Human Plasma IgG1 Repertoires are Simple, Unique, and Dynamic

Bondt, Albert; Hoek, Max; Tamara, Sem; Graaf, Bastiaan de; Peng, Weiwei; Schulte, Douwe; Boer, Maurits A. den; Greisch, Jean‐François; Varkila, Meri R. J.; Snijder, Joost; Cremer, Olaf L.; Bonten, Marc J. M.; Heck, Albert J. R.

doi:10.2139/ssrn.3749694

Cited by 7 publications

(13 citation statements)

References 24 publications

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“…Polarization of the repertoire was also observed by other groups in the context of the IgG1 repertoire [29] as well as autoimmunity [24]. Thus, a few antigen-specific clonal lineages seem to dominate both antigen-specific repertoires [21,24,28] and even the pan-IgG1 repertoire [29].…”

Section: Glossarysupporting

confidence: 54%

“…Furthermore, of the 80-120 antibody clonal lineages that typically constitute the serum titer to the tetanus toxoid vaccine, a single clonal lineage was found to account for as much as 15-30% of antigen-specific response [28]. Polarization of the repertoire was also observed by other groups in the context of the IgG1 repertoire [29] as well as autoimmunity [24]. Thus, a few antigen-specific clonal lineages seem to dominate both antigen-specific repertoires [21,24,28] and even the pan-IgG1 repertoire [29].…”

Section: Glossarymentioning

confidence: 71%

“…By contrast, intra-individual overlap across time in the measured IgG1 repertoires was high, even when the timespan largely exceeded the average antibody half-life of IgGs, indicating sustained secretion by a dedicated antibody-secreting cell (ASC) subset (e.g., terminally differentiated longlived plasma cells). Within a single donor, a large portion of the most abundant IgG1s remained present for up to several weeks [29]. Persisting clonal lineages (even over years) were also observed in autoimmunity [24,30] and vaccination settings [31] to varying degrees.…”

Section: Glossarymentioning

confidence: 95%

“…Intra-and inter-individual (longitudinal) clonal overlap Bondt and colleagues compared serum IgG1 Fab fragment (Box 1 and Figure 2) profiles between timepoints within one donor and between different donors. They found that virtually none of the Fab IDs overlapped between individuals when focusing on the top 30 most abundant IgG1s [29]. Cumulatively, in all eight patients, <10% of the detected IgG1s exhibited an inter-individual alikeness in mass and retention time, leading to the conclusion that each donor exhibited a unique IgG1 Fab molecular profile.…”

Section: Glossarymentioning

confidence: 99%

“…Although middle-down proteomics provides significantly improved coverage, sensitivity, and accuracy, it eschews the benefits of reduced sample processing and native chain pairing information [79]. Nevertheless, current middle-down efforts towards antibody discovery seem promising [29,105,139] In short, bottom-up proteomics can be utilized for antibody identification in complex mixtures, whereas top-down proteomics is used in (therapeutic) antibody PTM studies [140] and for tracking of single antibodies, which can provide information on the bioavailability and performance of therapeutic mAbs for quality control purposes [141]. Middle-down proteomics can be utilized as a complementary method to cover for the shortcomings of top-down or bottom-up methods.…”

Section: Microfluidicsmentioning

confidence: 99%

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Progress and challenges in mass spectrometry-based analysis of antibody repertoires

Snapkov

Chernigovskaya

Sinitcyn

et al. 2022

Trends in Biotechnology

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Humoral immunity is divided into the cellular B cell and protein-level antibody responses. High-throughput sequencing has advanced our understanding of both these fundamental aspects of B cell immunology as well as aspects pertaining to vaccine and therapeutics biotechnology. Although the protein-level serum and mucosal antibody repertoire make major contributions to humoral protection, the sequence composition and dynamics of antibody repertoires remain underexplored. This limits insight into important immunological and biotechnological parameters such as the number of antigen-specific antibodies, which are for example, relevant for pathogen neutralization, microbiota regulation, severity of autoimmunity, and therapeutic efficacy. High-resolution mass spectrometry (MS) has allowed initial insights into the antibody repertoire. We outline current challenges in MS-based sequence analysis of antibody repertoires and propose strategies for their resolution.

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Section: Glossarysupporting

confidence: 54%

Section: Glossarymentioning

confidence: 71%

Section: Glossarymentioning

confidence: 95%

Section: Glossarymentioning

confidence: 99%

Section: Microfluidicsmentioning

confidence: 99%

See 3 more Smart Citations

Progress and challenges in mass spectrometry-based analysis of antibody repertoires

Snapkov

Chernigovskaya

Sinitcyn

et al. 2022

Trends in Biotechnology

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Mass Spectrometry-Based De Novo Sequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme

2021

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Antibody sequence information is crucial to understanding the structural basis for antigen binding and enables the use of antibodies as therapeutics and research tools. Here, we demonstrate a method for direct de novo sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for de novo sequencing by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a bottom-up fashion. Furthermore, we apply a dual fragmentation scheme, using both stepped high-energy collision dissociation (stepped HCD) and electron-transfer high-energy collision dissociation (EThcD), on all peptide precursors. The method achieves full sequence coverage of the monoclonal antibody herceptin, with an accuracy of 99% in the variable regions. We applied the method to sequence the widely used anti-FLAG-M2 mouse monoclonal antibody, which we successfully validated by remodeling a high-resolution crystal structure of the Fab and demonstrating binding to a FLAG-tagged target protein in Western blot analysis. The method thus offers robust and reliable sequences of monoclonal antibodies.

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Mass spectrometry-basedde novosequencing of the anti-FLAG-M2 antibody using multiple proteases and a dual fragmentation scheme

Peng

Snijder

2021

Preprint

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Antibody sequence information is crucial to understanding the structural basis for antigen binding and enables the use of antibodies as therapeutics and research tools. Here we demonstrate a method for direct de novo sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for de novo sequencing by LC-MS/MS in a bottom-up fashion. Furthermore, we apply a dual fragmentation scheme, using both stepped high-energy collision dissociation (stepped HCD) and electron transfer high-energy collision dissociation (EThcD) on all peptide precursors. The method achieves full sequence coverage of the monoclonal antibody Herceptin, with an accuracy of 99% in the variable regions. We applied the method to sequence the widely used anti-FLAG™-M2 mouse monoclonal antibody, which we successfully validated by remodeling a high-resolution crystal structure of the Fab and demonstrating binding to a FLAG™-tagged target protein in Western blot analysis. The method thus offers robust and reliable sequences of monoclonal antibodies.

show abstract

Human Plasma IgG1 Repertoires are Simple, Unique, and Dynamic

Cited by 7 publications

References 24 publications

Progress and challenges in mass spectrometry-based analysis of antibody repertoires

Progress and challenges in mass spectrometry-based analysis of antibody repertoires

Mass Spectrometry-Based De Novo Sequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme

Mass spectrometry-basedde novosequencing of the anti-FLAG-M2 antibody using multiple proteases and a dual fragmentation scheme

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