Program

Poster Presentations

Day 3, May 17（Thu.）　　Poster

3P-16　　PDF

Charge-Mounted Positional Separation by Ion Exchange Chromatography for Protein N-Terminal Peptide Enrichment

(Kyoto University)
^oChih-hsiang Chang, Eito Yamamoto, Hsin-yi Chang, Naoyuki Sugiyama, Yasushi Ishihama

Mass spectrometry-based proteomics have been introduced to provide comprehensive identification of translation initiation sites, revealing unexpected regions such as upstream open reading frames (uORFs) and non-canonical translation site. In past studies, the enrichment methods of protein N-termini required huge sample amount, chemical modification steps and multidimensional chromatography. In this study, we aim to develop a simple and straight-forward method for protein N-terminome analysis. To generate peptide mixtures derived from protein N-termini, we build up a protein N-terminal peptide enrichment method named CHArge-Mounted Positional separation by ION exchange chromatography (CHAMPion). N-terminal RK proteases, Tryp-N, was introduced to generate digested peptides with doubly charged N-terminus. Without additional chemical derivatization, the enzymatic hydrolysis provides protein N-terminal peptides with singly charged or uncharged N-terminus and the internal peptides with doubly charged N-terminus. We found that strong cation exchange (SCX) chromatography can recognize the difference in the charge state at peptide N-termini and separate protein N-terminal peptides from the internal peptides. This method was applied to enriching protein N-terminal peptides from HEK293T human cell lysate. We identified 2,782 unique protein N-terminal peptides including 2,490 acetylated and 292 free N-terminal peptides (over 95% specificity of protein N-termini achieved by quantitative peak area) in triplicated LC/MS/MS analysis.