A perspective toward mass spectrometry-based de novo sequencing of endogenous antibodies

Abstract

A key step in therapeutic and endogenous humoral antibody characterization is identifying the amino acid sequence. So far, this task has been mainly tackled through sequencing of B-cell receptor (BCR) repertoires at the nucleotide level. Mass spectrometry (MS) has emerged as an alternative tool for obtaining sequence information directly at the – most relevant – protein level. Although several MS methods are now well established, analysis of recombinant and endogenous antibodies comes with a specific set of challenges, requiring approaches beyond the conventional proteomics workflows. Here, we review the challenges in MS-based sequencing of both recombinant as well as endogenous humoral antibodies and outline state-of-the-art methods attempting to overcome these obstacles. We highlight recent examples and discuss remaining challenges. We foresee a great future for these approaches making de novo antibody sequencing and discovery by MS-based techniques feasible, even for complex clinical samples from endogenous sources such as serum and other liquid biopsies.

N-linked glycosylation of the M-protein variable region: Glycoproteogenomics reveals a new layer of personalized complexity in multiple myeloma

“…Proteogenomics, the integration of genomics and proteomics, is an emerging technological development in the multi-omics field and has the potential to improve cancer characterization, diagnosis, and even advance cancer therapy( [37][38][39] ). Furthermore, such integration efforts are also being used to sequence antibody repertoires by MS 40 . With glycoproteogenomics, we can now relate genomic alterations to detailed glycosylation phenotypes in a clinical setting.…”

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“…With the increasing sensitivity of mass-spectrometric analysis workflows, this can also allow protein sequencing by mass-spectrometry de novo (from the protein without the antibody sequence), a technology which has already been reported for the discovery of antigens. 102,103…”

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“…Through advances in mass spectrometry it has become within reach to sequence recombinant antibodies de novo by mass spectrometry using either protein-centric (top-down) or peptide-centric (shotgun or bottom-up) proteomics approaches (Tran, Rahman et al 2016, Guthals, Gan et al 2017, Sen, Tang et al 2017, Peng, Pronker et al 2021, de Graaf, Hoek et al 2022, Schulte, Peng et al 2022, or combinations thereof. However, to do this on endogenous serum clones, in a background of many highly similar antibodies, represents still an arduous undertaking.…”