Abstract

1. Enhanced Protein Nα-acetylation Analysis by SCX and Dimethyl Labeling and Its Application to Discrimination of Protein Isoforms Sin-Hong Chen1,Ming-Hui Liao1 Jue-Liang Hsu2* 1 Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan 2Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan *jlhsu@mail.npust.edu.tw Abstract Result 3：Statistical analysis of identified Nα-acetylated peptides. 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 Nα-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 Nα-acetylated tryptic peptidesviadimethyl 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 Nα-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. (A) (A) (B) Fig 3 : (A) The subcellular distribution of the identified Nα-acetylated proteins. (B) Sequence conservation analysis of the first five residues of 351 Nα-acetylated termini by iceLogo. Result 4：Discrimination of protein isoforms based on the identification of unique Nα-acetylated peptides. Methods Flow chart of Nα-acetylated peptide enrichment Result 1：Binding affinity comparison of N-acetylated, dimethylated and native peptides by SCX fractionation 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. Result 5：Expression profiling of Nα-acetylated proteins from HepG2 cells upon oxidative damage (A) (A) Fig 1. SCX binding affinity comparison of Nα-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. (B) Result 2：Enhanced protein Nα-acetylation analysis of HepG2 cells (A) Fig 5 : Varied quantitation results between Nα-acetylated and internal peptides derived from beta-actin with and without oxidative stress. (A) Ratio of Nα-acetylated peptides from the SCX flow-through fraction; (B) Ratio of internal peptides identified from the SCX elute fraction. Conclusion 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. (B) References 1.Thomas Arnesen, Petra Van Damme et al. PNAS 2009,Vol.6, No.20, 8157-8162 2. Nikolai Mischerikow and Albert J. R. Heck. Proteomics 2011. 11, 571–589 3. Mann, M., and Jensen, O.N. Proteomic analysis of post-translational modifications. Nat. Biotechnol. 2003, 21, 255–261.. Fig 2 : (A) Specificity comparison of different methods for the analysis of Nα-acetylated peptides. (B) The number of Nα-acetylated peptides identified from each SCX fraction.