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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.
Keywords: Ac-LysargiNase; De novo sequencing; Enzyme catalysis*; Mass Spectrometry; Mirror proteases; Peptide mass fingerprinting; Protein engineering; Trypsin.
Higher activity and stability of newly developed Ac-LysargiNase. A , SDS-PAGE analysis… Fig. 1. Higher activity and stability of newly developed Ac-LysargiNase. A, SDS-PAGE analysis of the purified wild-type LysargiNase (WT) and its Acetylated form (Ac-LysargiNase). 15% SDS-PAGE was used. B, C, The MWs of LysargiNase and Ac-LysargiNase were determined with mass spectrometry. D, Self-digestion of LysargiNase and Ac-LysargiNase. The arrow indicates a chromatograph peak for an autolyzed peptide KIPVVVH of LysargiNase and Ac-LysargiNase. E, Intensity of the same autolyzed peptide from LysargiNase and Ac-LysargiNase. The digestion time is shown as indicated. F, Comparison of the activity of LysargiNase and Ac-LysargiNase using yeast TCLs as substrate. The ratios of protease to substrate are shown as indicated. The samples with an asterisk below were analyzed using LC-MS/MS. G, Comparison of protein identification and self-digestion with the same yeast TCL digested using LysargiNase and Ac-LysargiNase. The same samples were concurrently analyzed as indicated by the asterisk in panel f.
Fig. 2.
Mirror protease strategy for de…
Fig. 2.
Mirror protease strategy for de novo sequencing. A , The mirror spectra… Fig. 2. Mirror protease strategy for de novo sequencing. A, The mirror spectra improved the quality of the matched peptide-spectrum pairs by providing a continuous and complete set of product ions and distinguishable directions of most ions (marked with blue circles, e.g. vertices 2, 3, 4 and 5). Two dotted peaks denote that b1 and y6 were missing in the trypsin spectrum. B, Workflow of the pNovoM algorithm based on the mirror protease strategy for de novo sequencing. Mirror spectrum pairs from trypsin- and Ac-LysargiNase-digested samples were found and sequenced using pNovoM. Then, the two spectra in each pair were integrated into one merged spectrum graph for de novo sequencing.
Fig. 3.
Superior precision de novo sequencing…
Fig. 3.
Superior precision de novo sequencing of monoclonal antibody PXL1 based on the mirror… Fig. 3. Superior precision de novo sequencing of monoclonal antibody PXL1 based on the mirror protease strategy. A, Workflow of the analysis of two antibody data sets (PXL1 and PXL2) generated from trypsin- and Ac-LysargiNase-digested samples. B, Comparison of the specificity and miss cleavage of the spectra sequenced from trypsin or Ac-LysargiNase-digested antibody PXL1. C, The distribution of ion coverage of trypsin, Ac-LysargiNase and mirror spectra. D, Identification rates of the spectra for each peptide from pNovoM, pNovo+ and PEAKS. The blue numbers indicate the average percentage of the correctly sequenced mirror spectrum pairs, and the blue bar chart denotes the number of mirror spectrum pairs for each mirror peptide pair. E, The peptides sequenced by three algorithms on the heavy and light chains of PXL1. The three different color lines were the results of pNovoM (green), pNovo+ (blue) and PEAKS (red). The asterisks denote incorrect residues. The false amino acids matching was listed as the number showed.
Fig. 4.
Proteome-level de novo sequencing of…
Fig. 4.
Proteome-level de novo sequencing of yeast based on the mirror protease strategy. … Fig. 4. Proteome-level de novo sequencing of yeast based on the mirror protease strategy. A, Workflow for the de novo sequencing and FDR evaluation at the proteome level. B, FDR curves of pNovoM, pNovo+ and PEAKS at the spectrum level. C, Venn diagram of the spectra sequenced with pNovo+, PEAKS and pNovoM at 5% FDR at the spectrum level. The red numbers in parentheses denote the FDRs of the corresponding parts. D, FDR curves of pNovoM, pNovo+ and PEAKS at the peptide level. E, Venn diagram of the peptides sequenced using pNovo+, PEAKS and pNovoM at 5% FDR at the peptide level.
Fig. 5.
Mirror spectrum pairs achieved near-complete…
Fig. 5.
Mirror spectrum pairs achieved near-complete ion coverage. A , Percentages of matched … Fig. 5. Mirror spectrum pairs achieved near-complete ion coverage. A, Percentages of matched b and y ions in trypsin, Ac-LysargiNase and mirror spectra. B, The distributions of the intensities of the matched b and y ions in trypsin, Ac-LysargiNase and mirror spectra. C, The distributions of ion coverage in trypsin, Ac-LysargiNase and mirror spectra. D, In all 16,514 mirror spectrum pairs, the cumulative curves of the number of correctly matched spectra from top one to top ten candidates of pNovoM, pNovo+, pNovo+ (T+L), PEAKS and PEAKS (T+L). (T+L) denotes that both trypsin and Ac-LysargiNase spectra were considered. Numbers below the arrows indicate the increase from the top one sensitivity to the top two sensitivity.
Fig. 6.
Mirror protease strategy sequenced more…
Fig. 6.
Mirror protease strategy sequenced more long peptides and increased the ion coverage, especially… Fig. 6. Mirror protease strategy sequenced more long peptides and increased the ion coverage, especially for the N termini of peptides. A, Venn diagram of the correct peptides sequenced using pNovoM, pNovo+ and PEAKS. B, The distributions of the length of the peptides consistently sequenced using all three algorithms and uniquely sequenced using pNovoM. C, Relationship between the ion position and the matched ion ratio of the mirror peptide pairs (length ≤ 30). D, The distributions of the N- and C-terminal-most three-product ions for the 1,930 peptides uniquely sequenced using pNovoM. The blue numbers denote the percentage of the spectra without any ions at the corresponding ion position. All figures (7) See this image and copyright information in PMC
References
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