Direct mass spectrometry-based detection and antibody sequencing of Monoclonal Gammopathy of Undetermined Significance from patient serum – a case study

Peng, Weiwei; Boer, Maurits A. den; Tamara, Sem; Mokiem, Nadia J.; Lans, Sjors P. A. van der; Schulte, Douwe; Haas, Pieter-Jan; Minnema, Monique C.; Rooijakkers, Suzan H.M.; Zuilen, Arjan D. van; Heck, Albert J. R.; Snijder, Joost

doi:10.1101/2023.05.22.541697

Cited by 3 publications

(2 citation statements)

References 26 publications

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“…The Uniprot reviewed human protein database was used, with ∼20,300 entries (Release number 2018_05). An additional sequence of a dominant IGHG1 clone for P1 patient was added in the search, previously described by Peng et al 14 All downstream analyses were carried out in R. 15…”

Section: Journal Of Proteome Researchmentioning

confidence: 99%

Longitudinal Fluctuations in Protein Concentrations and Higher-Order Structures in the Plasma Proteome of Kidney Failure Patients Subjected to a Kidney Transplant

Kalaidopoulou Nteak,

Völlmy,

Lukassen

et al. 2024

J. Proteome Res.

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Using proteomics and complexome profiling, we evaluated in a yearlong study longitudinal variations in the plasma proteome of kidney failure patients, prior to and after a kidney transplantation. The post-transplant period was complicated by bacterial infections, resulting in dramatic changes in the proteome, attributed to an acute phase response (APR). As positive acute phase proteins (APPs), being elevated upon inflammation, we observed the well-described C-reactive protein and Serum Amyloid A (SAA), but also Fibrinogen, Haptoglobin, Leucine-rich alpha-2glycoprotein, Lipopolysaccharide-binding protein, Alpha-1-antitrypsin, Alpha-1-antichymotrypsin, S100, and CD14. As negative APPs, being downregulated upon inflammation, we identified the well-documented Serotransferrin and Transthyretin, but added Kallistatin, Heparin cofactor 2, and interalpha-trypsin inhibitor heavy chain H1 and H2 (ITIH1, ITIH2). For the patient with the most severe APR, we performed plasma complexome profiling by SEC-LC-MS on all longitudinal samples. We observed that several plasma proteins displaying alike concentration patterns coelute and form macromolecular complexes. By complexome profiling, we expose how SAA1 and SAA2 become incorporated into high-density lipid particles, replacing largely Apolipoprotein (APO)A1 and APOA4. Overall, our data highlight that the combination of in-depth longitudinal plasma proteome and complexome profiling can shed further light on correlated variations in the abundance of several plasma proteins upon inflammatory events.

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Section: Journal Of Proteome Researchmentioning

confidence: 99%

Longitudinal Fluctuations in Protein Concentrations and Higher-Order Structures in the Plasma Proteome of Kidney Failure Patients Subjected to a Kidney Transplant

Kalaidopoulou Nteak,

Völlmy,

Lukassen

et al. 2024

J. Proteome Res.

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“…27 Stitch has been shown to enable the accurate reconstruction of monoclonal antibody sequences, as well as the sequencing of isolated Fab fragments from patient serum, M-proteins in monoclonal gammopathies, antibody light chains from urine, and the profiling of whole IgG from COVID-19 patient sera. [13][14][15]26 Sequence accuracies of ∼99% can be obtained, which is sufficient to reverse engineer functional antibody products. 21,22,28,29 Remaining sequencing errors stem in large parts from common mass coincidences of isobaric residues like leucine/isoleucine but also from incomplete fragmentation spectra in which the order of two or more residues remains ambiguous due to lacking fragment ions for the intermediate positions.…”

Section: ■ Introductionmentioning

confidence: 99%

A Handle on Mass Coincidence Errors in De Novo Sequencing of Antibodies by Bottom-up Proteomics

Schulte,

Snijder

2024

J. Proteome Res.

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Antibody sequences can be determined at 99% accuracy directly from the polypeptide product by using bottom-up proteomics techniques. Sequencing accuracy at the peptide level is limited by the isobaric residues leucine and isoleucine, incomplete fragmentation spectra in which the order of two or more residues remains ambiguous due to lacking fragment ions for the intermediate positions, and isobaric combinations of amino acids, of potentially different lengths, for example, GG = N and GA = Q. Here, we present several updates to Stitch (v1.5), which performs template-based assembly of de novo peptides to reconstruct antibody sequences. This version introduces a mass-based alignment algorithm that explicitly accounts for mass coincidence errors. In addition, it incorporates a postprocessing procedure to assign I/L residues based on secondary fragments (satellite ions, i.e., w-ions). Moreover, evidence for sequence assignments can now be directly evaluated with the addition of an integrated spectrum viewer. Lastly, input data from a wider selection of de novo peptide sequencing algorithms are allowed, now including Casanovo, PEAKS, Novor.Cloud, pNovo, and MaxNovo, in addition to flat text and FASTA. Combined, these changes make Stitch compatible with a larger range of data processing pipelines and improve its tolerance to peptide-level sequencing errors.

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Reverse engineering the anti-MUC1 hybridoma antibody 139H2 by mass spectrometry-basedde novosequencing

Peng

Giesbers

Šiborová

et al. 2023

Preprint

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Mucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at different mucosal surfaces including breast and intestine. In the gastrointestinal tract, MUC1 has a barrier function against bacterial invasion, but can also serve as an entry receptor for pathogenic Salmonella bacteria. Moreover, MUC1 is well known for its aberrant expression and glycosylation in adenocarcinomas The MUC1 extracellular domain contains a variable number of tandem repeats (VNTR) of 20 amino acids, which are heavily O-linked glycosylated. Monoclonal antibodies against the MUC1 VNTR can be powerful tools because of their multiplicity of binding and possible applications in the diagnosis and treatment of MUC1-expressing cancers. One such antibody is the hybridoma mouse monoclonal 139H2, which is also widely used as a research tool to study non-cancer MUC1. Here we report direct mass spectrometry-based sequencing of hybridoma-derived 139H2 IgG, which enabled reverse engineering of a recombinant 139H2. The performance of the reverse engineered 139H2 IgG and its Fab fragment were validated by comparison to the hybridoma-derived product in Western blot and immunofluorescence microscopy. The reverse engineering of 139H2 allowed us to characterize binding to the VNTR peptide epitope by surface plasmon resonance (SPR) and solve the crystal structure of the 139H2 Fab fragment in complex with the MUC1 VNTR peptide. These analyses reveal the molecular basis for 139H2 binding specificity to MUC1 and its tolerance to O-glycosylation of the VNTR. The available sequence of 139H2 will allow further development of MUC1-related diagnostics, targeting and treatment strategies.

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Direct mass spectrometry-based detection and antibody sequencing of Monoclonal Gammopathy of Undetermined Significance from patient serum – a case study

Cited by 3 publications

References 26 publications

Longitudinal Fluctuations in Protein Concentrations and Higher-Order Structures in the Plasma Proteome of Kidney Failure Patients Subjected to a Kidney Transplant

Longitudinal Fluctuations in Protein Concentrations and Higher-Order Structures in the Plasma Proteome of Kidney Failure Patients Subjected to a Kidney Transplant

A Handle on Mass Coincidence Errors in De Novo Sequencing of Antibodies by Bottom-up Proteomics

Reverse engineering the anti-MUC1 hybridoma antibody 139H2 by mass spectrometry-basedde novosequencing

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