Abstract
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, which provide mutual complementarity of each other, and allow 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 sample 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 precision 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.
Highlights
Authors Hao Yang, Yan-Chang Li, Ming-Zhi Zhao, Fei-Lin Wu, Xi Wang, Wei-Di Xiao, Yi-Hao Wang, Jun-Ling Zhang, Fu-Qiang Wang, Feng Xu, Wen-Feng Zeng, Christopher M
After transforming the pXPZ1 into E. coli BL21 (DE3) and induced by isopropyl -Dthiogalactoside (IPTG), the high-purity LysargiNase was obtained through purification and activation processes (Fig. 1A)
The LC-MS/MS analysis showed an envelope of the isotope peaks for molecular weights of the modified LysargiNase
Summary
Authors Hao Yang, Yan-Chang Li, Ming-Zhi Zhao, Fei-Lin Wu, Xi Wang, Wei-Di Xiao, Yi-Hao Wang, Jun-Ling Zhang, Fu-Qiang Wang, Feng Xu, Wen-Feng Zeng, Christopher M. From a large-scale yeast proteome sample digested with trypsin and AcLysargiNase individually, 48% of all tandem mass spectra formed mirror spectrum pairs, 97% of which contained full coverage of ion series, resulting in precision 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. Muth et al [27] reported that less than 40% of the full-length peptide sequences were consistent with the results from database search regardless of which de novo sequencing algorithm was tested
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