title: Improved N(α)-acetylated peptide enrichment following dimethyl labeling and SCX.
abstract: Protein N-terminal acetylation is one of the most common modifications occurring co- and post-translationally on either eukaryote or prokaryote proteins. However, compared to other protein modifications, the physiological role of protein N-terminal acetylation is relatively unclear. To explore the biological functions of protein N-terminal acetylation, a robust and large-scale method for qualitative and quantitative analysis of this modification is required. Enrichment of N(α)-acetylated peptides or depletion of the free N-terminal and internal tryptic peptides prior to analysis by mass spectrometry are necessary based on current technologies. This study demonstrated a simple strong cation exchange (SCX) fractionation method to selectively enrich N(α)-acetylated tryptic peptides via dimethyl labeling without the need for tedious protective labeling and depleting procedures. This method was introduced for the comprehensive analysis of N-terminal acetylated proteins from HepG2 cells. Several hundred N-terminal acetylation sites were readily identified in a single SCX flow-through fraction. Moreover, the N(α)-acetylated peptides of some protein isoforms were simultaneously observed in the SCX flow-through fraction, which indicated that this approach can be utilized to discriminate protein isoforms with very similar full sequences but different N-terminal sequences, such as β-actin/γ-actin, ERK1/ERK2, α-centractin/β-centractin, and ADP/ATP translocase 2 and 3. Compared to other methods, this method is relatively simple and can be directly implemented in a two-dimensional separation (SCX-RP)-mass spectrometry scheme for quantitative N-terminal proteomics using stable-isotope dimethyl labeling.
authors.affiliation: Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan.
authors.forename: Sin-Hong
authors.initials: SH
authors.lastname: Chen
authors.forename: Chiy-Rong
authors.initials: CR
authors.lastname: Chen
authors.forename: Shu-Hui
authors.initials: SH
authors.lastname: Chen
authors.forename: Ding-Tzai
authors.initials: DT
authors.lastname: Li
authors.forename: Jue-Liang
authors.initials: JL
authors.lastname: Hsu
mesh_terms: D000107:Acetylation; D002411:Cation Exchange Resins / Q000737:chemistry*; D056945:Hep G2 Cells; D006801:Humans; D007553:Isotope Labeling; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry
chemical_list: D002411:Cation Exchange Resins; D010455:Peptides; D011506:Proteins