Mass spectrometry-based detection and assignment of protein posttranslational modifications

Abstract

Recent advances in mass spectrometry (MS)-based proteomics allow the identification and quantitation of thousands of posttranslational modification (PTM) sites in a single experiment. This follows from the development of more effective class enrichment strategies, new high performance instrumentation and bioinformatic algorithms with rigorous scoring strategies. More widespread use of these combined capabilities have led to a vast expansion in our knowledge of the complexity of biological processes mediated by PTMs. The classes most actively pursued include phosphorylation, ubiquitination, O-GlcNAcylation, methylation, and acetylation. Very recently succinylation, SUMOylation, and citrullination have emerged. Among the some 260 000 PTM sites that have been identified in the human proteome thus far, only a few have been assigned to key regulatory and/or other biological roles. Here, we provide an update of MS-based PTM analyses, with a focus on current enrichment strategies coupled with revolutionary advances in high performance MS. Furthermore, we discuss examples of the discovery of recently described biological roles of PTMs and address the challenges of defining site-specific functions.

Figure 1

Overview of PTM analysis at the protein and peptide level. The principal steps for protein level PTM enrichment and MS analysis are represented on the left. Peptide level phosphorylation enrichment strategies are illustrated on the right. Isomeric isobars are proteins or peptides that may reveal the same amino acid sequence with equal numbers of PTMs but with different PTM configurations. The development of algorithms that search for non-redundant ions representing all possible PTM configurations enable the identification, quantification, and localization of all PTMs.

Figure 2

Examples of PTM-mediated biological processes. (A) Arginine methylation-mediated Smad signaling pathway. (B) p53 interaction with the CBP/p300 complex through methylation and acetylation exchange after DNA damage. P53 is subsequently stabilized and transactivates pro-apoptotic genes. The lower portion of panel B represents a protein-mediated histone crosstalk between methylation and acetylation, leading to chromatin remodeling favorable to transcriptional activation. (C) PTM-mediated cellular trafficking of RAS.