Development And Application Of Visualization Tools For Protein Hydroxylation Modification

Background Post-translational modifications regulate protein functions and accordingly affect the cell cycle.Hydroxylation of proteins is an oxidative modification of specific amino acids catalyzed by 2-ketoglutarate-dependent dioxygenases.Hydroxylation plays an important role in tumorigenesis and development.Due to the lack of the related pan-antibody which can enrich hydroxylation,the investigation of hydroxylation has been restricted and only hundreds of modified sites are reported.We attempted to find novel hydroxylation sites from the public mass spectrometry big data.However,we face a few difficulties.1)non-enriched hydroxylated peptides are low-abundant and difficult to identify.2)Hydroxylation can occur on a variety of amino acid residues and does not significantly change their physicochemical properties.So coeluted modified peptides often occur.3)the oxidative modifications of a few amino acids caused by ROS(reactive oxygen species)increase the difficulty of hydroxylation identification.In addition,although the LS-MS/MS(Liquid Chromatography with tandem mass spectrometry)technology is the mainstream method for large-scale identification of post-translational modifications,the current search algorithm and modification localization algorithm have limitations for the localization of non-enriched modifications.To better determine the correctness of the modified sites,manual investigation of the spectrums is an important part,and the corresponding non-modified peptide spectrum is a very important reference.The current spectrum generation software has not yet achieved an automatic batch output of modified and unmodified spectrum pairs,which facilitates manual verification.Method To solve this problem,we developed MMS2 plot,an R package for visualizing multiple MS/MS spectra for groups of modified and non-modified peptides.The main idea is to use the corresponding spectra of unmodified peptides as a reference to help judge whether the corresponding spectra of modified peptides are correct.MMS2 plot has a good performance in both the display of spectrum information and the reliability of manual verification of modification sites.It can directly compare fragment ion spectra and retention time between modified and unmodified peptide pairs or groups;It supports the output of different ion types and has built-in parameters to display images of different specifications.MMS2 plot features a batch mode and generates the output images in vector graphics file format that facilitate evaluation and publication of the PSM assignment.In addition,we also developed a function to determine the similarity between modified and unmodified spectra,which can be combined with MMS2 plot to quantify the similarity between modified and unmodified spectra.Results Some spectra corresponding to modified peptides were generated by MMS2 plot.They assist the identification of the spectra with co-eluted modified peptides and the spectra with miscleavaged peptides.The non-miscleavaged modified peptides and the corresponding miscleavaged modified peptides may prove that the modified site has high confidence.After filtering a large number of Raw MS files,we found the hydroxylation sites that have been reported before,whose related spectra were generated by MMS2 plot.Conclusion MMS2 plot is not limited to a specific modification type but can be applied to any kind of modification type.It can facilitate the identification of protein hydroxylation and other post-translational modifications.