Improved detection of tryptic immunoglobulin variable region peptides by chromatographic and gas-phase fractionation techniques
peer-reviewed · Cell Reports Methods · 2024
| Date | 2024-06-01 |
| Type | peer-reviewed |
| Venue | Cell Reports Methods |
| Publisher | Elsevier BV |
| Contribution | downstream-application |
| DOI | 10.1016/j.crmeth.2024.100795 |
| Citations (OpenAlex) | 0 |
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.
Methods and tools
- Serum immunoglobulin variable-region detection: FAIMS or 2D chromatography increased detection of antibody variable-region peptides in serum, matched by combining de novo sequencing with BLAST.
Methods it uses
- PEAKS: Commercial DP-based de novo