Precision De Novo Peptide Sequencing Using Mirror Proteases of Ac-LysargiNase and Trypsin for Large-scale Proteomics
peer-reviewed · Molecular & Cellular Proteomics · 2019
| Date | 2019-04-01 |
| Type | peer-reviewed |
| Venue | Molecular & Cellular Proteomics |
| Publisher | Elsevier BV |
| Contribution | algorithm |
| DOI | 10.1074/mcp.tir118.000918 |
| Citations (OpenAlex) | 40 |
| Venue 2-year citedness | 4.69 |
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.
Methods and tools
- pNovoM: De novo sequencing from mirror spectrum pairs: digesting the same sample with Ac-LysargiNase and with trypsin yields two spectra whose b and y ion series complement each other, which pins down the sequence far more confidently than either alone.
Data deposited
- E. coli mirror LC-MS/MS - Precision de novo peptide sequencing using mirror proteases of Ac-LysargiNase and trypsin for (as deposited) · PXD008688
- Monoclonal antibody LC-MS/MS - Precision de novo peptide sequencing using mirror proteases of Ac-LysargiNase and trypsin (as deposited) · PXD008690
- Yeast mirror LC-MS/MS - Precision de novo peptide sequencing using mirror proteases of Ac-LysargiNase and trypsin for la (as deposited) · PXD011562
Data used
Cites (27)
- Complete de novo sequencing of antimicrobial peptides in the venom of the scorpion Isometrus maculatus (2017) both
- De novo peptide sequencing by deep learning (2017) both
- Combining De Novo Peptide Sequencing Algorithms, A Synergistic Approach to Boost Both Identifications and Confidence in Bottom-up Proteomics (2017) both
- Comprehensive de Novo Peptide Sequencing from MS/MS Pairs Generated through Complementary Collision Induced Dissociation and 351 nm Ultraviolet Photodissociation (2017) both
- Evaluating de novo sequencing in proteomics: already an accurate alternative to database-driven peptide identification? (2017) both
- Open-pNovo: De Novo Peptide Sequencing with Thousands of Protein Modifications (2017) both
- UVnovo: A de Novo Sequencing Algorithm Using Single Series of Fragment Ions via Chromophore Tagging and 351 nm Ultraviolet Photodissociation Mass Spectrometry (2016) both
- Novor: Real-Time Peptide de Novo Sequencing Software (2015) both
- A Framework of De Novo Peptide Sequencing for Multiple Tandem Mass Spectra (2015) both
- Sequencing-Grade De novo Analysis of MS/MS Triplets (CID/HCD/ETD) From Overlapping Peptides (2013) both
- pNovo+: De Novo Peptide Sequencing Using Complementary HCD and ETD Tandem Mass Spectra (2013) both
- De Novo Sequencing and Homology Searching (2012) both
- pNovo: De novo Peptide Sequencing and Identification Using HCD Spectra (2010) both
- De novo sequencing of peptides by MS/MS (2010) both
- De novo peptide sequencing by tandem MS using complementary CID and electron transfer dissociation (2009) both
- Straightforward and de Novo Peptide Sequencing by MALDI-MS/MS Using a Lys-N Metalloendopeptidase (2009) both
- Automated de novo protein sequencing of monoclonal antibodies (2008) both
- A comparative study of the accuracy of several de novo sequencing software packages for datasets derived by matrix‐assisted laser desorption/ionisation and electrospray (2008) both
- De Novo Peptide Sequencing and Identification with Precision Mass Spectrometry (2007) both
- Performance Evaluation of Existing De Novo Sequencing Algorithms (2006) both
- De novo peptide sequencing using exhaustive enumeration of peptide composition (2006) both
- De Novo Sequencing of Neuropeptides Using Reductive Isotopic Methylation and Investigation of ESI QTOF MS/MS Fragmentation Pattern of Neuropeptides with N-Terminal Dimethylation (2005) both
- NovoHMM: A Hidden Markov Model for de Novo Peptide Sequencing (2005) crossref
- PepNovo: de novo peptide sequencing via probabilistic network modeling (2005) both
- A Hidden Markov Model for de Novo Peptide Sequencing (2004) semanticscholar
- PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry (2003) both
- Automated de novo sequencing of proteins by tandem high-resolution mass spectrometry (2000) both
Cited by (10)
- Mass Spectrometry-based Antibody Sequencing Technologies (2026) crossref
- DiNovo enables high-coverage and high-confidence de novo peptide sequencing via mirror proteases and deep learning (2026) both
- Cumulating MS Signal enables polyclonal antibody analysis (2025) both
- DiNovo: high-coverage, high-confidence de novo peptide sequencing using mirror proteases and deep learning (2025) crossref
- Mirror-image trypsin digestion and sequencing of D-proteins (2024) crossref
- Algorithms for de-novo sequencing of peptides by tandem mass spectrometry: A review (2023) crossref
- Classification of B and Y Ions in Peptide MS/MS Spectra Based on Machine Learning (2023) crossref
- Multienzyme deep learning models improve peptide de novo sequencing by mass spectrometry proteomics (2023) both
- Full-length Protein Sequencing Based on Continuous Digestion Using Non-specific Proteases (2021) crossref
- Flying blind, or just flying under the radar? The underappreciated power of de novo methods of mass spectrometric peptide identification (2020) crossref