Experimental procedure for PTM proteomics. Antibody-based affinity purification for lysine acetylated peptides is used as an example. The proteomics study of a PTM typically involves preparation of a protein sample, proteolytic digestion of proteins into peptides, enrichment of PTM peptides using an appropriate method, HPLC/MS/MS analysis of the enriched PTM peptides, and protein sequence database searching using the MS/MS data for identifying peptide sequences, mapping PTM sites, and quantification. When necessary, a protein/peptide fractionation step is included prior to PTM peptide enrichment in order to reduce the sample complexity and to enhance the yield of enrichment. Different methods can be selected for protein fractionation, peptide fractionation, and enrichment of PTM peptides. Abbreviation: SAX, strong anion exchange.

Example strategies for enriching PTM peptides. (A) Antibody-based affinity purification for isolation of lysine acetylated peptides. An antibody immobilized to a solid support can bind ^AcLys peptide for enrichment. (B) IMAC for enriching phosphopeptides. Ferric ion (Fe³⁺) immobilized in solid beads (indicated by a solid green circle) bind specifically phosphate group in a phosphopeptide. (C) Isolation of GPI-APs by enzyme-catalyzed specific release. The plasma membrane fraction is isolated. The GPI-APs are specifically released from the membrane when phosphatidylinositol phospholipase hydrolyzes the phosphatidylinositol. (D) Chemical derivatization and subsequent enzymatic cleavage for isolation of glycosylated proteins. To isolate glycoproteins, glycoproteins are first oxidized and conjugated to hydrazide beads, while non-glycoproteins can be removed. The glycoproteins are then released by PNGase F. Abbreviations: PI-PLC, phosphatidylinositol phospholipase C; PNGase F, peptide-N-glycosidase F.