Madden, Benjamin J.

ABSTRACT Background Membranous nephropathy (MN) is a leading cause of nephrotic syndrome (NS). Since the identification of anti–phospholipase A2 receptor (anti-PLA2R) antibodies in 2009, the use of laser microdissection and tandem mass spectrometry (LMD/MS) has allowed the discovery of several target antigens in MN. Methods In this retrospective cohort study, adult patients evaluated at the Division of Nephrology at Brotzu Hospital (Cagliari, Italy) with biopsy-proven MN and a negative serological test for anti-PLA2R antibody underwent LMD/MS, performed at the Department of Laboratory Medicine and Pathology of Mayo Clinic (Rochester, MN, USA). Results Twenty-four cases of biopsy-proven MN were available for antigen detection by LMD/MS studies. High total spectral counts of PLA2R were detected in 12 out of 24 (50%) cases. In addition, high spectral counts of THSD7A and NELL1 were detected in two cases each, and EXT1/EXT2 and NCAM1 in one case each. Five putative antigens have been detected: SULF1, PGLYRP, HYAL1, THBS and SEZ6L2. Conclusions Our study highlights at least two interesting considerations. First, the determination of PLA2R on renal tissue in the diagnosis of PLA2R-associated MN is emphasized since 50% of our cases were falsely diagnosed with PLA2R-negative MN based on the serum anti-PLA2R antibodies determination. Second, our study shows six patients with MN likely associated with putative antigens, two of them showing new antigens never described before in literature (HYAL1 and THBS1). This high prevalence of putative antigens in our cohort is not easily explainable and paves the way for evaluating specific factors in the Sardinian population that could explain this evidence.

AbstractBackgroundTandem mass spectrometry (MS/MS) has become a common clinical laboratory testing modality has demonstrated success in distinguishing between small protein variations in transthyretin amyloidosis. Since many common clinically significant RBC antigens are also small protein variations, this study aimed to determine if MS/MS could correctly detect common RBC antigens within the Rh, Kell, Duffy, MNS, Kidd, Diego, and Lutheran blood group systems.Study Design and MethodsResidual samples from serotyped/genotyped blood donors at a hospital‐based blood donation center from February to August 2021 were analyzed. RBC membrane protein preparations underwent protease digestion prior to MS/MS analysis for RhD, RhCE, Kell, atypical chemokine receptor 1 (Duffy), glycophorin A (MNS), glycophorin B (MNS), urea transporter 1 (Kidd), band 3 anion transport protein (Diego), and basal cell adhesion molecule (Lutheran). Untargeted liquid chromatography (LC)‐MS/MS detected protein‐specific peptides, while targeted LC‐selected reaction monitoring (LC‐SRM) detected RBC antigen‐containing peptides.ResultsThrough the use of multiple proteases, untargeted LC–MS/MS detected protein‐specific peptides in all but glycophorin B, with band 3 anion transport protein, basal cell adhesion molecule, Kell, and glycophorin A having the greatest protein sequence coverage. Targeted LC‐SRM detected antigen‐specific peptides for Cw, Dia/Dib, Kpb, and Wra/Wrb, which agreed with the donors' previous typing results.DiscussionMS/MS can successfully detect peptides from several blood group systems but only detected a subset of their common RBC antigens. Further sample enrichment and MS/MS detection improvements will need to occur before MS/MS could be considered a clinical RBC phenotyping modality.

Introduction: Extracranial carotid artery pathology accounts for 15-20% of ischemic strokes. Advancements in magnetic resonance angiography (MRA) with vessel wall imaging (VWI) have enabled the identification of vulnerable plaques, aiding in risk stratification for neurovascular events. This pilot study aimed to identify proteins in plaques with and without vulnerable features on MRA with VWI. Hypothesis: There is a differential proteomic profile of vulnerable and non-vulnerable, thus, these can serve as potential biomarkers for disease progression, stroke prevention, and therapeutic targets. Methods: Consecutive patients undergoing carotid endarterectomy (CEA) were included in the study cohort with pre-operative MRA with VWI. A retrospective chart review was conducted to extract pertinent clinical data including cardiovascular risk factors and medications. The proteomic analysis involved Tandem Mass Tag (TMTpro) labeling of peptides, basic pH HPLC fractionation, and NanoLC-tandem mass spectrometry. Results: A total of sixteen plaques were retrieved from fifteen patients. There were 73.3% male patients. The most common comorbidities were hyperlipidemia (93.3%) and hypertension (80%). 31.2% of patients had symptomatic disease. Eleven plaques were vulnerable in preoperative MRI with VWI. From those, intraplaque hemorrhage was the most common feature (81.1%) . Proteomic analysis revealed 23 relevant proteins significantly elevated in vulnerable plaques, including Proteinase 3 (PRTN3), Phospholipid Transfer Protein (PLTP), and S100 Calcium-Binding Protein A12 (S100A12), with increased abundance exceeding two-fold changes or above (P<0.001). Conversely, three proteins exhibited reduced abundance in vulnerable plaques including Dynamin-3 (DNM3), Transmembrane Protein 181 (TMEM181), and Adducin-3 (ADD3) (P<0.05). All results are summarized in Figure 1A-C and Table I and II. Conclusion: This study contributes to the understanding of protein biomarkers associated with carotid plaque vulnerability, offering insights into disease progression, stroke prevention, and identification of potential therapeutic targets (Figure 1D) . Furthermore, these patients could benefit from early surgical intervention if these biomarkers were to be detected. We present a comprehensive study of multiple proteins with significant bioavailability in explanted plaque specimens that can serve as a foundation for identifying blood biomarkers for future validation studies.

AbstractBackgroundUFMylation is an understudied ubiquitin‐like post‐translational modification (PTM). Like ubiquitin, UFM1 is conjugated to substrates via a catalytic cascade involving a UFM1‐specific E1 (UBA5), E2 (UFC1), and an E3 ligase complex (UFL1, DDRGK1 and CDK5RAP3). UFMylation is reversible, and this is mediated by UFSP2. UFMylation plays essential roles in several biological processes including the DNA damage response, endoplasmic reticulum homeostasis, unfolded protein response, autophagic functions, and immune response. Importantly, UFMylation is also crucial for healthy brain development. Alzheimer’s disease (AD), a complex neurodegenerative disorder, is characterized by the accumulation of pathologic tau and beta‐amyloid proteins and has been associated with abnormalities in the aforementioned processes. Intriguingly, the potential link between AD and UFMylation has not been investigated yet. Considering that pathological tau plays a critical role in AD, and tau also plays an important role for the DNA damage response in neurons under physiological conditions, we speculated that tau itself could be modified with UFM1.MethodWe performed Western blot and Meso Scale Discovery based ELISA to measure the protein level of UFM1, UFSP2 and other UFMylation pathway components in RIPA‐soluble and insoluble fraction in human post‐mortem frontal cortex. To test whether tau can be modified by UFM1, we performed co‐immunoprecipitation (Co‐IP), Proximity Ligation Assays (PLA) and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS).ResultWe found a significant reduction of soluble UFSP2, but an increase of soluble total UFM1 in AD compared to controls. We found that tau interacts with UFL1 and DDRGK1. In addition, using immunoprecipitation of denaturing lysates we found that tau can indeed be covalently modified by UFM1 when overexpressed. We detected strong signal of HA‐UFM1‐tau in neurons but not in negative controls using PLA. Furthermore, we immunoprecipitated denatured UFM1 from human autopsy brain, and detected two specific UFM1‐tau bands with tau antibodies. Together, this data strongly indicates that tau is UFMylated under endogenous conditions. Using LC‐MS/MS, we identified 17 UFMylation sites in tau proteins.ConclusionUFMylation is dysregulated in AD; tau protein can be modified by UFM1 both in vitro and in vivo, which enables a more comprehensive understanding of tau PTMs.