Sulfonation is important for bioactivity across species—substrates range from small molecules to proteins, in which almost exclusively tyrosine sulfonation (sTyr) has been investigated. Although sulfoalkyl-hydroxyl amino acid residues in peptides and proteins have rarely been found, we will argue that it is highly likely that sSer and sThr are much more abundant than is currently evident. In the context of the recent detection of sThr-containing adipokinetic hormones in insects, the literature was screened to answer questions about the relative stability (lability) of the sulfate moiety at Tyr, Ser and Thr that may explain the historic abundance of sTyr reports; to ascertain if there are special grounds for finding sSer and sThr peptides in insects, and whether specific enzymes (sulfotransferases and sulfatases) involved in the biochemistry of sulfonation are documented in insects. Finally, we assessed detection methods for sThr and sSer-containing peptides to achieve greater accuracy. Our literature screening could not supply a special reason for the preferential formation of sThr/sSer in insects. Although the analysis of sulfonated molecules is challenging because of the lability of the sulfate group, sThr/sSer do not seem to be at a greater disadvantage compared to sTyr; it was even experimentally found that they are more stable than commonly reported. For untargeted studies of sulfoproteomes, special enrichment strategies are still required to identify these sulfonated peptides. Mass spectrometry is very valuable for the detection of the modification, but methods need to be adjusted, because sulfur trioxide is easily lost during measurement.

Abstract Background The male breast cancer is a rare malignancy comprising for less than 1% of all breast cancers. The late and incorrect diagnosis of this cancer causes increased incidence rates of metastatic spread within patients. In Pakistan, the male breast cancer is frequently diagnosed at an average age of 55 to 58 years. Additionally, different risk factors including old age, hormonal imbalances, as well as family history showed association with this cancer. Moreover, recent literature showed that the neoadjuvant chemotherapy can result in pathologic complete responses in male breast cancer patients, yet its use in real-world practice is low, highlighting the gap in clinical implementation. Therefore, we present a clinical case report of male breast cancer to identify the critical importance of considering this malignancy in young males with breast masses and to discuss the unique challenges in their management. Case presentation We present the case of a 55-year-old normotensive and normoglycemic male office clerk with a four-months history of mild pain and lump in the left breast. He did not have a family history of breast cancer. Physical examination revealed a hard lump with overlying skin changes suggestive of malignancy. Incisional biopsies confirmed invasive ductal carcinoma, grade II with ER-positive, PR-negative and HER2/neu-negative status with no lymphovascular invasion. Imaging studies showed lobulated mass in the left breast with axillary lymphadenopathy in the ipsilateral axilla, renal calculus and benign lytic lesions. The patient was initially treated with planned neoadjuvant chemotherapy including four cycles of doxorubicin and cyclophosphamide scheduled over twelve weeks. However, a follow-up biopsy performed after nine weeks showed persistent invasive carcinoma. Consequently, the treatment plan was reevaluated subsequently. Because of the tumor’s ER-positive status and suboptimal response to neoadjuvant chemotherapy, the treatment plan was changed to endocrine therapy such as tamoxifen (10 mg twice daily). The patient was clinically stable during discharge and follow-up was advised. Conclusion This case report of male breast cancer highlights the unique aspects and challenges of diagnosing and treating cancer emphasizing the importance of early diagnosis, hormone receptor status for treatment planning and ongoing research to manage cancer effectively. Moreover, it also highlights the necessity of imaging in such cases as clinical examination may fail to pick significant findings. Similarly, absence of lymphovascular invasion on biopsy is not always associated with absent lymph node involvement on imaging. Moreover, this case report also demonstrated the standard diagnostic and therapeutic pathways for a typical case of ER-positive invasive ductal carcinoma in a male patient. This clinical case report highlights the crucial in recognizing chemotherapy-resistant disease and supporting the clinicians in the timely initiation of the endocrine therapy.

Emerging evidence suggests the involvement of the renin-angiotensin system (RAS) in the pathogenesis and progression of autoimmune dermatological diseases. In a small exploratory study, we investigated angiotensin-converting enzyme (ACE) activity in blood obtained from male probands with psoriasis vulgaris (n = 4) and atopic dermatitis (AD, n = 5). The degradation capacity of dabsylated synthetic bradykinin (DBK) with and without inhibition was determined using a thin-layer chromatography (TLC)-based neuropeptide reporter assay. We observed a significantly reduced capacity for cleavage of DBK in psoriasis patients compared with that in AD patients and controls. In patient samples, the variation in the measured values was generally greater than that in healthy controls. We could not confirm the increased ACE activity in the circulation in psoriasis patients reported by others, likely because of different study designs and detection methods. We did not include samples of female patients and focused on younger men to avoid hormonal effects and minimize age-related factors. This preliminary study of the hypothesis-generating nature lacks power, but it certainly adds a question to the current view of the role of ACE in psoriasis. Treatments targeting specific components of the RAS could ameliorate inflammatory responses; thus, research in this area is becoming increasingly important.

Peptides of the adipokinetic hormone family are responsible for metabolic roles in insects, regulating release of energy metabolites from the fat body. We report on adipokinetic hormone octapeptide sequences bearing a rarely identified post-translational modification (sulfation of a threonine residue) in two beetle subfamilies (Cetoniinae and Dynastinae) of the large superfamily of Scarabaeoidea (dung beetles, rhinoceros beetles and flower beetles), and in a bug species (family Coreidae). In the cetonids Pachnoda sinuata, Dicronorhina derbyana derbyana, Tropinota hirta, Protaetia cuprea, Cetonia aurata and Oxytherea funesta sulfated Pacsi-AKH is found (pQINLTsTGW amide), while sulfated Penid-AKH (pQVNISsTGW amide) occurs in the dynastid beetles Pentodon idiota, Xylotrupes gideon and Syrichthodontus spurius. Sulfated Schgr-AKH-II (pQLNFSsTGW amide) is found in the twig wilter Holopterna alata. Sequence elucidation was achieved by mass spectrometry, however, due to the labile nature of the sulfate group under mass spectrometric conditions, the modified amino acid could not be easily identified. Edman degradation and comparative mass spectrometry evaluations with synthetic sulfopeptide standards were therefore employed for sequence validation. This type of sulfation was previously only reported present on the protein backbone of very few proteins from vertebrates (including humans), a mollusc and a protozoan parasite.

BACKGROUND:Acute kidney injury (AKI) is a common complication in critically ill patients and frequently progresses to persistent renal dysfunction. However, mechanisms underlying renal recovery versus persistent dysfunction remain poorly understood. Plasma proteomic profiling may identify proteins and biological pathways associated with the development of acute kidney disease (AKD) subsequent to AKI. MATERIAL AND METHODS:We conducted a plasma proteomics study within a prospective, single-centre observational cohort of 205 critically ill adult patients in Germany with moderate AKI (KDIGO stage 2) and predefined risk factors for AKI progression (mechanical ventilation and/or vasopressor therapy). Plasma samples from 195 patients were available for analysis in sufficient quantity. The primary outcome was AKD, defined as persistent AKI (KDIGO stage 2 or 3) or death seven days after AKI diagnosis. High-resolution mass spectrometry quantified protein abundances in non-depleted and immunodepleted plasma samples. Proteins associated with AKD were assigned using biostatistical analyses and selected candidates were validated by enzyme-linked immunosorbent assay (ELISA) in the entire cohort. RESULTS:Out of 195 patients, 103 developed AKD (52.8%). Proteomic profiling assigned 29 proteins in undepleted plasma and 38 proteins in depleted plasma that were significantly associated with development of AKD. In undepleted plasma, gelsolin and Zinc-alpha-2-glycoprotein were prominent candidates. In depleted plasma, S100A9, a component of the Calprotectin complex, showed the strongest association with AKD development. ELISA validation demonstrated higher plasma concentrations of Calprotectin (S100A8/A9) in patients who progressed from AKI to AKD. CONCLUSIONS:Unbiased plasma proteomics revealed several proteins associated with development of AKD following moderate AKI in critically ill patients. Elevated Calprotectin (S100A8/A9) levels were independently associated with AKD development in ELISA analyses, suggesting a potential role in AKI chronification. Further studies are warranted to validate these findings and explore their mechanistic and clinical implications.

Maximal safe tumor resection is crucial for the treatment of high-grade gliomas (HGG). 5-aminolevulinic acid (5-ALA)-mediated fluorescence-guided surgery enhances tumor visualization by inducing protoporphyrin IX (PpIX) accumulation. However, current fluorescence-based observation devices lack the sensitivity for detecting tumor cells in low-density infiltrative zones. Hyperspectral imaging (HI) offers a potential solution. In this study, HI-derived PpIX measurements were compared to those obtained from reversed-phase liquid chromatography coupled to mass spectrometry (LC-MS), a method that delivers accurate concentrations. Additionally, we investigated coproporphyrins (Cp) I and III, since they potentially interfere with PpIX determination. Pig brain was used as a surrogate for protocol development and acquisition of comparative HI and LC-MS reference data, which were subsequently used to evaluate the results obtained from 27 biopsies from nine patients undergoing 5-ALA-mediated tumor resection. During sample preparation for LC-MS, 80% PpIX and 45% combined Cp I & III were recovered from brain tissue. For LC-MS quantification of PpIX, accuracy ranged from 98 to 137%, and coefficient of variation was 5-14%, indicating sufficient precision. For HI, the values were 77-121% and 11-31%, respectively. Notably, HI significantly overestimated PpIX concentrations compared to those determined by LC-MS. This study highlights LC-MS as a reliable method for porphyrin quantification and suggests that HI workflows need further optimization for accurate tumor delineation in HGG.

Multiple sclerosis (MS) is the most common inflammatory and demyelinating disease affecting the central nervous system (CNS). While immune-modulating drugs can prevent new lesions by targeting lymphocyte activity, treating relapse-independent disease progression remains challenging. Persisting CNS inflammation, leading to axonal and neuronal injury along with failure of compensatory mechanisms, such as brain plasticity and remyelination, drives disease progression. Thus, identifying neuroprotective and/or remyelination-promoting compounds is urgently needed. We developed an in vitro platform utilizing human-induced pluripotent stem cell (iPSC)-derived neurons and oligodendrocytes to assess neuroprotective and potentially promyelinating effects of selected compounds. We established assays mimicking MS pathophysiologies, such as neuronal loss and axonal injury. Proteomic analysis revealed modulation of molecular mechanisms. Findings were validated in an acute cuprizone (CPZ) mouse model. We demonstrated that pioglitazone and minocycline protected against glutamate-induced axonal injury, rotenone-induced neuronal death and promoted oligodendrocyte differentiation. Proteomic analyses suggest that pioglitazone's neuroprotective effect may involve reducing mitochondrial reactive oxygen species (ROS) production via PGC-1α and stabilizing axonal transport through GSK3β phosphorylation. Minocycline mainly impacted glutathione metabolism. In the cuprizone model, both compounds displayed neuroprotective effects but did not reduce demyelination or oligodendroglial loss. In summary, our findings demonstrate that human preclinical IPSC platforms can be used to characterize the neuroprotective properties of compounds and thus may aid the selection of drugs for clinical trials. Moreover, the platform's flexibility allows for the easy incorporation of additional disease-specific phenotypic assays.

Branched-chain C5-dicarboxylic acids (e.g., citramalate, mesaconate or methylsuccinate) and their CoA-esters are important intermediates in bacterial metabolism, while itaconate is an antimicrobial agent, a potent immunomodulator and a growth substrate for many bacteria. The itaconate degradation pathway consists of three reactions catalyzed by itaconate CoA transferase, itaconyl-CoA hydratase and (S)-citramalyl-CoA lyase encoded in a cluster, which in saprophytic bacteria contains two additional genes for a putative acyl-CoA dehydrogenase and a protein of the MmgE/PrpD family. Here, we heterologously produced the corresponding proteins from Cupriavidus necator and Pseudomonas aeruginosa and showed that they catalyze the (RS)-methylsuccinyl-C4-CoA dehydrogenase and an (S)-(R)-methylsuccinate isomerase reaction, respectively. Together with itaconate CoA transferase, which is highly active with (R)-methylsuccinate but has low activity with (S)-methylsuccinate, these enzymes allow the utilization of both stereoisomers of methylsuccinate. Our bioinformatic analysis revealed that 1.6% of the sequenced prokaryotes (mainly Betaproteobacteria) possess an identified methylsuccinate isomerase. Analysis of the conserved amino acids of methylsuccinate isomerase and other MmgE/PrpD proteins suggests that they share a common catalytic mechanism via the formation of an enolate intermediate. The presence of specific methylsuccinate utilization genes in the itaconate degradation cluster, which is widespread in saprophytic bacteria, suggests the importance of methylsuccinate in the environment.

Beetles are the largest animal group, in general. Phylogenetically, beetles belong to the order Coleoptera, the most species-rich of the Insecta. Coleoptera is divided into four suborders: Polyphaga, Adephaga, Archostemata, and Myxophaga. Specimens from the latter two are difficult to obtain, hence, we have focused our research into the adipokinetic hormone (AKH) peptide family on the former two suborders. Data on the Adephaga were concluded in 2017. The "core Polyphaga" consists of three series: Elateriformia, Staphyliniformia, and Cucujiformia; the latter was concluded in 2019. Here, we report on the AKH sequence(s) of 23 species of beetles from 4 families of Elateriformia, namely, the Buprestidae, Cantharidae, Elateridae, and Lampyridae; and 4 families of Staphyliniformia, namely, the Hydrophilidae, Silphidae, Lucanidae, and Scarabaeidae. Sequence elucidation by mass spectrometry or Edman degradation revealed 13 octapeptides: 5 are novel, 12 are beetle-specific and Schgr-AKH-II is produced in the basal Polyphaga (Elateriformia, in Hydrophilididae and Silphidae). Since Schgr-AKH-II is also found in Adephaga, this confirms the ancestral AKH of Coleoptera. The first change in sequence is recorded in Staphylinoidea with two different residues, notably, the switch from Phe4 to Tyr4. Duplication of AKH peptides is first seen in Lucanidae, as well as the appearance of atypical AKH sequences, such as Phe2, Met4, Leu4, or Phe7 encountered in the Scarabaeoidea. The vast majority of the pest beetles do not have beetle-specific AKHs or share the same AKH as nonharmful beetles. Ideas for finding a lead compound for green insecticides are discussed.

Strophanthus sarmentosus is recognised for various ethnomedicinal applications, including treatment after snakebites. However, only limited scientific evidence exists on its antivenomous capabilities. This study investigates the efficacy of methanol and ethylacetate extracts from S. sarmentosus leaves and roots against Echis ocellatus venom. A non-toxic range for the extracts was determined in rats, and assays were performed to test their anti-hemorrhagic and anti-hemolytic activity as well as their influence on venom-induced blood clotting. In all of these experiments, the extracts demonstrated significant positive effects equal to or better than antivenom. Moreover, the extracts strongly inhibited and even abolished the digestion of the vasoactive neuropeptide bradykinin by snake venom metalloproteinases. Strophantus plants are known for their high content of cardiac glycosides, one of which is the commercially available ouabain, that by itself also considerably inhibited venom-induced bradykinin cleavage. Although ouabain is only present in low amounts in S. sarmentosus when compared to other cardenolides of similar structure, it can be hypothesized that members of this substance class may also have inhibitory properties against venom proteases. S. sarmentosus additionally contains bioactive substances such as flavonoids, terpenoids, tannins, saponins, and alkaloids, which contribute to its protective effects. The study provides scientific data to explain the success of the traditional use of S. sarmentosus plant extracts as a first aid against envenomation in rural Africa.

Arabinogalactan protein (AGP) was isolated from the fruits of Crataegus monogyna and C. laevigata (Rosaceae) with 3.9 % polypeptides and a carbohydrate moiety composed of Gal (57 %), Ara (35 %), Rha (1 %), and GluA (3 %). Linkage analysis, enzymatic digestion with exo-β-(1,3)-galactanase and NMR studies indicate the presence of a highly branched 1,3,6-β-D-galactose backbone with 1,6-galactan side chains, modified by the presence of α-1,5- and 1,3-linked arabinose residues. The molecular weight was determined by size exclusion chromatography with 100.6 kDa and molecular dispersity Đ of 1.71 was calculated. Data indicate the presence of a high molecular and heterodispers AGP. PNGase F treatment generated proteins of about 30 kDa, indicating that most of the carbohydrate chains are linked via asparagine and GlcNac of oligomannose or hybrid N-glycans. Mass spectrometric analysis of the tryptic digest assigned peptides of fasciclin-like arabinogalactans, which represent a subclass of AGP, responsible for cell-cell adhesion, cell sorting and embryogenesis.

Campylobacter jejuni is the leading cause of severe enteritis worldwide. Identification of natural products against C. jejuni that inhibit early-stage host-pathogen interactions is a promising strategy, particularly by blocking bacterial adhesion to and invasion into the host cells. This should help to reduce the bacterial load in vector animals. From the seeds of Trigonella foenum-graecum , an aqueous extract (fenugreek high molecular fraction) of high-molecular compounds (polysaccharides, proteins) was obtained. The polysaccharide was characterized as galactomannan (1,4-mannose backbone, substituted at O -6 with single galactose residues and galactose oligosaccharide chains). The protein part consisted of 50 kDa vicilin as the main compound. The fenugreek high molecular fraction did not influence proliferation of C. jejuni and viability of Caco-2 cells (1 - 1000 mu g/mL). The fenugreek high molecular fraction reduced bacterial adhesion to Caco-2 significantly (500 - 1000 mu g/mL), which was due to an interaction with the bacterial adhesin JlpA, preventing the interaction of this outer membrane protein to its ligand HSP90 alpha (IC 50 = 23.4 mu g/mL). Bacterial invasion was reduced significantly. Both polysaccharides as well as vicilin contribute to the observed antiadhesive effect. As vicilin-like proteins are widely found in plants from the Fabaceae family, a vicilin-enriched protein preparation from Pisum sativum was investigated for antiadhesive activity. These findings suggest that fenugreek seeds or vicilin-rich plant extracts could be used to develop novel strategies to control C. jejuni infections in food-producing animals, ultimately helping to decrease the prevalence of campylobacteriosis in humans.

Background: Bradykinin (BK) is an inflammatory mediator. The degradation of labeled synthetic BK in biofluids can be used to report on the activity of angiotensin-converting enzyme (ACE) and basic carboxypeptidases N and CBP2, for which the neuropeptide is a substrate. Clinical studies have shown significant changes in the serum activity of these enzymes in patients with inflammatory diseases. Methods: Here, we investigated variation in the cleavage of dabsylated synthetic BK (DBK) in serum and the formation of the major enzymatic fragments using a thin-layer chromatography-based neuropeptide reporter assay (NRA) in a large cohort of healthy volunteers from the international human Personal Omics Profiling consortium based at Stanford University. Results: Four major outcomes were reported. First, a set of NRA reference data for the healthy population was delivered, which is important for future investigations of patient sera. Second, it was shown that the measured serum degradation capacity for DBK was significantly higher in males than in females. There was no significant correlation of the NRA results with ethnicity, body mass index or overnight fasting. Third, a batch effect was noted among sampling sites (HUPO conferences). Thus, we used subcohorts rather than the entire collection for data mining. Fourth, as the low-cost and robust NRA is sensitive to enzyme activity, it provides such a necessary quick test to eliminate degraded and/or otherwise questionable samples. Conclusions: The results reiterate the critical importance of a high level of standardization in pre-analytical sample collection and processing—most notably, sample quality should be evaluated before conducting any large and expensive omics analyses.

Desulfurella acetivorans is a strictly anaerobic sulfur-reducing deltaproteobacterium that can grow heterotrophically by oxidation of acetate or autotrophically with molecular hydrogen. Here we show that D. acetivorans possesses a putative operon encoding enzymes of the methylcitrate cycle of propionate oxidation and demonstrate that this bacterium is capable of propionate growth. However, activities of the methylcitrate cycle enzymes could not be detected in extracts of propionate-grown cells, and experiments with [U-13C3]propionate and comparative proteomic analysis of acetate- and propionate-grown cells suggested that the methylcitrate cycle is not active during propionate growth. Instead, propionyl-CoA assimilation proceeds via its carboxylation to methylmalonyl-CoA, which is further converted to succinyl-CoA. The latter is directed to the tricarboxylic acid (TCA) cycle, where it is converted to oxaloacetate and condenses with acetyl-CoA (produced by decarboxylation of another oxaloacetate molecule) to form citrate, which is oxidized in the TCA cycle. These results highlight the uncertainty of genomic predictions in the analysis of microbial metabolic pathways and the need for their experimental confirmation.

High-grade gliomas (HGG) carry a dismal prognosis. Diagnosis comprises MRI followed by histopathological evaluation of tissue; no blood biomarker is available. Patients are subjected to serial MRIs and, if unclear, surgery for monitoring of tumor recurrence, which is laborious. MRI provides only limited diagnostic information regarding the differentiation of true tumor progression from therapy-associated side effects. 5-aminolevulinic acid (5-ALA) is routinely used for induction of protoporphyrin IX (PpIX) accumulation in malignant glioma tissue, enabling improved tumor visualization during fluorescence-guided resection (FGR). We investigated whether PpIX can also serve as a serum HGG marker to monitor relapse. Patients (HGG: n = 23 primary, pHGG; n = 5 recurrent, rHGG) undergoing FGR received 5-ALA following standard clinical procedure. The control group of eight healthy volunteers (HCTR) also received 5-ALA. Serum was collected before and repeatedly up to 72 h after drug administration. Significant PpIX accumulation in HGG was observed after 5-ALA administration (ANOVA: p = 0.005, post-hoc: HCTR vs. pHGG p = 0.029, HCTR vs. rHGG p = 0.006). Separation of HCTR from pHGG was possible when maximum serum PpIX levels were reached (CI95% of tMax). ROC analysis of serum PpIX within CI95% of tMax showed successful classification of HCTR and pHGG (AUCROC 0.943, CI95% 0.884–1.000, p < 0.001); the optimal cut-off for diagnosis was 1275 pmol PpIX/ml serum, reaching 87.0% accuracy, 90.5% positive predictive and 84.0% negative predictive value. Baseline PpIX level was similar in patient and control groups. Thus, 5-ALA is required for PpIX induction, which is safe at the standard clinical dosage. PpIX is a new target for liquid biopsy in glioma. More extensive clinical studies are required to characterize its full potential.

The genus Strophantus belongs to the Apocynaceae family of flowering plants which grows primarily in tropical Africa. The plants are widely used in traditional herbal medicine. S. sarmentosus, in particular, is used for the treatment of, e.g., joint pain and rheumatoid arthritis, wound infections, head lice, diarrhea, snake bite, and eye conditions. Despite its widespread use, dedicated research characterizing its bioactive plant components is scarce. Investigations have focused mainly on its cardenolides because of their cardioactivity and historical use as cardiotonic. There are also studies concerning the antibacterial, antioxidant, and anti-inflammatory activity of plant extracts. This review summarizes the present knowledge surrounding the biochemical and analytical research on Strophantus, in general, and S. sarmentosus, in particular, and describes the current state of the field based on the available scientific literature.

Myasthenia gravis is a chronic antibody-mediated autoimmune disease disrupting neuromuscular synaptic transmission. Informative biomarkers remain an unmet need to stratify patients with active disease requiring intensified monitoring and therapy; their identification is the primary objective of this study. We applied mass spectrometry-based proteomic serum profiling for biomarker discovery. We studied an exploration and a prospective validation cohort consisting of 114 and 140 anti-acetylcholine receptor antibody (AChR-Ab)-positive myasthenia gravis patients, respectively. For downstream analysis, we applied a machine learning approach. Protein expression levels were confirmed by ELISA and compared to other myasthenic cohorts, in addition to myositis and neuropathy patients. Anti-AChR-Ab levels were determined by a radio receptor assay. Immunohistochemistry and immunofluorescence of intercostal muscle biopsies were employed for validation in addition to interactome studies of inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3). Machine learning identified ITIH3 as potential serum biomarker reflective of disease activity. Serum levels correlated with disease activity scores in the exploration and validation cohort and were confirmed by ELISA. Lack of correlation between anti-AChR-Ab levels and clinical scores underlined the need for biomarkers. In a subgroup analysis, ITIH3 was indicative of treatment responses. Immunostaining of muscle specimens from these patients demonstrated ITIH3 localization at the neuromuscular endplates in myasthenia gravis but not in controls, thus providing a structural equivalent for our serological findings. Immunoprecipitation of ITIH3 and subsequent proteomics lead to identification of its interaction partners playing crucial roles in neuromuscular transmission. This study provides data on ITIH3 as a potential pathophysiological-relevant biomarker of disease activity in myasthenia gravis. Future studies are required to facilitate translation into clinical practice.

Background The clinical heterogeneity of myasthenia gravis (MG), an autoimmune disease de fi ned by antibodies (Ab) directed against the postsynaptic membrane, constitutes a challenge for patient strati fi cation and treatment decision making. Novel strategies are needed to classify patients based on their biological phenotypes aiming to improve patient selection and treatment outcomes. Methods For this purpose, we assessed the serum proteome of a cohort of 140 patients with anti-acetylcholine receptorAb-positive MG and utilised consensus clustering as an unsupervised tool to assign patients to biological pro fi les. For in-depth analysis, we used immunogenomic sequencing to study the B cell repertoire of a subgroup of patients and an in vitro assay using primary human muscle cells to interrogate serum-induced complement formation. Findings This strategy identi fi ed four distinct patient phenotypes based on their proteomic patterns in their serum. Notably, one patient phenotype, here named PS3, was characterised by high disease severity and complement activation as de fi ning features. Assessing a subgroup of patients, hyperexpanded antibody clones were present in the B cell repertoire of the PS3 group and effectively activated complement as compared to other patients. In line with their disease phenotype, PS3 patients were more likely to bene fi t from complement-inhibiting therapies. These fi ndings were validated in a prospective cohort of 18 patients using a cell-based assay. Interpretation Collectively, this study suggests proteomics-based clustering as a gateway to assign patients to a biological signature likely to bene fi t from complement inhibition and provides a strati fi cation strategy for clinical practice.