Modification site localization scoring integrated into a search engine

Abstract

Large proteomic data sets identifying hundreds or thousands of modified peptides are becoming increasingly common in the literature. Several methods for assessing the reliability of peptide identifications both at the individual peptide or data set level have become established. However, tools for measuring the confidence of modification site assignments are sparse and are not often employed. A few tools for estimating phosphorylation site assignment reliabilities have been developed, but these are not integral to a search engine, so require a particular search engine output for a second step of processing. They may also require use of a particular fragmentation method and are mostly only applicable for phosphorylation analysis, rather than post-translational modifications analysis in general. In this study, we present the performance of site assignment scoring that is directly integrated into the search engine Protein Prospector, which allows site assignment reliability to be automatically reported for all modifications present in an identified peptide. It clearly indicates when a site assignment is ambiguous (and if so, between which residues), and reports an assignment score that can be translated into a reliability measure for individual site assignments.

Fig. 1.

Distribution of correct and incorrect SLIP scores for the QTOF Micro data set. SLIP scores for analysis of the same data set of quadrupole CID data are plotted for Ascore (A); Mascot Delta Score (B), and PP Site Score (C). Correct site assignments are in blue; incorrect are plotted in red. Panel D plots a histogram of the local FLR for a given SLIP score. Panels A and B are reproduced from Savitski et al. (10).

Fig. 2.

FLR as a function of SLIP score for an ion trap CID data set. Histograms of FLR for a given SLIP score are plotted for the searches considering phosphoglutamate and phosphoproline modification.

Fig. 3.

Comparison of three different site assignment combinations for a doubly O-GlcNAc modified peptide from Ankyrin G. Assignments in common among the different assignments are in black, those that differentiate between different assignments are labeled in the color of the peptide.

Fig. 4.

Visual comparison of different modification site assignments. Protein Prospector allows superimposition of multiple interpretations onto the same spectrum. In this example, the reported modification site assignment result is compared with the correct identification. All peaks labeled in red are explained by both peptide interpretations; only the single green y8 2+ ion differentiates between the two interpretations.