Use of PASEF for Accelerated Protein Sequence Confirmation and De Novo Sequencing with High Data Quality
peer-reviewed · Methods in Molecular Biology · 2022
| Date | 2022-01-01 |
| Type | peer-reviewed |
| Venue | Methods in Molecular Biology |
| Publisher | Springer US |
| Contribution | algorithm |
| DOI | 10.1007/978-1-0716-1450-1_12 |
| Citations (OpenAlex) | 8 |
| Venue 2-year citedness | 1.14 |
Abstract
Biopharmaceutical sequences can be well confirmed by multiple protease digests-e.g., trypsin, elastase, and chymotrypsin-followed by LC-MS/MS data analysis. High quality data can be used for de novo sequencing as well. PASEF (Parallel Accumulation and Serial Fragmentation) on the timsTOF instrument has been used to accelerate proteome and protein sequence studies and increase sequence coverage concomitantly.Here we describe the protein chemical and LC-MS methods in detail to generate high quality samples for sequence characterization from only 3 digests. We applied PASEF to generate exhaustive protein sequence coverage maps by combination of results from the three enzyme digests using a short LC gradient. The data quality obtained was high and adequate for determining antibody sequences de novo.Nivolumab and dulaglutide were digested by 3 enzymes individually. For nivolumab, 94/94/90% sequence coverage and 86/84/85% fragment coverage were obtained from the individual digest analysis with trypsin/chymotrypsin/elastase, respectively. For dulaglutide, 96/100/90% sequence coverage and 92/90/83% fragment coverage were obtained. The merged peptide map from the 3 digests for nivolumab resulted in ∼550 peptides; enough to safely confirm the full sequences and to determine the nivolumab sequence de novo.
Methods and tools
- PASEF sequence confirmation and de novo: Uses PASEF on a timsTOF to accelerate the multi-protease digests (trypsin, elastase, chymotrypsin) that confirm a biopharmaceutical sequence, with data clean enough to drive de novo sequencing as well.