Database-independent Protein Sequencing (DiPS) Enables Full-length de Novo Protein and Antibody Sequence Determination
peer-reviewed · Molecular & Cellular Proteomics · 2017
| Date | 2017-06-01 |
| Type | peer-reviewed |
| Venue | Molecular & Cellular Proteomics |
| Publisher | Elsevier BV |
| Contribution | post-processor |
| DOI | 10.1074/mcp.o116.065417 |
| Citations (OpenAlex) | 28 |
| Venue 2-year citedness | 4.69 |
Abstract
Traditional “bottom-up” proteomic approaches use proteolytic digestion, LC-MS/MS, and database searching to elucidate peptide identities and their parent proteins. Protein sequences absent from the database cannot be identified, and even if present in the database, complete sequence coverage is rarely achieved even for the most abundant proteins in the sample. Thus, sequencing of unknown proteins such as antibodies or constituents of metaproteomes remains a challenging problem. To date, there is no available method for full-length protein sequencing, independent of a reference database, in high throughput. Here, we present Database-independent Protein Sequencing, a method for unambiguous, rapid, database-independent, full-length protein sequencing. The method is a novel combination of non-enzymatic, semi-random cleavage of the protein, LC-MS/MS analysis, peptide de novo sequencing, extraction of peptide tags, and their assembly into a consensus sequence using an algorithm named “Peptide Tag Assembler.” As proof-of-concept, the method was applied to samples of three known proteins representing three size classes and to a previously un-sequenced, clinically relevant monoclonal antibody. Excluding leucine/isoleucine and glutamic acid/deamidated glutamine ambiguities, end-to-end full-length de novo sequencing was achieved with 99-100% accuracy for all benchmarking proteins and the antibody light chain. Accuracy of the sequenced antibody heavy chain, including the entire variable region, was also 100%, but there was a 23-residue gap in the constant region sequence.
Methods and tools
- DiPS: Database-independent Protein Sequencing: de novo sequencing plus assembly that determines full-length protein and antibody sequences without a database.
Data deposited
- Next Generation Protein Sequencing (as deposited) · PXD003804
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- Homo-Tag-Assembler Assay for Full-Length Antibody Sequencing (2025) crossref
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- Simultaneous polyclonal antibody sequencing and epitope mapping by cryo electron microscopy and mass spectrometry (2025) crossref
- Metaproteomics Beyond Databases: Addressing the Challenges and Potentials of De Novo Sequencing (2025) both
- Systematic benchmarking of mass spectrometry-based antibody sequencing reveals methodological biases (2024) crossref
- Simplified and Rapid Workflow Enhances Throughput of De Novo Sequencing of COVID-19 Neutralizing Antibodies (2024) crossref
- A Handle on Mass Coincidence Errors in De Novo Sequencing of Antibodies by Bottom-up Proteomics (2024) crossref
- Comprehensive evaluation of peptide de novo sequencing tools for monoclonal antibody assembly (2023) both
- A perspective toward mass spectrometry-based de novo sequencing of endogenous antibodies (2022) both
- Template-Based Assembly of Proteomic Short Reads For De Novo Antibody Sequencing and Repertoire Profiling (2022) both
- Current state, existing challenges, and promising progress for de novo sequencing and assembly of monoclonal antibodies (2022) both
- Template-based assembly of proteomic short reads for de novo antibody sequencing and repertoire profiling (2022) crossref
- Highly Robust de Novo Full-Length Protein Sequencing (2021) crossref
- Mass Spectrometry-Based De Novo Sequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme (2021) both
- Full-length Protein Sequencing Based on Continuous Digestion Using Non-specific Proteases (2021) crossref
- Mass spectrometry-based de novo sequencing of the anti-FLAG-M2 antibody using multiple proteases and a dual fragmentation scheme (2021) both
- ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping (2020) both
- Flying blind, or just flying under the radar? The underappreciated power of de novo methods of mass spectrometric peptide identification (2020) crossref
- 2018 YPIC Challenge: A Case Study in Characterizing an Unknown Protein Sample (2019) crossref
- Multi-Enzymatic Limited Digestion: The Next-Generation Sequencing for Proteomics? (2019) crossref
- A potential golden age to come—current tools, recent use cases, and future avenues for de novo sequencing in proteomics (2018) crossref