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Benchmarking of mass spectrometry-based antibody repertoire profiling reveals extensive systematic biases

ABSTRACT: The serum antibody repertoire is crucial for long-term immune protection, holding significant immunological and biotechnological value. Bottom-up proteomics is the most widely used mass spectrometry (MS) technique for profiling the sequence diversity of serum antibodies. However, serum antibody bottom-up proteomics (Ab-seq) has not been thoroughly benchmarked. In this study, we quantified the replicability and robustness of Ab-seq using six monoclonal antibodies with known protein sequences in 70 concentration and polyclonality combinations with and without polyclonal blood IgG background. Each combination underwent four protease treatments and was analyzed across four experimental and three technical replicates (3,360 LC-MS/MS runs). We quantified the dependence of MS-based antibody sequence identification on antibody sequence as well as concentration, protease, background signal diversity, and bioinformatics setup. Integrating experimental replicates, multiple proteases, and bioinformatics tools enhanced antibody identification. De novo peptide sequencing further improved sequence identification, complementing database-dependent methods. Our work provides a foundational resource for the field of MS-based serum antibody profiling.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma, Blood Serum

SUBMITTER: Ingrid Maria Erika Ekman Stensland

LAB HEAD: Tuula Anneli Nyman

PROVIDER: PXD055846 | Pride | 2025-12-04

REPOSITORIES: Pride

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			Action	DRS
	1stEH3746-EH4086_txt.zip	Other
	1st_AspN_EH4090-EH4430_txt.zip	Other
	1st_Chym_trp_EH4439-EH4781_txt.zip	Other
	1st_trypsin_EH3414-EH3742_txt.zip	Other
	2d_submission_C_T_txt.zip	Other

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