Improved detection of tryptic immunoglobulin variable region peptides by chromatographic and gas-phase fractionation techniques.
Cell Rep Methods
; 4(6): 100795, 2024 Jun 17.
Article
em En
| MEDLINE
| ID: mdl-38861989
ABSTRACT
The polyclonal repertoire of circulating antibodies potentially holds valuable information about an individual's humoral immune state. While bottom-up proteomics is well suited for serum proteomics, the vast number of antibodies and dynamic range of serum challenge this analysis. To acquire the serum proteome more comprehensively, we incorporated high-field asymmetric waveform ion-mobility spectrometry (FAIMS) or two-dimensional chromatography into standard trypsin-based bottom-up proteomics. Thereby, the number of variable region (VR)-related spectra increased 1.7-fold with FAIMS and 10-fold with chromatography fractionation. To match antibody VRs to spectra, we combined de novo searching and BLAST alignment. Validation of this approach showed that, as peptide length increased, the de novo accuracy decreased and BLAST performance increased. Through in silico calculations on antibody repository sequences, we determined the uniqueness of tryptic VR peptides and their suitability as antibody surrogate. Approximately one-third of these peptides were unique, and about one-third of all antibodies contained at least one unique peptide.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
/
Tripsina
Limite:
Humans
Idioma:
En
Revista:
Cell Rep Methods
Ano de publicação:
2024
Tipo de documento:
Article
País de publicação:
EEUU
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ESTADOS UNIDOS
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ESTADOS UNIDOS DA AMERICA
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EUA
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UNITED STATES
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UNITED STATES OF AMERICA
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US
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USA