Use of PASEF for Accelerated Protein Sequence Confirmation and De Novo Sequencing with High Data Quality.
Dulaglutide
Mass Spectrometry
Monoclonal antibodies
Nivolumab
PASEF (Parallel Accumulation Serial Fragmentation)
Reversed Phase HPLC
Sequence confirmation
TIMS—Trapped Ion Mobility Spectrometry
Trypsin digestion
de novo sequencing
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
3
9
2021
pubmed:
4
9
2021
medline:
14
1
2022
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 34478140
doi: 10.1007/978-1-0716-1450-1_12
doi:
Substances chimiques
Peptides
0
Proteome
0
Nivolumab
31YO63LBSN
Chymotrypsin
EC 3.4.21.1
Pancreatic Elastase
EC 3.4.21.36
Trypsin
EC 3.4.21.4
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
207-217Informations de copyright
© 2022. Springer Science+Business Media, LLC, part of Springer Nature.
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