Protein posttranslational modifications (PTMs) play essential roles in the regulation of almost all biological processes, ranging from DNA replication and gene expression to metabolism. The dysregulation of proteins PTMs often leads to human diseases such as cancer and neurodegenerative diseases. Therefore, it is of great importance to reveal the biological significance of protein PTMs and their regulatory mechanisms. Benefiting from the rapid development of mass spectrometry technology, the number of known PTMs has increased rapidly. However, only a handful of protein PTMs have been extensively studied, while the cellular mechanisms and functions of many other PTMs, particularly those newly identified ones, remains poorly understood. In this talk, I will present the new strategies and discoveries on the biological significance and regulation mechanism of several novel protein PTMs.