Confident Phosphorylation Site Localization Using the Mascot Delta Score

Abstract

Large scale phosphorylation analysis is more and more getting into focus of proteomic research. Although it is now possible to identify thousands of phosphorylated peptides in a biological system, confident site localization remains challenging. Here we validate the Mascot Delta Score (MD-score) as a simple method that achieves similar sensitivity and specificity for phosphosite localization as the published Ascore, which is mainly used in conjunction with Sequest. The MD-score was evaluated using liquid chromatography-tandem MS data of 180 individually synthesized phosphopeptides with precisely known phosphorylation sites. We tested the MD-score for a wide range of commonly available fragmentation methods and found it to be applicable throughout with high statistical significance. However, the different fragmentation techniques differ strongly in their ability to localize phosphorylation sites. At 1% false localization rate, the highest number of correctly assigned phosphopeptides was achieved by higher energy collision induced dissociation in combination with an Orbitrap mass analyzer followed very closely by low resolution ion trap spectra obtained after electron transfer dissociation. Both these methods are significantly better than low resolution spectra acquired after collision induced dissociation and multi stage activation. Score thresholds determined from simple calibration functions for each fragmentation method were stable over replicate analyses of the phosphopeptide set. The MD-score outperforms the Ascore for tyrosine phosphorylated peptides and we further show that the ability to call sites correctly increases with increasing distance of two candidate sites within a peptide sequence. The MD-score does not require complex computational steps which makes it attractive in terms of practical utility. We provide all mass spectra and the synthetic peptides to the community so that the development of present and future localization software can be benchmarked and any laboratory can determine MD-scores and localization probabilities for their individual analytical set up.

Signal Transduction in Cerebral Arteries after Subarachnoid Hemorrhage—A Phosphoproteomic Approach

“…Phosphorylation site localization was aided by the use of a mascot delta score48 (ref. 13) combined with manual validation. Peptide ratios were normalized by the median of the unmodified peptides.…”

Modulation of Protein Phosphorylation, N-Glycosylation and Lys-Acetylation in Grape (Vitis vinifera) Mesocarp and Exocarp Owing to Lobesia botrana Infection

“…The MS and MS/MS data were searched with a peptide ion mass tolerance of 10 ppm and a fragment ion mass tolerance of Ϯ 0.6 Da (Ϯ 0.05 Da for high-resolution HCD spectra). Only high-confidence peptides with Mascot ion score Ն 23, phosphopeptides with MDscore Ն 9 (CID-MSA activation) or MD-score Ն 13 (HCD activation) (31), and N-glycopeptides with deamidation sites within the PNGase consensus sequence (N-X-S/T/C, X P) were considered for further analysis. Lys-acetylated peptides were confirmed by manual inspection of the MS/MS spectra based on the presence of the immonium ion of the acetylated lysine (m/z 143.1179) and/or its derivative with an ammonia loss (m/z 126.0913).…”

Identification of Glycopeptides with Multiple Hydroxylysine O-Glycosylation Sites by Tandem Mass Spectrometry

“…Many methods have been proposed for the localization of PTMs, such as A-score, 50 PTM score, 51 PhosphoRS 52 and Mascot Delta Score. 53 These methods compute a PTM localization score based on the similarity scores between an MS/MS spectrum and a peptide with different PTM sites. Similarly, the proposed algorithm can be modified to report the similarity scores between an MS/MS spectrum and a glycopeptide with different glycosylation sites, which can be utilized to compute localization scores of glycosylation sites.…”