De Novo Sequencing of Antibody Light Chain Proteoforms from Patients with Multiple Myeloma

Dupré, Mathieu; Duchateau, Magalie; Sternke-Hoffmann, Rebecca; Boquoi, Amelie; Malosse, Christian; Fenk, Roland; Haas, Rainer; Buell, Alexander K.; Rey, Martial; Chamot‐Rooke, Julia

doi:10.26434/chemrxiv.14541711.v1

Cited by 4 publications

(18 citation statements)

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“…We set out to perform a comprehensive characterisation of the thermodynamic and aggregation properties of the patient-derived light chains with the aim to combine this information with the sequences that we obtained from a parallel sequencing effort 41 . To this end, we have combined a wide array of state of the art biophysical and biochemical methods into the ThAgg-Fip approach.…”

Section: Resultsmentioning

confidence: 99%

“…This workflow is based on the combination of bottom-up and top-down proteomics experiments with appropriate data analysis. It is important to note that the sample numbers are not identical between the present manuscript and the manuscript with the sequencing results 41 , but the latter contains a correspondence table. Briefly, light chain samples were solubilized in 8 M urea, then reduced (5 mM TCEP, 30 min) and alkylated (10 mM iodoacetamide, 30 min in the dark).…”

Section: Lc Sequence Characterization and Protease Identificationmentioning

confidence: 93%

“…All LC sequences were determined using a dedicated mass spectrometry workflow described in 41 . This workflow is based on the combination of bottom-up and top-down proteomics experiments with appropriate data analysis.…”

Section: Lc Sequence Characterization and Protease Identificationmentioning

confidence: 99%

“…AUC allows the analysis under native conditions in solution and mass spectrometry allows unique identification of protein monomers and multimers. A detailed discussion of the dimerisation behaviour, including the observation of mixed dimers formed from two IgLC isoforms, can be found in 41 . The fractions of monomer and dimer displayed in Figure 1 A are extracted from sedimentation velocity c(s) profiles.…”

Section: Dimerisation Of the Light Chainsmentioning

confidence: 99%

“…In order to investigate the link between light chain sequence and aggregation behaviour, we have developed a complete de novo protein sequencing workflow based on a combination of top-down 40 and bottom-up proteomics with specific data analysis for patient-derived light chains. The details of this workflow are published separately 41 . The light chains investigated in this study were derived from 9 patients presenting light chains in their urine (1 isotype lambda and 8 isotype kappa) and represent a sub-set of a larger collection from a patient cohort at the University Hospital Düsseldorf, which has already been subjected to an initial biochemical and biophysical characterisation 19 .…”

Section: Introductionmentioning

confidence: 99%

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Universal amyloidogenicity of patient-derived immunoglobulin light chains

Sternke-Hoffmann

Pauly

Norrild

et al. 2021

Preprint

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The deposition of immunoglobulin light chains (IgLCs) in the form of amorphous aggregates or amyloid fibrils in different tissues of patients can lead to severe and potentially fatal organ damage, requiring transplantation in some cases. There has been great interest in recent years to elucidate the origin of the very different in vivo solubilities of IgLCs, as well as the molecular determinants that drive either the formation of ordered amyloid fibrils or disordered amorphous aggregates. It is commonly thought that the reason of this differential aggregation behaviour is to be found in the amino acid sequences of the respective IgLCs, i.e. that some sequences display higher intrinsic tendencies to form amyloid fibrils. Here we perform in depth Thermodynamic and Aggregation Fingerprinting (ThAgg-Fip) of 9 multiple myeloma patient-derived IgLCs, the amino acid sequences of all of which we have solved by de novo protein sequencing with mass spectrometry. The latter technique was also used for one IgLc from a patient with AL amyloidosis. We find that all samples also contain proteases that fragment the proteins under physiologically relevant mildly acidic pH conditions, leading to amyloid fibril formation in all cases. Our results suggest that while every pathogenic IgLC has a unique ThAgg fingerprint, all sequences have comparable amyloidogenic potential. Therefore, extrinsic factors, in particular presence of, and susceptibility to, proteolytic cleavage is likely to be a strong determinant of in vivo aggregation behaviour. The important conclusion, which is corroborated by systematic analysis of our sequences, as well as many sequences of IgLCs from amyloidosis patients reported in the literature, challenges the current paradigm of the link between sequence and amyloid fibril formation of pathogenic light chains.

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Section: Resultsmentioning

confidence: 99%

Section: Lc Sequence Characterization and Protease Identificationmentioning

confidence: 93%

Section: Lc Sequence Characterization and Protease Identificationmentioning

confidence: 99%

Section: Dimerisation Of the Light Chainsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Universal amyloidogenicity of patient-derived immunoglobulin light chains

Sternke-Hoffmann

Pauly

Norrild

et al. 2021

Preprint

Self Cite

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Template-based assembly of proteomic short reads forde novoantibody sequencing and repertoire profiling

Schulte

Peng

Snijder

2022

Preprint

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Antibodies can target a vast molecular diversity of antigens. This is achieved by generating a complementary diversity of antibody sequences though somatic recombination and hyper mutation. A full understanding of the antibody repertoire in health and disease therefore requires dedicated de novo sequencing methods. Next generation cDNA sequencing methods have laid the foundation of our current understanding of the antibody repertoire, but these methods share one major limitation in that they target the antibody-producing B-cells, rather than the functional secreted product in bodily fluids. Mass spectrometry-based methods offer an opportunity to bridge this gap between antibody repertoire profiling and bulk serological assays, as they can access antibody sequence information straight from the secreted polypeptide products. In a step to meeting the challenge of MS-based antibody sequencing, we present a fast and simple software tool (Stitch) to map proteomic short reads to user-defined templates with dedicated features for both monoclonal antibody sequencing and profiling of polyclonal antibody repertoires. We demonstrate the use of Stitch by fully reconstructing 2 monoclonal antibody sequences with >98% accuracy (including I/L assignment); sequencing a Fab from patient serum isolated by reversed-phase LC fractionation against a high background of homologous antibody sequences; sequencing antibody light chains from urine of multiple-myeloma patients; and profiling the IgG repertoire in sera from patients hospitalized with COVID-19. We demonstrate that Stitch assembles a comprehensive overview of the antibody sequences that are represented in the dataset and provides an important first step towards analyzing polyclonal antibodies and repertoire profiling.

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A multi-parameter optimization in middle-down analysis of monoclonal antibodies by LC-MS/MS

Dhenin

Dupré

Druart

et al. 2022

Preprint

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In antibody-based drug research, regulatory agencies request a complete characterization of antibody proteoforms covering both the amino acid sequence and all post-translational modifications. The usual mass spectrometry-based approach to achieve this goal is bottom-up proteomics, which relies on the digestion of antibodies, but does not allow the diversity of proteoforms to be assessed. Middle-down and top-down approaches have recently emerged as attractive alternatives but are not yet mastered and thus used in routine by many analytical chemistry laboratories. The work described here aims at providing guidelines to achieve the best sequence coverage for the fragmentation of intact light and heavy chains generated from a simple reduction of intact antibodies using Orbitrap mass spectrometry. Three parameters were found crucial to this aim: the use of an electron-based activation technique, the multiplex selection of precursor ions of different charge states and the combination of replicates.

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De Novo Sequencing of Antibody Light Chain Proteoforms from Patients with Multiple Myeloma

Cited by 4 publications

References 9 publications

Universal amyloidogenicity of patient-derived immunoglobulin light chains

Universal amyloidogenicity of patient-derived immunoglobulin light chains

Template-based assembly of proteomic short reads forde novoantibody sequencing and repertoire profiling

A multi-parameter optimization in middle-down analysis of monoclonal antibodies by LC-MS/MS

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