TEMPLATE DESIGN © Abstract Result 3 ： Statistical analysis of identified N α -acetylated peptides. Methods Conclusion Result 1 ： Binding affinity comparison of N-acetylated, dimethylated and native peptides by SCX fractionation Result 4 ： Discrimination of protein isoforms based on the identification of unique N α -acetylated peptides. Result 5 ： Expression profiling of N α -acetylated proteins from HepG2 cells upon oxidative damage Result 2 ： Enhanced protein N α -acetylation analysis of HepG2 cells References Enhanced Protein N α -acetylation Analysis by SCX and Dimethyl Labeling and Its Application to Discrimination of Protein Isoforms (A) Sin-Hong Chen 1, Ming-Hui Liao 1 Jue-Liang Hsu 2* 1 Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan 2 Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan 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 post-translational modifications (PTMs), 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 PTM 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 tedious protective labeling and depleting procedures. This method was introduced for the comprehensive analysis of N-terminal acetylated proteins from HepG2 cells under oxidative damage by tert -butyl peroxide ( t -BHP). Several hundreds of N-terminal acetylation sites were readily identified in a single SCX flow- through fraction and the protein N-terminal acetylation patterns with and without oxidative damage were simultaneously determined when the stable isotope dimethyl labeling was introduced. 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. 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-termini proteomics using stable-isotope dimethyl labeling. (B) Fig 3 : (A) The subcellular distribution of the identified N α -acetylated proteins. (B) Sequence conservation analysis of the first five residues of 351 N α -acetylated termini by iceLogo. Fig 2 : (A) Specificity comparison of different methods for the analysis of N α -acetylated peptides. (B) The number of N α -acetylated peptides identified from each SCX fraction. 1.Thomas Arnesen, Petra Van Damme et al. PNAS 2009, Vol.6, No.20, Nikolai Mischerikow and Albert J. R. Heck. Proteomics , 571– Mann, M., and Jensen, O.N. Proteomic analysis of post-translational modifications. Nat. Biotechnol. 2003, 21, 255–261.. In this study, we demonstrated that SCX-SPE coupled with dimethyl labeling can specifically isolate N-acetylated peptides in the SCX flow-through fraction due to the increase in charge differences between N-acetylated peptides and internal peptides. This characteristic dramatically enhances the analysis of protein N-acetylation. The relative abundances of N-acetylated proteins from HepG2 cells with and without t-BHP treatment were simultaneously obtained when stable- isotope dimethyl labeling was introduced. Beyond the enhanced analysis of protein N-acetylation, this approach is well-suited for the discrimination of protein isoforms with very similar full sequences but different N-terminal sequences, and is feasible for circumventing the quantitation interferences caused by the same internal peptides from isoforms. Fig 1. SCX binding affinity comparison of N α -acetylated, native and dimethylated peptides. The mixture of native, dimethylated and acetylated peptides (denoted P, DM-P and Ac-P, respectively) was fractionated by SCX and each fraction was analyzed by LC-MS. (A) Flow-through fraction, (B) 10% of 0.5 M NaCl, and (C) 20% of 0.5 M NaCl. (A) Fig 5 : Varied quantitation results between N α -acetylated and internal peptides derived from beta- actin with and without oxidative stress. (A) Ratio of N α -acetylated peptides from the SCX flow- through fraction; (B) Ratio of internal peptides identified from the SCX elute fraction. Hit No. Protein accessionProtein nameN-acetylated peptide% identity in residues overlap 1ADT2_HUMANADP/ATP translocase 2Ac-MTDAAVSFA*K 92.6% identity in 296 residues overlap ADT3_HUMANADP/ATP translocase 3Ac-MTEQAISFA*K 2 DX_HUMANATP-dependent RNA helicase DDX Ac- AEQDVENDLLDYDEEEEPQAPQEST PAPP*K 89.7% identity in 428 residues overlap DX39B_HUMANSpliceosome RNA helicase DDX39B Ac- AENDVDNELLDYEDDEVETAAGGD GAEAPA*K 3 ACTB_HUMANActin, cytoplasmic 1 Ac-DDDIAALVVDNGSGMC*K 98.9% identity in 375 residues overlap ACTG_HUMANActin, cytoplasmic 2 Ac-EEEIAALVIDNGSGMC*K 4 ACTY_HUMANBeta-centractin Ac-MESYDIIANQPVVIDNGSGVI*K 90.4% identity in 376 residues overlap ACTZ_HUMANAlpha-centractin Ac-MESYDVIANQPVVIDNGSGVI*K 5 ST1_HUMANSulfotransferase 1 Ac-MELIQDTSRPPLEYV*K 92.9% identity in 295 residues overlap ST3_HUMANSulfotransferase 3/4 Ac-MELIQDTSRPPLEYV*K 6 RAB_HUMANRas-related protein Rab Ac-SSMNPEYDYLF*K 93.1% identity in 202 residues overlap RAB1B_HUMANRas-related protein Rab-1B Ac-MNPEYDYLF*K 7 COF1_HUMANCofilin-1 Ac-ASGVAVSDGVI*K 80.7% identity in 166 residues overlap COF2_HUMANCofilin-2 Ac-ASGVTVNDEVI*K 8 MK01_HUMANMitogen-activated protein kinase 1 Ac-AAAAAAGAGPEMV*R 88.2% identity in 346 residues overlap MK03_HUMANMitogen-activated protein kinase 3 Ac-AAAAQGGGGGEP*R Flow chart of N α -acetylated peptide enrichment (A) Fig 4 : MS/MS spectra of N-terminal acetylated peptides derived from (A) beta-actin and (B) gamma-actin. Table 1. Summary of identified protein isoforms with a greater than 80% sequence identity. Peptide(from β-actin)D/H ratio m/zcharge Ac-DDDIAALVVDNGSGMC*K Ac-DDDIAALVVDNGSGMC*K (Oxidation M) Peptide(from β-actin)D/H ratio m/zcharge GILTLK AGFAGDDAPR GYSFTTTAER QEYDESGPSIVHR (B)