Abstract Human antibody discovery relies on accessing in vivo -matured repertoires, yet conventional single-cell B cell receptor sequencing (scBCR-seq) often overlooks the most relevant, functional antibodies secreted by plasma cells. Here, we introduce AbDirect, a protein-centric discovery platform that obtains antibody sequences directly from small-volume biofluids. In this proof-of-concept, we apply AbDirect to potent COVID-19 plasma from which an early-pandemic scBCR-seq study did not identify neutralizers. Upfront reactivity screening of anti-SARS-CoV-2 spike protein repertoires revealed diverse clonal profiles with distinct cross-reactivity and subunit specificity. Targeted de novo sequencing via standalone integrative proteomics yielded 14 IgG1 and 4 IgA1 clones that diverged markedly from peripheral B cell counterparts in germline usage and phylogeny, indicating distinct immunological compartments. Validation via recombinant mAbs demonstrated superior binding and highly potent neutralization for multiple sequenced clones (three with IC 50 ≤1.4 nM). AbDirect thus yielded potent antibodies overlooked by scBCR-seq, demonstrating serological discovery as a powerful complementary approach for uncovering functional repertoires that may be inaccessible to cell-based methods.

Blunted first-phase insulin secretion and insulin deficiency are indicators of beta cell dysfunction and diabetes manifestation. Therefore, insights into molecular mechanisms that regulate insulin homeostasis might provide entry sites to replenish insulin content and restore beta cell function. Here, we identify the insulin inhibitory receptor (inceptor; encoded by the gene IIR/ELAPOR1) as an insulin-binding receptor that regulates insulin stores by lysosomal degradation. Using human induced pluripotent stem cell (SC)-derived islets, we show that IIR knockout (KO) results in enhanced SC beta cell differentiation and survival. Strikingly, extended in vitro culture of IIR KO SC beta cells leads to greatly increased insulin content and glucose-stimulated insulin secretion (GSIS). We find that inceptor localizes to clathrin-coated vesicles close to the plasma membrane and in the trans-Golgi network as well as in secretory granules, where it acts as a sorting receptor to direct proinsulin and insulin towards lysosomal degradation. Targeting inceptor using a monoclonal antibody increases proinsulin and insulin content and improves SC beta cell GSIS. Altogether, our findings reveal the basic mechanisms of beta cell insulin turnover and identify inceptor as an insulin degradation receptor.

Serum proteomics has matured and is now able to monitor hundreds of proteins quantitatively in large cohorts of patients. However, the fine characteristics of some of the most dominant proteins in serum, the immunoglobulins, are in these studies often ignored, due to their vast, and highly personalized, diversity in sequences. Here, we focus exclusively on these personalized features in the serum proteome and distinctively chose to study individual samples from a low diversity population: elderly donors infected by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2). By using mass spectrometry-based methods, immunoglobulin IgG1 and IgA1 clonal repertoires were monitored quantitatively and longitudinally in more than 50 individual serum samples obtained from 17 Corona virus disease 2019 patients admitted to intensive care units. These clonal profiles were used to examine how each patient reacted to a severe SARS-CoV-2 infection. All 17 donors revealed unique polyclonal repertoires and substantial changes over time, with several new clones appearing following the infection, in a few cases leading to a few, very high, abundant clones dominating their repertoire. Several of these clones were de novo sequenced through combinations of top-down, middle-down, and bottom-up proteomics approaches. This revealed sequence features in line with sequences deposited in the SARS-CoV-specific antibody database. In other patients, the serological Ig profiles revealed the treatment with tocilizumab, that subsequently dominated their serological IgG1 repertoire. Tocilizumab clearance could be monitored, and a half-life of approximately 6 days was established. Overall, our longitudinal monitoring of IgG1 and IgA1 repertoires of individual donors reveals that antibody responses are highly personalized traits of each patient, affected by the disease and the chosen clinical treatment. The impact of these observations argues for a more personalized and longitudinal approach in patients' diagnostics, both in serum proteomics as well as in monitoring immune responses.

Monoclonal gammopathy of undetermined significance (MGUS) is a plasma cell disorder characterized by the presence of a predominant monoclonal antibody (i.e., M-protein) in serum, without clinical symptoms. Here we present a case study in which we detect MGUS by liquid-chromatography coupled with mass spectrometry (LC-MS) profiling of IgG1 in human serum. We detected a Fab-glycosylated M-protein and determined the full heavy and light chain sequences by bottom-up proteomics techniques using multiple proteases, further validated by top-down LC-MS. Moreover, the composition and location of the Fab-glycan could be determined in CDR1 of the heavy chain. The outlined approach adds to an expanding mass spectrometry-based toolkit to characterize monoclonal gammopathies such as MGUS and multiple myeloma, with fine molecular detail. The ability to detect monoclonal gammopathies and determine M-protein sequences straight from blood samples by mass spectrometry provides new opportunities to understand the molecular mechanisms of such diseases.

The most abundant immunoglobulin present in the human body is IgA. It has the highest concentrations at the mucosal lining and in biofluids such as milk and is the second most abundant class of antibodies in serum. We assessed the structural diversity and clonal repertoire of IgA1-containing molecular assemblies longitudinally in human serum and milk from three donors using a mass spectrometry-based approach. IgA-containing molecules purified from serum or milk were assessed by the release and subsequent analysis of their Fab fragments. Our data revealed that serum IgA1 consists of two distinct structural populations, namely monomeric IgA1 (∼80%) and dimeric joining (J-) chain coupled IgA1 (∼20%). Also, we confirmed that IgA1 in milk is present solely as secretory (S)IgA, consisting of two (∼50%), three (∼33%) or four (∼17%) IgA1 molecules assembled with a J-chain and secretory component (SC). Interestingly, the serum and milk IgA1-Fab repertoires were distinct between monomeric, and J-chain coupled dimeric IgA1. The serum dimeric J-chain coupled IgA1 repertoire contained several abundant clones also observed in the milk IgA1 repertoire. The latter repertoire had little to no overlap with the serum monomeric IgA1 repertoire. This suggests that human IgA1s have (at least) two distinct origins; one of these produces dimeric J-chain coupled IgA1 molecules, shared in human serum and milk, and another produces monomeric IgA1 ending up exclusively in serum.

The most abundant immunoglobulin present in the human body is IgA1. It has the highest concentrations at the mucosal lining and in biofluids such as milk and is the second most abundant class of antibodies in serum. We assessed the structural diversity and clonal repertoire of IgA1-containing molecular assemblies longitudinally in human serum and milk from three donors using a mass spectrometry-based approach. IgA-containing molecules purified from serum or milk were assessed by the release and subsequent analysis of their Fab fragments. Our data revealed that serum IgA1 consists of two distinct structural populations, namely monomeric IgA1 (∼ 80%) and dimeric joining (J-) chain coupled IgA1 (∼ 20%). Also, we confirmed that IgA1 in milk is present solely as secretory (S)IgA, consisting of two (∼ 50%), three (∼ 33%) or four (∼ 17%) IgA1 molecules assembled with a J-chain and secretory component (SC). Interestingly, the serum and milk IgA1-Fab repertoires were distinct between monomeric, and J-chain coupled dimeric IgA1. The serum dimeric J-chain coupled IgA1 repertoire contained several abundant clones also observed in the milk IgA1 repertoire. The latter repertoire had little to no overlap with the serum monomeric IgA1 repertoire. This suggests that human IgA1s have (at least) two distinct origins; one of these produces dimeric J-chain coupled IgA1 molecules, shared in human serum and milk, and another produces monomeric IgA1 ending up exclusively in serum.

We investigated protein glycation in a complex milk system under controlled conditions representative of real-life consumer products, analysing intermediate and final products from skim milk powder production, and aged powder samples. We combined protein-centric LC-MS(/MS) with peptide-centric multi-protease LC-MS/MS focusing on the six most abundant bovine milk proteins. This strategy resulted in the identification of glycated proteoforms and of the extent of glycation per protein, high protein sequence coverage, and identification and relative occupancy of the glycation sites. We identified new glycation hot-spots additionally to the ones already described in literature. Primary sequence motif analysis revealed that glycation hot-spots were preceded N-terminally by a stretch rich in basic amino acids, and followed C-terminally by a stretch enriched in aliphatic and hydrophobic amino acids. Our study considerably extends the current understanding of milk protein glycation, discussing glycation hot-spots and their localisation in relation to the primary sequences and higher-order protein structures.

Here, by using mass spectrometry-based methods IgG1 and IgA1 clonal repertoires were monitored quantitatively and longitudinally in more than 50 individual serum samples obtained from 17 COVID-19 patients admitted to intensive care units because of acute respiratory distress syndrome. These serological clonal profiles were used to examine how each patient reacted to a severe SARS-CoV-2 infection. All 17 donors revealed unique polyclonal repertoires and changes after infection. Substantial changes over time in the IgG1 and/or IgA1 clonal repertoires were observed in individual patients, with several new clones appearing following the infection, in a few cases leading to a few very high abundant IgG1 and/or IgA1 clones dominating the repertoire. Several of these clones were de novo sequenced through combinations of top-down, middle-down and bottom-up proteomics approaches. This revealed several sequence features in line with sequences deposited in the SARS-CoV-specific database of antibodies. In other patients, the serological Ig profiles revealed the treatment with tocilizumab, as after treatment, this IgG1-mAb dominated the serological IgG1 repertoire. Tocilizumab clearance could be monitored and a half-life of approximately 6 days was established in these patients. Overall, our longitudinal monitoring of IgG1 and IgA1 repertoires of individual donors reveals that antibody responses are highly personalized traits of each patient, affected by the disease and the chosen clinical treatment. The impact of these observations argues for a more personalized and longitudinal approach in patients’ diagnostics, both in serum proteomics as well as in monitoring immune responses.

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.

Native mass spectrometry (MS) involves the analysis and characterization of macromolecules, predominantly intact proteins and protein complexes, whereby as much as possible the native structural features of the analytes are retained. As such, native MS enables the study of secondary, tertiary, and even quaternary structure of proteins and other biomolecules. Native MS represents a relatively recent addition to the analytical toolbox of mass spectrometry and has over the past decade experienced immense growth, especially in enhancing sensitivity and resolving power but also in ease of use. With the advent of dedicated mass analyzers, sample preparation and separation approaches, targeted fragmentation techniques, and software solutions, the number of practitioners and novel applications has risen in both academia and industry. This review focuses on recent developments, particularly in high-resolution native MS, describing applications in the structural analysis of protein assemblies, proteoform profiling of─among others─biopharmaceuticals and plasma proteins, and quantitative and qualitative analysis of protein-ligand interactions, with the latter covering lipid, drug, and carbohydrate molecules, to name a few.

Standard methods for mass analysis measure ensembles of thousand to millions of molecules. This approach enables analysis of monodisperse recombinant proteins, whereas some heterogeneous protein assemblies pose a significant challenge, whereby co-occurring stoichiometries, sub-complexes, and modifications hamper analysis using native mass spectrometry. To tackle the challenges posed by mass heterogeneity, single-particle methods may come to the rescue. Recently, two such approaches have been introduced, namely, mass photometry (MP) and Orbitrap-based charge detection mass spectrometry (CDMS). Both methods assess masses of individual molecules, albeit adhering to distinct physical principles. To evaluate these methods side by side, we analyzed a set of ribosomal particles, representing polydisperse ribonucleoprotein assemblies in the MDa range. MP and CDMS provide accurate masses for intact ribosomes and enable quantitative analysis of concomitant distinct particles within each ribosome sample. Here, we discuss pros and cons of these single-molecule techniques, also in the context of other techniques used for mass analysis.

Background: Since the outbreak of coronavirus disease 2019 (COVID-19), many put their hopes in the rapid availability of effective immunizations. Human milk, containing antibodies against syndrome coronavirus 2 (SARS-CoV-2), may serve as means of protection through passive immunization. We aimed to determine the presence and pseudovirus neutralization capacity of SARS-CoV-2 specific IgA in human milk of mothers who recovered from COVID-19, and the effect of pasteurization on these antibodies. Methods: This prospective case control study included lactating mothers, recovered from (suspected) COVID-19 and healthy controls. Human milk and serum samples were collected. To assess the presence of SARS-CoV-2 antibodies we used multiple complementary assays, namely ELISA with the SARS-CoV-2 spike protein (specific for IgA and IgG), receptor binding domain (RBD) and nucleocapsid (N) protein for IgG in serum, and bridging ELISA with the SARS-CoV-2 RBD and N protein for specific Ig (IgG, IgM and IgA in human milk and serum). To assess the effect of pasteurization, human milk was exposed to Holder (HoP) and High Pressure Pasteurization (HPP). Results: Human milk contained abundant SARS-CoV-2 antibodies in 83% of the proven cases and in 67% of the suspected cases. Unpasteurized milk with and without these antibodies was found to be capable of neutralizing a pseudovirus of SARS-CoV-2 in (97% and 85% of the samples respectively). After pasteurization, total IgA antibody levels were affected by HoP, while SARS-CoV-2 specific antibody levels were affected by HPP. Pseudovirus neutralizing capacity of the human milk samples was only retained with the HPP approach. No correlation was observed between milk antibody levels and neutralization capacity. Conclusions: Human milk from recovered COVID-19-infected mothers contains SARS-CoV-2 specific antibodies which maintained neutralization capacity after HPP. All together this may represent a safe and effective immunization strategy after HPP.

Although humans can produce billions of IgG1 variants through recombination and hypermutation, the diversity of IgG1 clones circulating in human blood plasma has largely eluded direct characterization. Here, we combined several mass-spectrometry-based approaches to reveal that the circulating IgG1 repertoire in human plasma is dominated by a limited number of clones in healthy donors and septic patients. We observe that each individual donor exhibits a unique serological IgG1 repertoire, which remains stable over time but can adapt rapidly to changes in physiology. We introduce an integrative protein- and peptide-centric approach to obtain and validate a full sequence of an individual plasma IgG1 clone de novo. This IgG1 clone emerged at the onset of a septic episode and exhibited a high mutation rate (13%) compared with the closest matching germline DNA sequence, highlighting the importance of de novo sequencing at the protein level. A record of this paper's transparent peer review process is included in the supplemental information.

Immunoglobulins A (IgA) include some of the most abundant human antibodies and play an important role in defending mucosal surfaces against pathogens. The unique structural features of the heavy chain of IgA subclasses (called IgA1 and IgA2) enable them to polymerize via the joining J-chain, resulting in IgA dimers but also higher oligomers. While secretory sIgA oligomers are dominant in milk and saliva, IgAs exist primarily as monomers in serum. No method currently allows disentangling the millions of unique IgAs potentially present in the human antibody repertoire. Obtaining unambiguous sequence reads of their hypervariable antigen-binding regions is a prerequisite for IgA identification. We here report a mass spectrometric method that uses electron capture dissociation (ECD) to produce straightforward-to-read sequence ladders of the variable parts of both the light and heavy chains of IgA1s, in particular, of the functionally critical CDR3 regions. We directly compare the native top-down ECD spectra of a heavily and heterogeneously N- and O-glycosylated anti-CD20 IgA1, the corresponding N-glycosylated anti-CD20 IgG1, and their Fab parts. We show that while featuring very different MS1 spectra, the native top-down ECD MS2 spectra of all four species are nearly identical, with cleavages occurring specifically within the CDR3 and FR4 regions of both the heavy and light chain. From the sequence-informative ECD data of an intact glycosylated IgA1, we foresee that native top-down ECD will become a valuable complementary tool for the de novo sequencing of IgA1s from milk, saliva, or serum.

The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo-microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. This unique architecture generates specificity for thousands of SPs based on the length of their hydrophobic segments.

Huntington’s disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo and in cell models that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 Å cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The exon 1 region of HTT is dynamic but shows greater conformational variety in the polyglutamine expanded mutant than wildtype exon 1. Our data provide a foundation for future functional and drug discovery studies targeting Huntington’s disease and illuminate the structural consequences of HTT polyglutamine expansion.

Huntington’s disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 Å cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass-spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The polyglutamine tract containing N-terminal exon 1 region of HTT is dynamic, but shows greater conformational variety in the mutant than wildtype exon 1. By providing novel insight into the structural consequences of HTT polyglutamine expansion, our data provide a foundation for future functional and drug discovery studies targeting Huntington’s disease.

Since the outbreak of coronavirus disease 2019 (COVID-19), many put their hopes in the rapid development of effective immunizations. As this intervention will not be available on a short notice, patient isolation, physical distancing, protective materials and good hygiene are currently the sole measures for prevention. Here we provide multiple lines of evidence on the presence of a variety of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) present in breastmilk from mothers who have recovered from COVID-19, with no such antibodies present in the controls. These antibodies are capable of neutralizing a clinical isolate of SARS-CoV-2 in vitro . We furthermore show that high pressure pasteurization hardly affects antibody levels and efficacy. We hypothesize that breastmilk, obtained from mothers who have recovered from COVID-19, may serve as a safe and widely applicable preventive strategy. Trial Registration: This research project was registered at the Dutch Trial Register on May 1st, 2020, number: NL 8575 Funding Statement: The study was funded by de Stichting Steun Emma Kinderziekenhuis. No payment by a pharmaceutical company or other agency was made. Declaration of Interests: The authors have no conflicts of interest to declare. Ethics Approval Statement: Ethical approval was obtained from the Medical Ethics Committee of Amsterdam UMC, location VUmc and written informed consent was obtained from all participants.

Although incredibly diverse in specificity, millions of unique Immunoglobulin G (IgG) molecules in the human antibody repertoire share most of their amino acid sequence. These constant parts of IgG do not yield any useful information in attempts to sequence antibodies de novo. Therefore, methods focusing solely on the variable regions and providing unambiguous sequence reads are strongly advantageous. We report a mass spectrometry-based method that uses electron capture dissociation (ECD) to provide straightforward-to-read sequence ladders for the variable parts of both the light and heavy chains, with a preference for the functionally important CDR3. We optimized this method on the therapeutic antibody Trastuzumab and demonstrate its applicability on two monoclonal quartets of the four IgG subclasses, IgG1, IgG2, IgG3 and IgG4. The method is based on proteolytically separating the variable F(ab')2 part from the conserved Fc part, whereafter the F(ab')2 portions are mass-analyzed and fragmented by ECD. Pure ECD, without additional collisional activation, leads to straightforward-to-read sequence tags covering the CDR3 of both the light and heavy chains. Using molecular modelling and structural analysis, we discuss and explain this selective fragmentation behavior and describe how structural features of the different IgG subclasses lead to distinct fragmentation patterns. Overall, we foresee that pure ECD on F(ab')2 or Fab molecules can become a valuable tool for the de novo sequencing of serum antibodies.

Grana are a characteristic feature of higher plants' thylakoid membranes, consisting of stacks of appressed membranes enriched in Photosystem II (PSII) and associated light-harvesting complex II (LHCII) proteins, together forming the PSII-LHCII supercomplex. Grana stacks undergo light-dependent structural changes, mainly by reorganizing the supramolecular structure of PSII-LHCII supercomplexes. LHCII is vital for grana formation, in which also PSII-LHCII supercomplexes are involved. By combining top-down and crosslinking mass spectrometry we uncover the spatial organization of paired PSII-LHCII supercomplexes within thylakoid membranes. The resulting model highlights a basic molecular mechanism whereby plants maintain grana stacking at changing light conditions. This mechanism relies on interactions between stroma-exposed N-terminal loops of LHCII trimers and Lhcb4 subunits facing each other in adjacent membranes. The combination of light-dependent LHCII N-terminal trimming and extensive N-terminal α-acetylation likely affects interactions between pairs of PSII-LHCII supercomplexes across the stromal gap, ultimately mediating membrane folding in grana stacks.