Isolation of acetylated and free N-terminal peptides from proteomic samples based on tresyl-functionalized...

Analysis of protein N-termini is of great importance in helping to figure out important posttranslational modifications (PTMs) occurred in N-termini. Those PTMs include initial methionine removal, proteolytic cleavage, peptide signal processing, or N-terminal acetylation, which are usually neglected by conventional shotgun proteomics strategies. Herein, we develop a protein N-terminal peptides enrichment method based on commercial tresyl-functionalized microspheres (TFM). TFM could specifically immobilize the non-N-terminal peptides (internal peptides) from the supernatant. We demonstrated the isolation by TFM was more fast and efficient than formyl or epoxy-functionalized microspheres. Furthermore, this method could simultaneously isolate not only naturally free but acetylated blocked N-terminus. That facilitates a more comprehensive acquisition of N-terminus. After being verified by three standard proteins, cytochrome C, ribonuclease B and bovine serum albumin, this method was applied to mouse liver protein sample. We identified 122 naturally acetylated N-terminus and 107 free N-terminus in the sample. With the good performance of TFM, this method is efficient and useful for N-termini recovery.