Improved detection of tryptic immunoglobulin variable region peptides by chromatographic and gas-phase fractionation techniques

Christoph Stingl*, Martijn M. VanDuijn, Thomas Dejoie, Peter A.E. Sillevis Smitt, Theo M. Luider

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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.

Original languageEnglish
Article number100795
Number of pages20
JournalCell Reports Methods
Volume4
Issue number6
Early online date10 Jun 2024
DOIs
Publication statusPublished - 17 Jun 2024

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© 2024 The Author(s)

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