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Mol Cell Proteomics. 2019 Jan 8. pii: mcp.TIR118.000918. doi: 10.1074/mcp.TIR118.000918. [Epub ahead of print]

Precision de novo peptide sequencing using mirror proteases of Ac-LysargiNase and trypsin for large-scale proteomics.

Author information

Institute of Computing Technology.
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Institute of Lifeomics, Beijing, China.
Beijing Proteome Research Center.
Institute of Computing Technology, Chinese Academy of Sciences, China.
Chinese Academy of Sciences.
Center for Blood Research, University of British Columbia, Canada.
Institute of Computing Technology, Chinese Academy of Sciences.
State Key Laboratory of Proteomics, National Center for Protein Sciences Beijing, China


De novo peptide sequencing for large-scale proteomics remains challenging because of the lack of full coverage of ion series in tandem mass spectra. We developed a mirror protease of trypsin, acetylated LysargiNase (Ac-LysargiNase), with superior activity and stability. The mirror spectrum pairs derived from the Ac-LysargiNase and trypsin treated samples can generate full b and y ion series. The b and y ion series provide mutual complementarity and examination of each other, which allowed us to develop a novel algorithm, pNovoM, for de novo sequencing. Using pNovoM to sequence peptides of purified proteins, the accuracy of the sequence was close to 100%. More importantly, from a large-scale yeast proteome samples digested with trypsin and Ac-LysargiNase individually, 48% of all tandem mass spectra formed mirror spectrum pairs, 97% of which contained full coverage of ion series, resulting in precise de novo sequencing of full-length peptides by pNovoM. This enabled pNovoM to successfully sequence 21,249 peptides from 3,753 proteins and interpreted 44-152% more spectra than pNovo+ and PEAKS at a 5% FDR at the spectrum level. Moreover, the mirror protease strategy had an obvious advantage in sequencing long peptides. We believe that the combination of mirror protease strategy and pNovoM will be an effective approach for precision de novo sequencing on both single proteins and proteome samples.


Ac-LysargiNase; De novo sequencing; Enzyme catalysis*; Mass Spectrometry; Mirror proteases; Peptide mass fingerprinting; Protein engineering; Trypsin

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