Systematic benchmarking of mass spectrometry-based antibody sequencing reveals methodological biases

The circulating antibody (Ab) repertoire is crucial for immune protection, holding significant immunological and biotechnological value. While bottom-up mass spectrometry (MS) is widely used for profiling the sequence diversity of circulating Abs (Ab repertoire sequencing [Ab-seq]), it has not been thoroughly benchmarked. We quantified the replicability and robustness of Ab-seq using six monoclonal Ab spike-ins in 70 combinations of concentration and oligoclonality, with and without polyclonal serum immunoglobulin G (IgG) background. Each combination underwent four protease treatments and was analyzed across four experimental and three technical replicates, totaling 3,360 liquid chromatography-tandem MS (LC-MS/MS) runs. We quantified the dependence of Ab-seq identification on Ab sequence, concentration, protease, presence of background IgGs, and bioinformatics methods. Integrating the data from experimental replicates, proteases, and bioinformatics tools enhanced Ab identification. De novo sequencing performed similarly to database-dependent methods at higher Ab concentrations, but de novo Ab reconstruction remains challenging. Our work provides a foundational resource for the field of MS-based Ab profiling. A record of this paper's transparent peer review process is included in the supplemental information.

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Publication Details

Journal
Cell Systems
Published
2025-11-01
DOI
https://doi.org/10.1016/j.cels.2025.101449
Primary Topic
Monoclonal and Polyclonal Antibodies Research
Type
article
Field-Weighted Citation Impact
0.00

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article

Systematic benchmarking of mass spectrometry-based antibody sequencing reveals methodological biases

Rowan Nelson, Pavel Sinitcyn, Jan Terje Andersen, Natalie Castellana et al.
Cell Systems
Monoclonal and Polyclonal Antibodies Research
article

Systematic benchmarking of mass spectrometry-based antibody sequencing reveals methodological biases

Rowan Nelson, Pavel Sinitcyn, Jan Terje Andersen, Natalie Castellana, Igor Snapkov, Sachin Kumar Singh, Timothy P. Jenkins, Geir Kjetil Sandve, William Stafford Noble, Anand Patel, Konstantinos Kalogeropoulos, Maria Chernigovskaya, Tuula A. Nyman, Melih Yilmaz, Stefano Bonissone, Stian Foss, Maria Stensland, Victor Greiff, Khang Lê Quý
article en

Abstract

The circulating antibody (Ab) repertoire is crucial for immune protection, holding significant immunological and biotechnological value. While bottom-up mass spectrometry (MS) is widely used for profiling the sequence diversity of circulating Abs (Ab repertoire sequencing [Ab-seq]), it has not been thoroughly benchmarked. We quantified the replicability and robustness of Ab-seq using six monoclonal Ab spike-ins in 70 combinations of concentration and oligoclonality, with and without polyclonal serum immunoglobulin G (IgG) background. Each combination underwent four protease treatments and was analyzed across four experimental and three technical replicates, totaling 3,360 liquid chromatography-tandem MS (LC-MS/MS) runs. We quantified the dependence of Ab-seq identification on Ab sequence, concentration, protease, presence of background IgGs, and bioinformatics methods. Integrating the data from experimental replicates, proteases, and bioinformatics tools enhanced Ab identification. De novo sequencing performed similarly to database-dependent methods at higher Ab concentrations, but de novo Ab reconstruction remains challenging. Our work provides a foundational resource for the field of MS-based Ab profiling. A record of this paper's transparent peer review process is included in the supplemental information.

Cell SystemsVol. 16(11)
Oslo University Hospital (NO), Norwegian Institute of Public Health (NO), University of Applied Sciences Utrecht (NL), University of Oslo (NO), Utrecht University (NL), University of Washington (US), Technical University of Denmark (DK)
Leona M. and Harry B. Helmsley Charitable Trust, European Federation of Pharmaceutical Industries and Associations, European Commission, Universitetet i Oslo, Norges Forskningsråd, Helse Sør-Øst RHF
Openalex Percentile: Top 51%
Monoclonal and Polyclonal Antibodies Research
DOIOpen Access PDFOpenAlex
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