Assessment method for deamidation in proteins using carboxylic acid derivatization-liquid chromatography-tandem mass spectrometry.
Carboxy group
Deamidation
Derivatization
LC–MS
Peptide
Protein
Journal
Journal of pharmaceutical and biomedical analysis
ISSN: 1873-264X
Titre abrégé: J Pharm Biomed Anal
Pays: England
ID NLM: 8309336
Informations de publication
Date de publication:
20 Mar 2020
20 Mar 2020
Historique:
received:
07
10
2019
revised:
01
01
2020
accepted:
02
01
2020
pubmed:
22
1
2020
medline:
1
12
2020
entrez:
22
1
2020
Statut:
ppublish
Résumé
An analytical method for the degree of protein deamidation has been developed by using carboxy group derivatization and liquid chromatography-tandem mass spectrometry (LCMS/MS). The fragment peptides (LGEYGFQNALIVR and YNGVFQECCQAEDK) obtained by digesting bovine serum albumin (BSA) with trypsin and their asparagine deamidated peptides (LGEYGFQDALIVR and YDGVFQECCQAEDK) were selected as model peptides, and their carboxy groups were derivatized with ethylamine. This derivatization enabled a clear distinction between natural peptides and deamidated peptides by mass, allowing for facile distinction by LCMS/MS before and after deamidation. Good linearity was confirmed for four peptides used in this study via isotope dilution mass spectrometry, showing that protein deamidation can be evaluated by the present method. To confirm the validity of this method for the evaluation of deamidation, natural peptides and deamidated peptides were mixed in arbitrary ratios, and degree of deamidation in these solution was analyzed. This confirmed that accurate evaluation was possible at deamidation degree values of ca. 10 %, 5 %, 2.5 %, and 1 %. Additionally, an accelerated storage test of BSA demonstrated that the deamidation of asparagine at position 404 of BSA progressed by 4 % in 9 weeks at 40 °C and pH 8 in the dark, and that the deamidation process can be traced over time.
Identifiants
pubmed: 31962249
pii: S0731-7085(19)32443-4
doi: 10.1016/j.jpba.2020.113095
pii:
doi:
Substances chimiques
Amides
0
Carboxylic Acids
0
Peptide Fragments
0
Glutamine
0RH81L854J
Serum Albumin, Bovine
27432CM55Q
Asparagine
7006-34-0
Types de publication
Journal Article
Validation Study
Langues
eng
Sous-ensembles de citation
IM
Pagination
113095Informations de copyright
Copyright © 2020 Elsevier B.V. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of Competing Interest This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. All study participants provided informed consent, and the study design was approved by the appropriate ethics review board. We have read and understood your journal’s policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare.