Post-translational modification (PTM) regulates protein functions and influence almost all aspects of normal cell biology and pathogenesis. Therefore, identifying and understanding PTMs is crucial in the study of cell biology and disease treatment and prevention. Recently, we have developed a few proteomics tools to facilitate the discovery of the new function of PTMs. A highly robust N-glycoproteomics (HRN) platform comprising an automated enrichment method, a stable microflow LC-MS/MS system, and a sensitive glycopeptide-spectra-deciphering software tool (i.e. Glyco-Decipher) is developed for large-scale quantitative N-glycoproteome analysis, which enabled the identification of over 20,000 unique site-specific glycans and discovered one with good performance in diagnosing early-stage gastric cancer. A methyl-specific metabolic labeling approach was developed for the identification of methylations on 8 types of residues. Interestingly, of the identified methylation events, His methylation is found to be preferably occurred in C3H1 zinc fingers (ZFs). The His methylation is found to stabilize the structure of ZFs. U2AF1 is used as a proof-of-concept to highlight the functional importance of His methylation in ZFs in RNA binding and RNA metabolism. A special limited proteolysis approach, "peptide-centric local stability assay"(PELSA), was developed to enable proteome-wide target identification and binding-region determination of diverse ligands. Beyond offering users unprecedented sensitivity for characterizing diverse target-ligand interactions, it can also be applied to identify the reader proteins of PTMs. This was demonstrated by identifying the target proteins of a synthetic phosphotyrosine peptide pYEEI via using PELSA. It was founs that 28 proteins were significantly stabilized, among which 9 proteins contain SH2 domains, the reading domains for tyrosine phosphorylation. Our results demonstrated that PELSA can be readily applied to investigate the recognition domains of PTMs, which is crucial for understanding the biological functions of the PTMs.