earticle

영어

O-GlcNAcylation is one of the post-translational modification that occurs at Ser/Thr residues of a protein by O-GlcNAc transferase (OGT), but the O-GlcNAcylation is regulated by O-GlcNAcase (OGA). The O-GlcNAcylation is involved in various cellular processes and diseases including diabetes, cancer and Alzheimer’s disease. Herein, we suggest a new method for identifying O-GlcNAcylated proteins including classification of O-GlcNAc and O-GalNAc with the same molecular weight. Using the fractionated raw files of LC-MS/MS from T24 cell lines, we developed an identification algorithm coded by python 3.9 as follows: 1.Selection of spectra with oxonium ions in ms2 file extracted from raw MS data obtained using LC-MS/MS; 2.Spectral matching with 20 ppm against a tryptic and O-GlcNAcylated peptide database generated from proteins identified using IP2 proteomic search result; 3.In the candidates, matching between tandem MS with b/y and c/z ion list and experimental HCD, ETD spectra, respectively; 4.Selection of the best scored O-GlcNAcylated peptide from the candidated peptides calculated by scoring system; 5.Classification of O-GlcNAc and O-GalNAc based on their corresponding fragment ions; 6.Evaluating a model using ROC curve comparing search results with manual validation. In addition, we also performed relative quantitative analysis in conjunction with the TMT Quantification method. In the glycoscience field, our study could be applied to various human disease treatments by identifying O-GlcNAcylated proteins and by engaging in cell signaling after regulating the function of them.