Introduction Idiopathic Multicentric Castleman Disease (iMCD) is a polyclonal lymphoproliferative disorder involving cytokine storms that can lead to organ failure and death. The cause of iMCD is unknown, but some clinical evidence suggests an autoimmune etiology. For example, connective tissue disorders (CTDs) and iMCD share many clinical features, and autoantibodies have been anecdotally reported in individual iMCD patients. This study investigates whether common autoantibodies are shared across iMCD patients.Methods We assembled custom bead-based protein arrays consisting of 52 autoantigens traditionally associated with CTDs and 38 full-length cytokines and screened serum samples from 101 iMCD patients for IgG autoantibodies. We also screened samples with a 1,103-plex array of recombinant human protein fragments to identify additional autoantibody targets. Finally, we performed receptor blocking assays on select samples with anti-cytokine autoantibodies (ACAs) identified by array.Results We found that an increased proportion of iMCD patients (47%) tested positive for at least one CTD-associated autoantibody compared to healthy controls (HC) (17%). Commonly detected CTD-associated autoantibodies were associated with myositis and overlap syndromes as well as systemic lupus erythematosus (SLE) and Sj & ouml;gren's Syndrome (SS). ACAs were also detected in a greater proportion of iMCD patients (38%) compared to HC (10%), while the protein fragment array identified a variety of other autoantibody targets. One iMCD sample tested positive for receptor blocking against interferon-omega (IFN omega).Discussion IgG autoantibodies binding autoantigens associated with common CTDs and cytokines are elevated in iMCD patients compared to HC, suggesting that autoimmunity may be involved in iMCD pathogenesis.

Receptor activity-modifying proteins (RAMPs) form complexes with G protein-coupled receptors (GPCRs) and may regulate their cellular trafficking and pharmacology. RAMP interactions have been identified for about 50 GPCRs, but only a few GPCR-RAMP complexes have been studied in detail. To elucidate a comprehensive GPCR-RAMP interactome, we created a library of 215 dual epitope-tagged (DuET) GPCRs representing all GPCR subfamilies and coexpressed each GPCR with each of the three RAMPs. Screening the GPCR-RAMP pairs with customized multiplexed suspension bead array (SBA) immunoassays, we identified 122 GPCRs that showed strong evidence for interaction with at least one RAMP. We screened for interactions in three cell lines and found 23 endogenously expressed GPCRs that formed complexes with RAMPs. Mapping the GPCR-RAMP interactome expands the current system-wide functional characterization of RAMP-interacting GPCRs to inform the design of selective therapeutics targeting GPCR-RAMP complexes.

Background Self-sampling of dried blood spots (DBS) offers new routes to gather valuable health-related information from the general population. Yet, the utility of using deep proteome profiling from home-sampled DBS to obtain clinically relevant insights about SARS-CoV-2 infections remains largely unexplored.Methods Our study involved 228 individuals from the general Swedish population who used a volumetric DBS sampling device and completed questionnaires at home during spring 2020 and summer 2021. Using multi-analyte COVID-19 serology, we stratified the donors by their response phenotypes, divided them into three study sets, and analyzed 276 proteins by proximity extension assays (PEA). After normalizing the data to account for variances in layman-collected samples, we investigated the association of DBS proteomes with serology and self-reported information.Results Our three studies display highly consistent variance of protein levels and share associations of proteins with sex (e.g., MMP3) and age (e.g., GDF-15). Studying seropositive (IgG+) and seronegative (IgG-) donors from the first pandemic wave reveals a network of proteins reflecting immunity, inflammation, coagulation, and stress response. A comparison of the early-infection phase (IgM+IgG-) with the post-infection phase (IgM-IgG+) indicates several proteins from the respiratory system. In DBS from the later pandemic wave, we find that levels of a virus receptor on B-cells differ between seropositive (IgG+) and seronegative (IgG-) donors.Conclusions Proteome analysis of volumetric self-sampled DBS facilitates precise analysis of clinically relevant proteins, including those secreted into the circulation or found on blood cells, augmenting previous COVID-19 reports with clinical blood collections. Our population surveys support the usefulness of DBS, underscoring the role of timing the sample collection to complement clinical and precision health monitoring initiatives. The COVID-19 pandemic has posed multiple challenges to healthcare systems. A significant gap that remains is a lack of understanding of the impact of SARS-CoV-2 on individuals who did not seek or require hospitalization. To address this, we distribute self-sampling devices to random citizens, aiming to analyze how blood protein levels are affected in people who have had COVID-19 but had no or mild symptoms. Conducting multiple molecular measurements in dried blood, our study confirms clinically known markers and their relationship to infection stages, even if the donors themselves collect the sample. Our work highlights the potential of combining self-sampling with laboratory methods to provide useful information on human health. This convenient patient-centric sampling approach may potentially be useful when studying other diseases. Fredolini et al. present a proteomics analysis of home-sampled dried blood spots taken from the general population in Stockholm during the COVID-19 pandemic. The study provides insights into the molecular effects of SARS-CoV-2 infection in non-hospitalized individuals and demonstrates the compatibility of self-sampled blood spots with proteomics.

Cholestatic itch is a severe and debilitating symptom in liver diseases with limited treatment options. The class A G proteincoupled receptor (GPCR) Mas-related GPCR subtype X4 (MRGPRX4) has been identified as a receptor for bile acids, which are potential cholestatic pruritogens. An increasing number of GPCRs have been shown to interact with receptor activity-modifying proteins (RAMPs), which can modulate different aspects of GPCR biology. Using a combination of multiplexed immunoassay and proximity ligation assay, we show that MRGPRX4 interacts with RAMPs. The interaction of MRGPRX4 with RAMP2, but not RAMP1 or 3, causes attenuation of basal and agonist-dependent signaling, which correlates with a decrease of MRGPRX4 cell surface expression as measured using a quantitative NanoBRET pulse-chase assay. Finally, we use AlphaFold Multimer to predict the structure of the MRGPRX4-RAMP2 complex. The discovery that RAMP2 regulates MRGPRX4 may have direct implications for future drug development for cholestatic itch.

G protein-coupled receptors (GPCRs) control critical cellular signaling pathways. Therapeutic agents including anti-GPCR antibodies (Abs) are being developed to modulate GPCR function. However, validating the selectivity of anti-GPCR Abs is challenging because of sequence similarities among individual receptors within GPCR sub-families. To address this challenge, we developed a multiplexed immunoassay to test >400 anti-GPCR Abs from the Human Protein Atlas targeting a customized library of 215 expressed and solubilized GPCRs representing all GPCR subfamilies. We found that-61% of Abs tested were selective for their intended target,-11% bound off -target, and-28% did not bind to any GPCR. Antigens of on-target Abs were, on average, significantly longer, more disordered, and less likely to be buried in the interior of the GPCR protein than the other Abs. These results provide important insights into the immunogenicity of GPCR epitopes and form a basis for designing therapeu-tic Abs and for detecting pathological auto-Abs against GPCRs.

Protein biomarkers in biological fluids represent an important resource for improving the clinical management of diseases. Current proteomics technologies are capable of performing high-throughput and multiplex profiling in different types of fluids, often leading to the shortlisting of tens of candidate biomarkers per study. However, before reaching any clinical setting, these discoveries require thorough validation and an assay that would be suitable for routine analyses. In the path from biomarker discovery to validation, the performance of the assay implemented for the intended protein quantification is extremely critical toward achieving reliable and reproducible results. Development of robust sandwich immunoassays for individual candidates is challenging and labor and resource intensive, and multiplies when evaluating a panel of interesting candidates at the same time. Here we describe a versatile pipeline that facilitates the systematic and parallel development of multiple sandwich immunoassays using a bead-based technology. 

Serological testing is essential to curb the consequences of the COVID-19 pandemic. However, most assays are still limited to single analytes and samples collected within healthcare. Thus, we establish a multianalyte and multiplexed approach to reliably profile IgG and IgM levels against several versions of SARS-CoV-2 proteins (S, RBD, N) in home-sampled dried blood spots (DBS). We analyse DBS collected during spring of 2020 from 878 random and undiagnosed individuals from the population in Stockholm, Sweden, and use classification approaches to estimate an accumulated seroprevalence of 12.5% (95% CI: 10.3%-14.7%). This includes 5.4% of the samples being IgG(+)IgM(+) against several SARS-CoV-2 proteins, as well as 2.1% being IgG(-)IgM(+) and 5.0% being IgG(+)IgM(-) for the virus' S protein. Subjects classified as IgG(+) for several SARS-CoV-2 proteins report influenza-like symptoms more frequently than those being IgG(+) for only the S protein (OR=6.1; p<0.001). Among all seropositive cases, 30% are asymptomatic. Our strategy enables an accurate individual-level and multiplexed assessment of antibodies in home-sampled blood, assisting our understanding about the undiagnosed seroprevalence and diversity of the immune response against the coronavirus. Here, Roxhed et al. develop a multiplexed approach to screen IgG and IgM levels against several SARS-CoV-2 proteins in home-sampled dried blood spots and estimate seroprevalence of 12.5% in Stockholm in spring of 2020.

Background: Precision medicine approaches aim to tackle diseases on an individual level through molecular profiling. Despite the growing knowledge about diseases and the reported diversity of molecular phenotypes, the descriptions of human health on an individual level have been far less elaborate. Methods: To provide insights into the longitudinal protein signatures of well-being, we profiled blood plasma collected over one year from 101 clinically healthy individuals using multiplexed antibody assays. After applying an antibody validation scheme, we utilized > 700 protein profiles for in-depth analyses of the individuals’ short-term health trajectories. Findings: We found signatures of circulating proteomes to be highly individual-specific. Considering technical and longitudinal variability, we observed that 49% of the protein profiles were stable over one year. We also identified eight networks of proteins in which 11–242 proteins covaried over time. For each participant, there were unique protein profiles of which some could be explained by associations to genetic variants. Interpretation: This observational and non-interventional study identifyed noticeable diversity among clinically healthy subjects, and facets of individual-specific signatures emerged by monitoring the variability of the circulating proteomes over time. To enable more personal hence precise assessments of health states, longitudinal profiling of circulating proteomes can provide a valuable component for precision medicine approaches. Funding: This work was supported by the Erling Persson Foundation, the Swedish Heart and Lung Foundation, the Knut and Alice Wallenberg Foundation, Science for Life Laboratory, and the Swedish Research Council.

Proteins are essential macromolecules that carry out complex functions in human cells, tissues, and organs. They regulate a diverse set of biological processes and protect against pathogens. However, dysregulation or malformation of proteins can cause disease. By characterizing proteins in health and disease, we can gain insights into disease aetiology and identify druggable targets to treat disorders. By bringing protein discoveries from the research lab into clinical practice, protein assays have been and will continue to be important tools for enabling and improving medical decision-making.

The work presented in this thesis concerns both exploratory and targeted affinity-based assays applied for the study of proteins. High-throughput and multiplexed suspension bead arrays have been the primary technology for measuring proteins with antibodies in samples such as human blood. Identification and validation of protein-protein interactions that may provide novel insights into the druggable proteome have also been carried out. Throughout the projects, methods for validating the observations have been pursued and include replication in independent sample sets, as well as the assessment of antibody selectivity via other proteomics assays or orthogonal methods such as genetic associations.

In Paper I, we used multiplexed exploratory antibody arrays comprising almost 1.500 affinity binders to study proteins that circulate in plasma. Here, the focus was to determine the longitudinal variability of proteins. We analysed samples from 101 clinically healthy individuals, collected each third month for one year. The protein data provided insights into inter-individual diversity and the unique molecular fingerprint of each participant. We found that 49% of the studied proteins were stable across one year, as these had low variability in each individual. Eight modules, each containing 11-242 proteins, were found to co-vary across one year. We also found genetic variations to influence 15 of the detected protein profiles and confirmed selected indications in an independent set of 3.000 subjects. In summary, we observed the existence of individual-specific protein profiles and found that short-term and continuous changes occurred in almost every participant.

In Paper II, we investigated blood-derived serum and plasma to identify age-associated proteins. We started from a large set of exploratory antibody bead arrays to screen 156 individuals aged 50-92 years. We found protein profiles of the histidine-rich glycoprotein (HRG) to be significantly associated with age. This association was further corroborated by the analysis of >4.000 individuals from eight additional and independent sets of blood samples. We further validated the HRG protein profiles by sandwich assays and protein microarrays developed in-house. Comparing genetic data and HRG profiles obtained by two independent antibodies, we observed strong but inverse associations to the genetic variants for two anti-HRG antibodies.

In Paper III, we applied multiplexed assays for the detection of autoantibodies against cancer-testis antigens (CTAs) in 133 non-small cell lung cancer (NSCLC) patients. We found reactivity against 29 unique CTAs exclusively in cases, compared to 57 matched controls with benign lung diseases. The presence of six CTAs was further confirmed in an independent set of 34 NSCLC cases. Analysis of longitudinal samples from seven patients demonstrated that the presence of CTA autoantibodies was stable over time for each of the individuals.

In Paper IV, we developed a novel multiplexed sandwich-immunoassay for the detection of interaction partners to G-protein coupled receptors (GPCRs). This pharmaceutically important family of membrane proteins is believed to be regulated by another group of receptor activity-modulating proteins (RAMPs) by the formation of protein complexes. We studied cell lysates expressing combinations of 23 GPCRs with three RAMPs. We confirmed most of the previously reported interaction pairs and additionally found evidence for 15 new GPCR-RAMP complexes. All interactions were validated using epitope tags that were engineered onto the proteins. Selected complexes were further validated by in situ proximity ligation assays performed in cell membranes.

In summary, the work included in this thesis describes the use of multiplexed affinity-based assays for research within plasma proteomics and the interrogation of protein complexes. The work highlights the method’s potential for the identification of circulating proteins that may aid and add to the current knowledge about human health and disease.

Proteiner är makromolekyler som utför essentiella funktioner i människans celler, vävnader, och organ. De deltar i många olika biologiska processer och kan exempelvis skydda mot patogen, så som bakterier och virus. Proteiner är en av kroppens viktigaste byggstenar och förändringar i deras aktivitet kan leda till sjukdom. Genom att studera proteiner i friska och sjuka individer kan vi få en bättre inblick om de bakomliggande molekylära processer som orsakar sjukdom, samt identifiera målprotein för läkemedelsutveckling. Proteinanalys har varit och kommer att fortsätta vara ett viktigt verktyg inom sjukvården.

Arbetet i denna avhandling berör affinitetsbaserade metoder för proteinanalys. Antikroppsarrayer med hög kapacitet att mäta många proteiner parallellt har tillämpats för att studera proteiner i blod. Dessutom har metoden använts för att identifiera och validera proteininteraktioner som kan vara relevanta för läkemedelsstrategier. Forskningsprojekten som presenteras här har ämnat att validera de undersökningarna som utförts genom att bland annat replikera resultaten i olika patientprov. Antikropparnas selektivitet har bekräftats genom jämförelser av olika proteinanalyser, antikroppsfria metoder, samt genetisk variation.

I Paper I tillämpades en antikroppsarray bestående av 1.500 antikroppar för att studera proteiner som cirkulerar i blodplasma. Huvudsyftet var att undersöka hur proteinnivåer varierar över tid. Vi analyserade prov från 101 kliniskt friska individer som donerat blod var tredje månad under ett års tid. Proteindata visade att det fanns en inter-individuell mångfald och att varje individ har ett unikt proteinbaserat ”fingeravtryck”. Vidare fann vi att 49% av de studerade proteiner hade låg variabilitet inom varje individ och var stabila under året. Vi identifierade åtta grupper bestående av 11-242 proteiner som samordnat varierade under ett år. Femton proteiner var associerade till genetisk variation och ett urval av dessa bekräftades i en separat studie som inkluderade 3.000 personer. Sammanfattningsvis fann vi att varje individ har en personlig och unik proteinprofil som är mestadels stabil under ett år, samt att det förkommer både kortsiktiga och långsiktiga förändringar i proteinuttrycket hos varje individ.

I Paper II analyserades proteiner som cirkulerar i blod och hur de är associerade till åldrande. Med hjälp av antikroppsarrayer undersöktes 156 individer i åldrarna 50-92 år och en association till åldrande identifierades för histidine-rich glycoprotein (HRG). Detta samband bekräftades även i en utökad analys av >4.000 blodprov från åtta separata kohorter. Vidare utvecklades en separat sandwich assay analysmetod för att utvärdera HRG-antikroppens affinitet. HRG-nivåer i blod som uppmättes med två olika antikroppar påvisade även stark association till en genetisk variant av HRG.

I Paper III studerades förekomsten av autoantikroppar mot cancer testis antigens (CTAs) i 133 patienter med icke-småcellig lungcancer (NSCLC). Vi identifierade reaktivitet mot 29 unika CTAs i patienter med NSCLC som ej påvisade reaktivitet i 57 prov från individer med godartade lungsjukdomar. Reaktiviteten för sex av dessa CTAs kunde bekräftas i ytterligare 34 NSCLC-patienter. Analys av longitudinella prover från sju patienter påvisade att uttrycket av CTA-autoantikroppar var stabilt under studieperioden för samtliga patienter.

I Paper IV utvecklades en ny antikroppsbaserad analysmetod för detektion av proteiner som bildar komplex med G-proteinkopplade receptorer (GPCRs). Denna familj av membranprotein är viktig för många läkemedel. Det finns underlag för att GPCRs funktioner kan regleras via receptor activity-modulating proteins (RAMPs), en annan grupp av proteiner som kan bilda komplex med GPCRs. Med den nya analysmetoden studerade vi 23 stycken GPCRs i kombination med tre stycken RAMPs i cellysat. Vi kunde bekräfta majoriteten av tidigare rapporterade komplex, och kunde vidare identifiera ytterligare 15 helt nya GPCR-RAMP-komplex. Ett urval av interaktionerna validerades med hjälp av epitoptaggar på proteinerna, samt med hjälp av in situ proximity ligation assays.

Sammanfattningsvis beskriver arbetet i denna avhandling användning av en affinitetsbaserad metod för proteinforskning i blodplasma, samt undersökning av proteininteraktioner. Studierna belyser metodens potential för identifikation av cirkulerande proteiner som kan komma att addera kunskap till det vi idag känner till om hälsa och sjukdom.