Expanding N-terminomics Coverage by Extending N-terminal Peptide Length
- 1Kyoto University
Abstract
In bottom-up proteomics, protease specificity determines peptide length and LC/MS/MS detectability; in N-terminomics, this is critical because each protein N-terminal peptide is unique and cannot be substituted by other peptides. CHromatographic AMplification of Protein N-terminal peptides (CHAMP-N) combines LysargiNase digestion with StageTip-based strong cation exchange chromatography (SCX), removing internal peptides by exploiting their newly generated N-terminal Lys/Arg residues and thereby selectively isolating protein N-terminal peptides. However, LysargiNase cleavage near the protein N-terminus can generate excessively short peptides. Here, we developed two complementary CHAMP-N approaches that modurate N-terminal peptide length while retaining the charge-based SCX separation principle. LysN digestion avoided cleavage at N-terminal-proximal Arg residues, whereas D3-acetylation blocked Lys cleavage by LysargiNase to produce ArgN-like specificity. Both approaches extended otherwise short N-terminal peptides while preserving N-terminal basic residues on internal peptides. Conventional LysargiNase, LysN, and ArgN-like CHAMP-N generated complementary sets of N-terminal peptides. Integration of the three workflows identified 2,552 protein N-termini, approximately twice the number identified by conventional CHAMP-N alone. The median relative standard deviations of N-terminal peptide intensities were approximately 15% for all three workflows, indicating comparable technical reproducibility. These results demonstrate that coordinating protease cleavage specificity with SCX selectivity reduces sequence-dependent losses and expands N-terminome coverage while preserving the simplicity and reproducibility of CHAMP-N.
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BibTeX
@misc{takeshita2026expanding,
title = {{Expanding N-terminomics Coverage by Extending N-terminal Peptide Length}},
author = {A. Takeshita and E. Kanao and K. Imami and Y. Ishihama},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.22.753420},
url = {https://www.biorxiv.org/content/10.64898/2026.09.22.753420v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Expanding N-terminomics Coverage by Extending N-terminal Peptide Length
AU - A. Takeshita
AU - E. Kanao
AU - K. Imami
AU - Y. Ishihama
PY - 2026
DA - 2026/09/22
PB - bioRxiv
DO - 10.64898/2026.09.22.753420
UR - https://www.biorxiv.org/content/10.64898/2026.09.22.753420v1
AB - In bottom-up proteomics, protease specificity determines peptide length and LC/MS/MS detectability; in N-terminomics, this is critical because each protein N-terminal peptide is unique and cannot be substituted by other peptides. CHromatographic AMplification of Protein N-terminal peptides (CHAMP-N) combines LysargiNase digestion with StageTip-based strong cation exchange chromatography (SCX), removing internal peptides by exploiting their newly generated N-terminal Lys/Arg residues and thereby selectively isolating protein N-terminal peptides. However, LysargiNase cleavage near the protein N-terminus can generate excessively short peptides. Here, we developed two complementary CHAMP-N approaches that modurate N-terminal peptide length while retaining the charge-based SCX separation principle. LysN digestion avoided cleavage at N-terminal-proximal Arg residues, whereas D3-acetylation blocked Lys cleavage by LysargiNase to produce ArgN-like specificity. Both approaches extended otherwise short N-terminal peptides while preserving N-terminal basic residues on internal peptides. Conventional LysargiNase, LysN, and ArgN-like CHAMP-N generated complementary sets of N-terminal peptides. Integration of the three workflows identified 2,552 protein N-termini, approximately twice the number identified by conventional CHAMP-N alone. The median relative standard deviations of N-terminal peptide intensities were approximately 15% for all three workflows, indicating comparable technical reproducibility. These results demonstrate that coordinating protease cleavage specificity with SCX selectivity reduces sequence-dependent losses and expands N-terminome coverage while preserving the simplicity and reproducibility of CHAMP-N.
ER -