing the performance of these different tools. Methods for representing ambiguity are reviewed and a discussion of how the approaches transfer to different data types and modifications is presented.

Fig. 1.

In an MS/MS spectrum modification site localization is enabled by the presence of one or more ions of unambiguously assignable ion type that are derived from fragmentation between two amino acids in the peptide that can bear the modification. A, The y5 and y6 ions enable the phosphate to be localized to Ser-3 rather than Thr-5. B, The absence of fragmentation between Ser-1 and Ser-2 prevents localization of the phosphate, however the y13++ ion indicates absence of localization to Ser-3. In peptide sequence, red forward slash indicates observation of y ion, blue backslash indicates observation of b ion and magenta vertical bar indicates observation of both b and y ion.

Fig. 2.

Comparison of different peak list generation methods. The peak lists from a spectrum of the phosphopeptide RGT(Phospho)VEGSVQEVQEEK, generated with a 20 + 20 approach and 4 peaks per 100 Th are compared. Peaks indicated in blue and red are b and y ions only included in one peak list or the other. In the 20 + 20 peak list the modified b4 ion localizes the modification site; in the 4 peaks per 100 Th peak list the modified y10 ion (combined with unmodified y7 ion) localizes the modification site.

Fig. 3.

Phosphosite localization agreement for SCX IMAC fraction 4 in the 2010 ABRF-iPRG study on identifying phosphopeptides and localizing phosphorylation sites. A, For 3932 spectra the identification was designated as certain by ≥ 2/22 participants. Of these, 3136 spectra had possible ambiguity in site localization that was designated as certain; 457 spectra had no possibility of site localization ambiguity (#phosphosites equal to #STY residues in peptide); 119 spectra had unanimous agreement on localization being ambiguous; and with 220 spectra only 1 participant designated the site localization as certain. B, For the 3136 spectra with >2/22 participants willing to designate localization as certain they unanimously agree on localization of phosphosites for 79% of spectra. C, In the remaining 21% of cases (649 spectra), the disagreement was not found to be limited to a single or select few participants. D, The participants who were most likely to make localization decisions (rather than declare ambiguity) were more likely to disagree with the consensus view. The x axis of (C) and (D) is sorted in descending order of # localized/# identified.

Fig. 4.

In the 2010 ABRF iPRG study 14/21 participants sensibly designated the phosphate site localization as ambiguous for this peptide spectrum match. Among seven inordinately aggressive participants the localization was split, 4 for Ser-8 (upper) and three for Thr-10 (lower). The spectrum has only a low intensity ion at m/z 1055.4 and an ion at m/z 879 that can be assigned as different fragment ion types depending on the localization of the phosphate to Ser-8 or Thr-10.

Fig. 5.

Visual comparison of modification site localization alternatives. Protein Prospector automatically displays the annotations of all potential site localizations within the employed SLIP score threshold. In this view the discriminating peaks between the different site localizations are indicated. Peaks labeled in red are explained by both interpretations, but the ion localizations are only displayed for the discriminating peaks. In this example, localizations of phosphorylation to serine at residue 12 or threonine 14 both explain 26 out of the 40 peak masses in the spectrum. With y7 ions corresponding to both interpretations present in the spectrum, this is probably a mixture spectrum of the peptide modified on the two different sites.