rPTMDetermine: A Fully Automated Methodology for Endogenous Tyrosine Nitration Validation, Site-Localization, and Beyond

Abstract

We present herein rPTMDetermine, an adaptive and fully automated methodology for validation of the identification of rarely occurring post-translational modifications (PTMs), using a semisupervised approach with a linear discriminant analysis (LDA) algorithm. With this strategy, verification is enhanced through similarity scoring of tandem mass spectrometry (MS/MS) comparisons between modified peptides and their unmodified analogues. We applied rPTMDetermine to (1) perform fully automated validation steps for modified peptides identified from an in silico database and (2) retrieve potential yet-to-be-identified modified peptides from raw data (that had been missed through conventional database searches). In part (1), 99 of 125 3-nitrotyrosyl-containing (nitrated) peptides obtained from a ProteinPilot search were validated and localized. Twenty nitrated peptides were falsely assigned because of incorrect monoisotopic peak assignments, leading to erroneous identification of deamidation and nitration. Five additional nitrated peptides were, however, validated after performing nonmonoisotopic peak correction. In part (2), an additional 236 unique nitrated peptides were retrieved and localized, containing 113 previously unreported nitration sites; 25 endogenous nitrated peptides with novel sites were selected and verified by comparison with synthetic analogues. In summary, we identified and confidently validated 296 unique nitrated peptidescollectively representing the largest number of endogenously identified 3-nitrotyrosyl-containing peptides from the cerebral cortex proteome of a Macaca fascicularis model of stroke. Furthermore, we harnessed the rPTMDetermine strategy to complement conventional database searching and enhance the confidence of assigning rarely occurring PTMs, while recovering many missed peptides. In a final demonstration, we successfully extended the application of rPTMDetermine to peptides featuring tryptophan oxidation.

Protein Tyrosine Nitration in Plant Nitric Oxide Signaling

“…However, there is abundant evidence pointing to the need of having extreme caution when making assignments based on MS/MS spectra alone, especially when MS/MS resolution or mass accuracy are not good enough. It has been reported an automated method for the validation of endogenous Tyr nitration called rPTMDetermine that enhances the verification through similarity scoring of tandem MS/MS comparisons between modified peptides and their unmodified analogs ( Dong et al, 2020 ). On the other hand, the low levels of nitrated proteins detected in vivo has been tried to overcome by exogenous treatment with nitrating agents that largely increase the number and quantity of detected nitrated proteins ( Table 1 ).…”

Unveiling the human nitroproteome: Protein tyrosine nitration in cell signaling and cancer

“… 2018 ( 149 ) NTyroSite https://biocomputer.bio.cuhk.edu.hk/NTyroSite/ . 2018 ( 185 ) rPTMDetermine https://pubs.acs.org/doi/abs/10.1021/acs.analchem.0c02148 2020 ( 186 ) PredNTS http://kurata14.bio.kyutech.ac.jp/PredNTS 2021 ( 187 ) PreNitro http://103.99.176.239/PredNitro 2022 ( 188 ) …”