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Isoaspartate (isoAsp) is a damaging amino acid residue formed in proteins as a result of spontaneous deamidation. IsoAsp disrupts protein structures, making them prone to aggregation. Here we strengthened the link between isoAsp and Alzheimer's disease (AD) by novel approaches to isoAsp analysis in human serum albumin (HSA), the most abundant blood protein and a major carrier of amyloid beta (Aβ) and phosphorylated tau (p-tau) in blood. We discovered a reduced amount of anti-isoAsp antibodies (P < 0.0001), an elevated isoAsp level in HSA (P < 0.001), more HSA aggregates (P < 0.0001), and increased levels of free Aβ (P < 0.01) in AD blood compared to controls. We also found that deamidation significantly reduces HSA capacity to bind with Aβ and p-tau (P < 0.05). These suggest the presence in AD of a bottleneck in clearance of Aβ and p-tau, leading to their increased concentrations in the brain and facilitating their aggregations there.

Spontaneous deamidation of asparaginyl residues in proteins, if not repaired or cleared, can set in motion a cascade that leads to deteriorated health. Previously, we have discovered that deamidated human serum albumin (HSA) is elevated in blood of patients with Alzheimer’s disease and other neurodegenerative diseases, while the level of endogenous antibodies against deamidated HSA is significantly diminished, creating an imbalance between the risk factor and the defense against it. Endogenous antibodies against deamidated proteins are still unexplored. In the current study, we employed the SpotLight proteomics approach to identify novel amino acid sequences in antibodies specific to deamidated HSA. The results provide new insights into the clearance mechanism of deamidated proteins, a possible avenue for prevention of neurodegeneration.

O-GalNAc type glycosylation is a common post-translational modification (PTM) of proteins catalyzed by polypeptide GalNAc transferases, but the substrate specificity of these transferases is poorly understood. Here we develop a strategy based on integral thermal proteome solubility profiling to identify and prioritize the protein substrates of polypeptide N-acetylgalactosaminyltransferase 1 (GALNT1). Combined with glycoprotein enrichment followed by HCD and soft EThcD gas-phase fragmentation technique, we uncover hundreds of novel GALNT1 substrates in two model human cell lines. GALNT1-mediated O-glycosylation is more common on Thr than Ser residues, with a strong preference for Pro at positions +3 and +4 in respect to O-glycosylation. These results implicate GALNT1 in potentially regulating proteins in several diverse pathways, including some unexpected processes, such as TCA cycle and DNA transcription. This study depicts a roadmap for identification of functional substrates for glycosyltransferases, facilitating fundamental insight into the role of glycosylation in homeostasis and disease.

Abs against phosphorylcholine (anti-PC) and Abs against malondialdehyde (anti-MDA) may be protective in chronic inflammation, like atherosclerosis and cardiovascular disease. It is not known how they develop early in life. Ab titers were measured using ELISA in healthy women (n = 105; born into life study) and their children. Plasma samples were collected from the mothers before conception and from the children at birth as well as at 1 and 2 y after birth. Extracted Abs were compared using a proteomics de novo sequencing approach. It was observed that children were born with very low levels of IgM anti-PC, whereas IgM anti-MDA was present at birth. Both IgM anti-PC and anti-MDA increased during the first 2 y of life, but IgM anti-PC in contrast to IgM anti-MDA was still significantly lower than in the mothers. IgG anti-PC decreased after 1 y but reached similar levels as mothers’ after 2 y, whereas IgG anti-MDA reached similar levels as mothers’ already after 1 y. Proteomics peptide sequencing analysis indicated large peptide sequence variation without specific clone expression during the early stage of life compared with the adult stage for which specific peptide sequences dominated. IgM anti-PC levels develop much slower than anti-MDA and are still relatively low at 2 y. We hypothesize that anti-PC is developed by a combination of preprogramming and exposure to the external world, in which infectious agents may play a role. For anti-MDA, preprogramming is likely to play a major role and at an earlier stage than for anti-PC.

Chemical modification of proteins is enormously useful for characterizing protein function in complex biological systems and for drug development. Selective labeling of native or endogenous proteins is challenging owing to the existence of distinct functional groups in proteins and in living systems. Chemistry for rapid and selective labeling of proteins remains in high demand. Here we have developed novel affinity labeling probes using benzotriazole (BTA) chemistry. We showed that affinity-based BTA probes selectively and covalently label a lysine residue in the vicinity of the ligand binding site of a target protein with a reaction half-time of 28-42 s. The reaction rate constant is comparable to the fastest biorthogonal chemistry. This approach was used to selectively label different cytosolic and membrane proteins in vitro and in live cells. BTA chemistry could be widely useful for labeling of native/endogenous proteins, target identification and development of covalent inhibitors.

This protocol describes the proteomics technique called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis or SIESTA 1,2. SIESTA can be used for universal discovery of enzyme substrates that shift in thermal stability or solubility upon post-translational modification (PTM). Experimental design, proteomics sample preparation and data analysis are the key stages of this protocol. Data analysis can be performed using our SIESTA package hosted on GitHub 3. When performed with classical thermal proteome profiling (TPP), the protocol will take 5 days for sample preparation and 14 days of sample analysis by mass spectrometry (the current protocol). If our high-throughput version of TPP called Proteome Integral Solubility Alteration assay (PISA) 4 is used instead, the sample analysis time by mass spectrometry is reduced to 1-2 days for the same number of conditions.

Despite the immense importance of enzyme–substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery., The global identification of enzyme substrates is still challenging. Here, the authors develop a method based on proteome-wide thermal shift assays to discover enzyme substrates directly from cell lysates, identifying known and novel oxidoreductase, kinase and poly-(ADP-ribose) polymerase substrates.

Background The risk of cardiovascular disease (CVD) and atherosclerosis is very high in SLE. This is a clinical problem, and could also shed light on immunity and atherosclerosis in general. IgM antibodies against phosphorylcholine (anti-PC) may be protective in atherosclerosis, cardiovascular disease (CVD) and systemic lupus erythematosus (SLE). We here study IgG1 and IgG2 anti-PC, with focus on atherosclerosis and SLE. Methods We determined anti-PC by ELISA in 116 SLE-patients and 110 age- and sex-matched controls. For functional studies, we used three in-house generated, fully human monoclonal IgG1 anti-PC (A01, D05, E01). Apoptosis was induced in Jurkat T-cells and pre-incubated with A01, D05, E01 or isotype control IgG1 and effects on efferocytosis by human macrophages studied. Anti-PC peptide/protein characterization was determined using a proteomics de novo sequencing approach. Results IgG1 but not IgG2 anti-PC levels were higher among SLE patients (p=0.02). IgG1 anti-PC was negatively associated with SLICC and SLEDAI (OR: 2,978 CI: 0.876–10.098, OR: 5.108 CI 1.3 20.067 respectively) and negatively associated with CVD, atherosclerotic plaques and echolucent (potentially vulnerable plaques) but the association for the two former was not significant after controlling for confounders. D05 had maximum effect on macrophage efferocytosis efficiency, followed by A01 and E01. The monoclonal antibodies showed differential binding specificity to PC and PC associated neo-epitopes. Peptide analysis showed difference in the CDR3 region of the three anti-PC IgG1 clones which are crucial for recognition of PC on apoptotic cell surface and other neo-epitopes. Conclusion Anti-PC IgG1 is negatively associated with disease activity, and disease damage in SLE, but the negative association with CVD is also dependent on confounding risk factors. One potential underlying mechanism could be increased clearance of dead cells. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Swedish Heart and Lung Foundation, Reumatikerfonden

IgG Fc-glycans affect IgG function and are altered in autoimmune diseases and autoantibodies. Anti-histidyl tRNA synthetase autoantibodies (anti-Jo1) are frequent in patients with idiopathic inflammatory myopathies (IIM) and anti-synthetase syndrome (ASS) with associated interstitial lung disease (ILD). Thus, we hypothesized that the total-IgG Fc-glycans from Jo1+ versus Jo1− patients and anti-Jo1-IgG would show characteristic differences, and that particular Fc-glycan features would be associated with specific clinical manifestations. By proteomics based mass spectrometry we observed a high abundance of agalactosylated IgG1 Fc-glycans in ASS/IIM patients (n = 44) compared to healthy age matched controls (n = 24). Using intra-individual normalization of the main agalactosylated glycan (FA2) of IgG1 vs FA2-IgG2, ASS/IIM and controls were distinguished with an area under the curve (AUC) of 79 ± 6%. For Jo1+ patients (n = 19) the AUCs went up to 88 ± 6%. Bisected and afucosylated Fc-glycans were significantly lower in Jo1+ compared to Jo1− patients. Anti-Jo1-IgG enriched from eleven patients contained even significantly lower abundances of bisected, afucosylated and galactosylated forms compared to matched total-IgG. ASS and ILD diagnosis, as well as lysozyme and thrombospondin correlated with Jo1+ characteristic Fc-glycan features. These results suggest that the anti-Jo1+ patient Fc-glycan profile contains phenotype specific features which may underlie the pathogenic role of Jo1 autoantibodies.

Characterising chronic lung diseases is challenging. New, less invasive diagnostics are needed to decipher disease pathologies and subphenotypes. Fc galactosylation is known to affect IgG function, and is altered in autoimmune disorders and under other pathological conditions. We tested how well Fc glycans in IgG from bronchoalveolar lavage fluid (BALF) and serum correlated, and if the Fc glycan profile could reveal pulmonary inflammation.A shotgun proteomics approach was used to profile Fc glycans in serum and BALF of controls (n=12) and sarcoidosis phenotypes (Löfgren's syndrome (LS), n=11; and non-LS, n=12). Results were further validated in severe asthma (SA) (n=20) and published rheumatoid arthritis (RA) patient data (n=13) including clinical information.Intra-individually, Fc-galactosylation status of IgG1 (R2=0.87) and IgG4 (R2=0.95) correlated well between matrixes. Following GlycoAge-index correction, the ratio between agalactosylated and digalactosylated Fc glycans of IgG4 could distinguish sarcoidosis and SA from healthy and RA subjects with a mean±se area under the curve (AUC) of 78±6%. The AUC increased to 83±6% using the more chronic lung disease types (non-LS and SA) and most strikingly, to 87±6% for the SA subgroup.The results indicate that the Fc galactosylation status of IgG4 is a potential blood test marker for chronic lung inflammation.

Background:Autoantibodies that target aminoacyl transfer(t) RNA synthetases (aaRS) represent the serological marker of the anti-synthetase syndrome (ASS), a major subgroup of the idiopathic inflammatory myopathies (IIM) (1). Among the anti-aaRS, anti-histidyl tRNA synthetase (HisRS) autoantibodies (anti-Jo1) are the most common. Up to 90% of IIM/ASS patients diagnosed with interstitial lung disease (ILD) harbor anti-Jo1 autoantibodies (2).Objectives:Reactivity and affinity of anti-Jo1 autoantibodies from serum and broncheoalveolar lavage fluid (BALF) were investigated against HisRS autoantigen. Associations with clinical data from patients IIM/ASS were addressed.Methods:Total IgGs were purified by affinity chromatography. Samples and clinical data were obtained from: i) 26 anti-Jo1+patients (19 at diagnosis, 16/19 at follow-up, 7 BALF/matching serum at baseline; ii) 29 anti-Jo1-(25 serum at diagnosis, 4 BALF/matching serum at baseline); iii) 24 age/gender matched healthy controls. Anti-Jo1 IgG and IgA response against HisRS was evaluated by ELISA and western blot. Affinity was measured by surface plasmon resonance. HisRS full-length (HisRS-FL), two HisRS domains (ABD and CD), and two HisRS splice variants (WHEP and WHEP + ABD splice variant (SV)) were tested. Correlations between autoantibody reactivity and clinical data, at baseline and over disease course, were evaluated.Results:Anti-Jo1 autoantibodies from serum and lung bound HisRS-FL, WHEP and SV with high reactivity and affinity already at diagnosis and recognized both conformational and linear HisRS epitopes (Fig. 1). Levels of autoantibodies (against HisRS-FL, -domains and -splice variants) varied among patients and overtime. Patients with ILD, arthritis and less skin involvement presented higher anti-Jo1 titers compared to those with lower anti-Jo1 titers and to the anti-Jo1 negative group (Fig. 2). Anti-WHEP reactivity in BALF strongly correlated with poor pulmonary function.Conclusion:High reactivity and affinity at time of diagnosis indicates that autoimmunity against HisRS is most likely initiated before IIM/ASS diagnosis. Reactivity to specific splice variants of HisRS may be employed as diagnostic and prognostic markers.References:[1]Marguerie C, Bunn CC, Beynon HL, Bernstein RM, Hughes JM, So AK, Walport MJ: Polymyositis, pulmonary fibrosis and autoantibodies to aminoacyl-tRNA synthetase enzymes. Q J Med 1990, 77(282):1019-1038[2]Richards TJ, Eggebeen A, Gibson K, Yousem S, Fuhrman C, Gochuico BR, Fertig N, Oddis CV, Kaminski N, Rosas IO et al: Characterization and peripheral blood biomarker assessment of anti-Jo-1 antibody-positive interstitial lung disease. Arthritis Rheum 2009, 60(7):2183-2192.Fig. 1.Anti-Jo1 reactivity in total IgG purified from the first available serum sampleFig. 2.Reactivity of total anti-Jo1+ IgG purified from the first available serum close to IIM/ASS diagnosis in relation to clinical dataDisclosure of Interests:Antonella Notarnicola: None declared, Charlotta Preger: None declared, Susanna Lundström: None declared, Nuria Renard: None declared, Edvard Wigren: None declared, Eveline Van Gompel: None declared, Angeles Shunashy Galindo-Feria: None declared, Helena Persson: None declared, Maryam Fathi: None declared, Johan Grunewald: None declared, Per-Johan Jakobsson Shareholder of: Gesynta Pharma, Grant/research support from: Gesynta Pharma, AstraZeneca,, Susanne Gräslund: None declared, Ingrid E. Lundberg Grant/research support from: Bristol Meyer Squibb, Corbus Pharmaceuticals, Inc and Astra Zeneca, Catia Cerqueira: None declared

Various agents, including drugs as well as nonmolecular stimuli, induce alterations in the physicochemical properties of proteins in cell lysates, living cells, and organisms. These alterations can be probed by applying a stability- and solubility-modifying factor, such as elevated temperature, to a varying degree. As a second dimension of variation, drug concentration or agent intensity/concentration can be used. Compared to standard approaches where curves are fitted to protein solubility data acquired at different temperatures and drug concentrations, Proteome Integral Solubility Alteration (PISA) assay increases the analysis throughput by 1 to 2 orders of magnitude for an unlimited number of factor variation points in such a scheme. The consumption of the compound and biological material decreases in PISA by the same factor. We envision widespread use of the PISA approach in chemical biology and drug development.

Background: IgM antibodies against phosphorylcholine (anti-PC) may be protective in atherosclerosis, cardiovascular disease (CVD) and systemic lupus erythematosus (SLE). Less is known about other anti-PC isotypes and subclasses. In this study, we study the role of IgG1 and IgG2 anti-PC, with focus on atherosclerosis and SLE. Objectives: To study the role of IgG1 and IgG2 anti-PC, with focus on atherosclerosis and SLE, both in a clinical setting and by experimental studies, where we use our in-house produced monoclonal IgG1 anti-PC. Methods: We determined IgG1 and IgG2 anti-PC in our SLE cohort study (SLEVIC), among 116 SLE-patients from Karolinska University Hospital Huddinge and 110 population controls matched for age and gender. The level of antibodies was measured by ELISA. For functional studies, we used three of our in-house generated, fully human monoclonal IgG1 anti-PC (A01, D05, E01). Primary human macrophages were derived from peripheral blood. Apoptosis was induced in Jurkat T-cells and pre-incubated with A01, D05, E01 or isotype control IgG1 and effect on phagocytosis by marcophages studied. Anti-PC peptide/protein characterization was determined in anti-PC clones compared to isotype control using a proteomics de novo sequencing approach. Results: IgG1 but not IgG2 anti-PC levels were higher among SLE patients than controls (p=0.02). IgG1 anti-PC was negatively associated with prevalence of atherosclerotic plaques, below 10thpercentile, (OR: 2.48, CI: 0.69-9.00). IgG1 Anti-PC was negatively associated with CVD, SLICC and SLEDAI (OR: 4.74 CI: 1.29-17.39, OR: 2,978 CI: 0.876-10.098, OR: 5.108 CI 1.3 20.067 respectively). Monoclonal D05 had maximum effect on macrophage efferocytosis efficiency, followed by A01 and E01. This is because anti-PC IgG1s bind to phosphorylcholine exposed on apoptotic cells and facilitate the uptake by macrophage. The in-house produced monoclonal antibodies showed differential binding specificity to PC and PC associated neo-epitopes. Peptide analysis showed difference in the CDR3 region of the three anti-PC IgG1 clones which are crucial for recognition of the phosphorylcholine on the apoptotic cell surface and other neo-epitopes. Conclusion: anti-PC IgG1 is negatively associated with disease activity and atherosclerosis in SLE. One potential underlying mechanism could be increased phagocytosis and clearance of dead cells. Our findings raise the possibility of prevention and/or treatment of SLE (and atherosclerosis) to raise anti-PC levels, by passive or active immunization. References: [1] Frostegard J. Immunity, atherosclerosis and cardiovascular disease. BMC Med. 2013;11:117. [2] Anania C, Gustafsson T, Hua X, Su J, Vikstrom M, de Faire U, Heimburger M, Jogestrand T and Frostegard J. Increased prevalence of vulnerable atherosclerotic plaques and low levels of natural IgM antibodies against phosphorylcholine in patients with systemic lupus erythematosus. Arthritis Res Ther. 2010;12:R214. Disclosure of Interests: Divya Thiagarajan: None declared, Nina Oparina: None declared, Johanna Steen: None declared, Mizanur Rahman: None declared, Max Vikstrom: None declared, Roman Zubarev: None declared, Susanna Lundstrom: None declared, Johan Frostegard Shareholder of: Minor shareholder and inventor in startup-company Athera Biotechnologies, but they do not produce drugs yet and rheumatology is not in their focus.

Objective Immunoglobulin M antibodies against phosphorylcholine (anti‐PCs) may be protective in atherosclerosis, cardiovascular disease (CVD), and systemic lupus erythematosus (SLE). We study immunoglobulin G1 (IgG1) and immunoglobulin G2 (IgG2) anti‐PCs, with a focus on atherosclerosis and SLE. Methods We determined anti‐PCs by using the enzyme‐linked immunosorbent assay in 116 patients with SLE and 110 age‐ and sex‐matched controls. For functional studies, we used three in‐house–generated, fully human monoclonal IgG1 anti‐PCs (A01, D05, and E01). Apoptosis was induced in Jurkat T cells and preincubated with A01, D05, E01, or IgG1 isotype control, and effects on efferocytosis by human macrophages were studied. Anti‐PC peptide/protein characterization was determined using a proteomics de novo sequencing approach. Results IgG1, but not IgG2, anti‐PC levels were higher among patients with SLE (P = 0.02). IgG1 anti‐PCs were negatively associated with Systemic Lupus International Collaborating Clinics (SLICC) damage index and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores (odds ratio [OR]: 2.978 [confidence interval (CI): 0.876‐10.098] and OR: 5.108 [CI 1.3‐20.067], respectively) and negatively associated with CVD, atherosclerotic plaques, and echolucent plaques (potentially vulnerable plaques), but the association for the two former was not significant after controlling for confounders. D05 had a maximum effect on macrophage efferocytosis efficiency, followed by A01 and E01. The monoclonal antibodies showed differential binding specificity to PC and PC‐associated neoepitopes. A peptide analysis showed a difference in the complementarity‐determining region 3 of the three IgG1 anti‐PC clones that are crucial for recognition of PC on apoptotic cell surfaces and other neoepitopes. Conclusion IgG1 anti‐PCs are negatively associated with disease activity and disease damage in SLE, but the negative association with CVD is also dependent on confounding risk factors. One potential underlying mechanism could be increased clearance of dead cells., What is already known about this subject? •Low levels of immunoglobulin M antibodies against phosphorylcholine (anti‐PCs) is more common in patients with systemic lupus erythematosus (SLE) compared with controls and is associated with increased prevalence of vulnerable plaque among patients with SLE. What does this study add? Immunoglobulin G1 (IgG1) anti‐PCs are negatively associated with disease activity, disease damage, cardiovascular disease, and measures of atherosclerosis in SLE.We have produced in‐house, fully human monoclonal antibodies of the IgG1 isotype that increase apoptotic cell uptake efficiently and reduce inflammation induced by lipopolysaccharide. Effects varied depending on the clone used.A peptide analysis showed a difference in the complementarity‐determining region 3 of the three IgG1 anti‐PC clones that are crucial for the recognition of phosphorylcholine (PC) on apoptotic cell surfaces and other neoepitopes. How might this impact clinical practice or future developments? Measurement of IgG1 anti‐PCs, along with other autoantibodies, could improve prevention in patients with SLE with vascular implications.Anti‐PCs could be developed as a novel treatment in SLE, either as monoclonal antibodies or as a vaccine with PC.

Background:Phosphorylcholine (PC) is an important component in cellular membranes and in lipoproteins that is exposed and recognized by the immune system, when cells undergo apoptosis or lipoproteins like LDL undergo oxidation. PC is also exposed in some microorganisms including nematodes and bacteria (non-self). We reported that IgM anti-PC is associated with protection in atherosclerosis, SLE, RA and other chronic inflammatory conditions.1We also reported potential underlying protective mechanisms: 1: increase in clearance of human dead cells,22: inhibition of uptake of oxLDL in macrophages, 3: inhibition of cell death.14: anti-inflammatory; 5: promotion of polarization of T regulatory cells in SLE-patients´ T cells from a low level and also in plaque T cells.3We generated in-house fully human IgG1 anti-PC clones for experimental studies to study anti-PC properties in humans. In contrast to mice, anti-PC are not germ-line encoded with a dominant clone.4Objectives:We here study IgG1 and IgG2 anti-PC, with focus on atherosclerosis and SLE and properties of fully human IgG1 clones, in relation to SLE.Methods:We determined anti-PC by ELISA in 116 SLE-patients and 110 age- and sex-matched controls. For functional studies, we used three in-house generated, fully human monoclonal IgG1 anti-PC (A01, D05, E01). Apoptosis was induced in Jurkat T-cells and pre-incubated with A01, D05, E01 or isotype control IgG1 and effects on efferocytosis by human macrophages studied. Anti-PC peptide/protein characterization was determined using a proteomics de novo sequencing approach.Results:IgG1 but not IgG2 anti-PC levels were higher among SLE patients (p=0.02). IgG1 anti-PC was negatively associated with SLICC and SLEDAI (OR: 2,978 CI: 0.876-10.098, OR: 5.108 CI 1.3 20.067 respectively) and negatively associated with CVD, atherosclerotic plaques and echolucent (potentially vulnerable plaques) but the association for the two former was not significant after controlling for confounders. D05 had maximum effect on macrophage efferocytosis efficiency, followed by A01 and E01. The monoclonal antibodies showed differential binding specificity to PC and PC associated neo-epitopes. Peptide analysis showed difference in the CDR3 region of the three anti-PC IgG1 clones which are crucial for recognition of PC on apoptotic cell surface and other neo-epitopes.Conclusion:Anti-PC IgG1 is negatively associated with disease activity, and disease damage in SLE, but the negative association with CVD is also dependent on confounding risk factors. One potential underlying mechanism could be increased clearance of dead cells.References:[1]Frostegard J. Immunity, atherosclerosis and cardiovascular disease.BMC Med. 2013;11:117.[2]Rahman M, Sing S, Golabkesh Z, Fiskesund R, Gustafsson T, Jogestrand T, Frostegard AG, Hafstrom I, Liu A and Frostegard J. IgM antibodies against malondialdehyde and phosphorylcholine are together strong protection markers for atherosclerosis in systemic lupus erythematosus: Regulation and underlying mechanisms.Clin Immunol. 2016;166-167:27-37.[3]Sun J, Lundstrom SL, Zhang B, Zubarev RA, Steuer J, Gillgren P, Rahman M, Ajeganova S, Liu A and Frostegard J. IgM antibodies against phosphorylcholine promote polarization of T regulatory cells from patients with atherosclerotic plaques, systemic lupus erythematosus and healthy donors.Atherosclerosis. 2018;268:36-48.[4]Fiskesund R, Steen J, Amara K, Murray F, Szwajda A, Liu A, Douagi I, Malmstrom V and Frostegard J. Naturally occurring human phosphorylcholine antibodies are predominantly products of affinity-matured B cells in the adult.J Immunol. 2014;192:4551-9.Disclosure of Interests: :Divya Thiagarajan: None declared, Roland Fiskesund: None declared, Johanna Steen: None declared, Mizanur Rahman: None declared, Susanna Lundström: None declared, Johan Frostegård Grant/research support from: Unconditional competitive grant from Amgen, related only to PCSK9, not the topic of this abstract

Background:IgM antibodies against phosphorylcholine (anti-PC) and malondialdehyde (anti-MDA) may have protective properties in both atherosclerosis and rheumatic disease, especially anti-PC. Low levels of IgM anti-PC is associated with SLE itself and also with atherosclerotic plaques1and with being a non-responder to biologics in RA.1We determined mechanisms by which anti-PC (and to some extent anti-MDA) could be protective: 1: anti-inflammatory; 2: inhibition of uptake of oxLDL in macrophages, 3: inhibition of cell death.14: increase in clearance of human dead cells,25: anti-PC promotes polarization of T regulatory cells in SLE-patients´ T cells from a low level and also in cells from atherosclerotic plaques.3Objectives:To compare systemic rheumatic diseases in relation to natural anti-PC and anti-MDA, to develop novel classifications but also potential treatment against rheumatic disease. We here determine anti-PC and anti-MDA in different systemic rheumatic conditions and study there potential role properties.Methods:Anti-PC and anti-MDA was measured using ELISA in patients with SLE (374), RA (354), Mixed connective tissue disease (MCTD, 77), Systemic sclerosis (SSc, 331), Sjögren’s syndrome (SjS, 324), primary antiphospholipid syndrome (PAPs, 65), undifferentiated connective tissue disease (UCTD, 118) and 515 matched healthy controls (HC). Cardiovascular score (CV) was broadly defined based on clinical disease symptoms. Anti-PC and anti-MDA peptide/protein characterization were compared using a proteomics de novo sequencing approach. anti-MDA and anti-PC were extracted from total IgM. The proportion of Treg cells was determined by flow cytometry.Results:The maximal difference between cases and controls was shown for MCTD: significantly lower IgM Anti-PC but not anti-MDA among patients (median 49.3RU/ml vs 70.4 in healthy controls, p(t-test)=0.0037). IgM low levels were more prevalent in MCTD, SLE, SjS, SSc and UCTD. IgM anti-PC variable region profiles were different from and more homologous than anti-MDA. Anti-PC but not anti-MDA were significantly negatively correlated with CV in the whole patient group. In contrast to IgM anti-PC, anti-MDA did not promote polarization of Tregs.Conclusion:Anti-PC is decreased in MCTD and also in SLE, SjS and SSc but not in other studied diseases. Anti-PC may thus differentiate between these. In contrast, anti-MDA did not show these differences between diseases studied. Anti-PC level is negatively correlated with CV in the patient group cohort. In contrast to anti-PC, anti-MDA did not promote Treg polarization. These findings could have both diagnostic and therapeutic implications, one possibility being active or passive immunization with PC in some rheumatic conditions.References:[1]Frostegard J. Immunity, atherosclerosis and cardiovascular disease.BMC Med. 2013;11:117.[2]Rahman M, Sing S, Golabkesh Z, Fiskesund R, Gustafsson T, Jogestrand T, Frostegard AG, Hafstrom I, Liu A and Frostegard J. IgM antibodies against malondialdehyde and phosphorylcholine are together strong protection markers for atherosclerosis in systemic lupus erythematosus: Regulation and underlying mechanisms.Clin Immunol. 2016;166-167:27-37.[3]Sun J, Lundstrom SL, Zhang B, Zubarev RA, Steuer J, Gillgren P, Rahman M, Ajeganova S, Liu A and Frostegard J. IgM antibodies against phosphorylcholine promote polarization of T regulatory cells from patients with atherosclerotic plaques, systemic lupus erythematosus and healthy donors.Atherosclerosis. 2018;268:36-48.Acknowledgments:Preciseads Clinical ConsortiumDisclosure of Interests:Divya Thiagarajan: None declared, Susanna Lundström: None declared, Roman Zubarev: None declared, jitong Sun: None declared, Marta Alarcon-Riquelme: None declared, Johan Frostegård Grant/research support from: Unconditional competitive grant from Amgen, related only to PCSK9, not the topic of this abstract

Background:Antibodies against phosphorylcholine (anti-PC) have potentially protective properties in both atherosclerosis and rheumatic disease. IgM anti-PC could play a role in SLE being associated with protection, also in relation to atherosclerotic plaques and vulnerable plaques in SLE1and being a non-responder to biologics in RA.1We reported potential mechanisms by which anti-PC could be protective: 1:anti-inflammatory; 2: inhibits uptake of oxLDL in macrophages, 3: inhibits cell death.14: anti-PC (and anti-MDA) increases clearance of human dead cells which could be of importance not especially in SLE;25: anti-PC increases T regulatory cells in SLE-patients´ T cells from a low level and also in atherosclerosis, with implications for both conditions.3Also antibodies against malondialdehyde (anti-MDA) have interesting propertiesObjectives:It is not known how these antibodies develop early in life and what may cause low levels. The objective is to determine this.Methods:Antibodies were studied by ELISA in healthy pregnant women (n=105; Born into life study) and their newborn children. Women were recruited before conception. Informed consent, questionnaires from parents and plasma sample was collected from children at birth from cord blood, at 1-year and 2 years after birth. Extracted antibodies were compared using a proteomics de novo sequencing approach.Results:Children were born with very low levels of IgM anti-PC, while IgM anti-MDA was present at birth,. Both IgM anti-PC and anti-MDA increased during the first two years of life, but IgM anti-PC in contrast to IgM anti-MDA was still significantly lower than mothers´. IgG anti-PC decreased after 1 year, but reached similar levels as mothers´ after 2 years while IgG anti-MDA reached similar levels as mothers´ already after one year. Proteomics peptide sequencing analysis indicates large peptide sequence variation without specific clone expression during early stage of life compared to the adult stage for which specific peptide sequences dominated.Conclusion:IgM anti-PC levels develop much slower than anti-MDA and are still relatively low at 2 years. We hypothesize that anti-PC is developed by a combination of pre-programming and exposure to the external world, where infectious agents may play a role. For anti-MDA pre-programming is likely to play a major role and at an earlier stage than for anti-PC.References:[1]Frostegard J. Immunity, atherosclerosis and cardiovascular disease.BMC Med. 2013;11:117.[2]Rahman M, Sing S, Golabkesh Z, Fiskesund R, Gustafsson T, Jogestrand T, Frostegard AG, Hafstrom I, Liu A and Frostegard J. IgM antibodies against malondialdehyde and phosphorylcholine are together strong protection markers for atherosclerosis in systemic lupus erythematosus: Regulation and underlying mechanisms.Clin Immunol. 2016;166-167:27-37.[3]Sun J, Lundstrom SL, Zhang B, Zubarev RA, Steuer J, Gillgren P, Rahman M, Ajeganova S, Liu A and Frostegard J. IgM antibodies against phosphorylcholine promote polarization of T regulatory cells from patients with atherosclerotic plaques, systemic lupus erythematosus and healthy donors.Atherosclerosis. 2018;268:36-48.Disclosure of Interests:Divya Thiagarajan: None declared, Susanna Lundström: None declared, Göran Pershagen: None declared, Catharina Almqvist Malmros: None declared, Ellika Andolf: None declared, Anna Hedman: None declared, Oscar Berg: None declared, Nina Oparina: None declared, Johan Frostegård Grant/research support from: Unconditional competitive grant from Amgen, related only to PCSK9, not the topic of this abstract

Objective: Anti-citrullinated protein antibodies (ACPAs) develop many years before the clinical onset of rheumatoid arthritis (RA). This study was undertaken to address the molecular basis of the specificity and cross-reactivity of ACPAs from patients with RA. Methods: Antibodies isolated from RA patients were expressed as monoclonal chimeric antibodies with mouse Fc. These antibodies were characterized for glycosylation using mass spectrometry, and their cross-reactivity was assessed using Biacore and Luminex immunoassays. The crystal structures of the antigen-binding fragment (Fab) of the monoclonal ACPA E4 in complex with 3 different citrullinated peptides were determined using x-ray crystallography. The prevalence of autoantibodies reactive against 3 of the citrullinated peptides that also interacted with E4 was investigated by Luminex immunoassay in 2 Swedish cohorts of RA patients. Results: Analysis of the crystal structures of a monoclonal ACPA from human RA serum in complex with citrullinated peptides revealed key residues of several complementarity-determining regions that recognized the citrulline as well as the neighboring peptide backbone, but with limited contact with the side chains of the peptides. The same citrullinated peptides were recognized by high titers of serum autoantibodies in 2 large cohorts of RA patients. Conclusion: These data show, for the first time, how ACPAs derived from human RA serum recognize citrulline. The specific citrulline recognition and backbone-mediated interactions provide a structural explanation for the promiscuous recognition of citrullinated peptides by RA-specific ACPAs.

Various factors, including drugs as well as non-molecular influences, induce alterations in the stability of proteins in cell lysates, living cells and organisms. These alterations can be probed by applying a stability-modifying agent, such as elevated temperature, to a varying degree. As a second dimension of variation, drug concentration or factor intensity can be used. However, the corresponding analysis scheme has a low throughput and high cost. Additionally, since traditional data analysis employs curve fitting, proteins with unusual behavior are frequently ignored. The novel Proteome Integral Stability Alteration (PISA) assay avoids these issues altogether, increasing the analysis throughput by one to two orders of magnitude for unlimited number of parameter variation points. The consumption of the compound and biological material decreases by the same factor. We envision widespread use of the PISA approach in chemical biology and drug development.

Despite the immense importance of enzyme-substrate reactions, there is a lack of generic and unbiased tools for identifying and prioritizing substrate proteins which are modulated in the structural and functional levels through modification. Here we describe a high-throughput unbiased proteomic method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that enzymatic post-translational modification of substrate proteins might change their thermal stability. SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems in up to a depth of 7179 proteins. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, open new opportunities in investigating the effect of PTMs on signal transduction, and facilitate drug discovery.

Autoreactive B cells have a central role in the pathogenesis of rheumatoid arthritis (RA), and recent findings have proposed that anti-citrullinated protein autoantibodies (ACPA) may be directly pathogenic. Herein, we demonstrate the frequency of variable-region glycosylation in single-cell cloned mAbs. A total of 14 ACPA mAbs were evaluated for predicted N-linked glycosylation motifs in silico and compared to 452 highly-mutated mAbs from RA patients and controls. Variable region N-linked motifs (N-X-S/T) were strikingly prevalent within ACPA (100%) compared to somatically hypermutated (SHM) RA bone marrow plasma cells (21%), and synovial plasma cells from seropositive (39%) and seronegative RA (7%). When normalized for SHM, ACPA still had significantly higher frequency of N-linked motifs compared to all studied mAbs including highly-mutated HIV broadly-neutralizing and malaria-associated mAbs. The Fab glycans of ACPA-mAbs were highly sialylated, contributed to altered charge, but did not influence antigen binding. The analysis revealed evidence of unusual B-cell selection pressure and SHM-mediated decreased in surface charge and isoelectric point in ACPA. It is still unknown how these distinct features of anti-citrulline immunity may have an impact on pathogenesis. However, it is evident that they offer selective advantages for ACPA+ B cells, possibly also through non-antigen driven mechanisms.

Endo-β-N-acetylglucosaminidase (EndoS) is a family 18 glycosyl hydrolase secreted by Streptococcus pyogenes. Recombinant EndoS hydrolyzes the β-1,4-di-N-acetylchitobiose core of the N-linked complex type glycan on the asparagine 297 of the γ-chains of IgG. Here, we report that EndoS and IgG hydrolyzed by EndoS induced suppression of local immune complex (IC)-mediated arthritis. A small amount (1 µg given i.v. to a mouse) of EndoS was sufficient to inhibit IgG-mediated arthritis in mice. The presence of EndoS disturbed larger IC lattice formation both in vitro and in vivo, as visualized with anti-C3b staining. Neither complement binding in vitro nor antigen-antibody binding per se were affected. Thus, EndoS could potentially be used for treating patients with IC-mediated pathology.

Amyloid formation is associated with a number of neurodegenerative diseases that affect the independence and quality of life of aging populations. One of rather atypical, occurring at a young age amyloidosis is hereditary cystatin C amyloid angiopathy (HCCAA) related to aggregation of L68Q variant of human cystatin C (hCC). Human cystatin C plays a very important role in many aspects of human health; however, its amyloidogenic properties manifested in HCCAA present a real, lethal threat to some populations and any work on factors that can affect possible influencing hCC aggregation is not to overestimate. It was proved that interaction of hCC with monoclonal antibodies suppresses significantly hCC dimerization process. Therefore, immunotherapy seems to be the right approach toward possible HCCAA treatment. In this work, the hCC fragment encompassing residue 60-70 (in 2 variants: linear peptide and multiple antigenic peptide) was used as an immunogen in rabbit immunization. As a result, specific anti-hCC antibodies were found in both rabbit sera. Surprisingly, rabbit antibodies were obtained after immunization with only a short peptide. The obtained antibodies were characterized, and their influence on the aggregation propensity of the hCC molecules was evaluated. The antibodies turned out not to have any significant influence on the cystatin C dimerization process. Nevertheless, we hope that antibodies elicited in rabbits by other hCC fragments could lead to elaboration of effective treatment against HCCAA.

Background IgM antibodies against Phosphorylcholine (anti-PC) are negatively associated with atherosclerosis, cardiovascular disease (CVD) and systemic lupus erythematosus (SLE) where the risk of CVD and atherosclerosis is very high. We here study effects of IgM anti-PC on Th17 and T regulatory cells (Tregs). Objectives Immunomodulation in atherosclerosis and SLE could have a huge impact on disease prevention and treatment. Methods Mononuclear leukocytes were isolated from peripheral blood (PBMC) obtained from healthy blood donors, from six SLE patients with age- and sex-matched controls and from symptom-giving human atherosclerotic plaques. The proportion of Th17 (CD4+CCR6+) and Treg (CD4+CD25+CD127dim/-) cells were determined by flow cytometry in CD4+T cells after 6 days culture with Th17 or Treg-polarizing cytokines, with PMA and Ionomycin stimulation. IgM anti-PC were extracted from total IgM, with flow-through IgM as controls. Dendritic cells (DC) were differentiated from PBMC. Antibody peptide/protein characterization was done by a proteomics de novo sequencing approach. Results IgM anti-PC increased significantly the proportion of Tregs from healthy donors, SLE patients and from atherosclerotic plaque cells while control antibodies did not. T cells from SLE patients had a significantly lower proportion of Tregs and higher proportion of Th17 cells as compared to matched controls. IgM anti-PC but not control antibodies significantly reduced production of IL-17 and TNF-alpha in cell culture from SLE patients and from atherosclerotic plaque cells. IgM anti-PC interacted with CD40 and kept DCs in an immature stage potentially being tolerogenic. We identify differences on the IgM peptide expression level in anti-PC compared to control antibodies. Conclusions IgM anti-PC increase Tregs and having low levels could contribute to both SLE and atherosclerosis (and CVD) and could thus represent a novel underlying mechanism in these conditions. This finding could also have therapeutic implications. Disclosure of Interest None declared

Sarcoidosis is a systemic interstitial lung disease of unknown aetiology. Less invasive diagnostics are needed to decipher disease pathology and to distinguish sub-phenotypes. Here we test if SpotLight proteomics, which combines de novo MS/MS sequencing of enriched IgG and co-extracted proteins with subsequent label-free quantification of new and known peptides, can differentiate controls and sarcoidosis phenotypes (L&ouml, fgrens and non-L&ouml, fgrens syndrome, LS and nonLS). Intra-individually matched IgG enriched from serum and bronchial lavage fluid (BALF) from controls (n = 12), LS (n = 11) and nonLS (n = 12) were investigated. High-resolution mass-spectrometry SpotLight proteomics and uni- and multivariate-statistical analyses were used for data processing. Major differences were particularly observed in control-BALF versus sarcoidosis-BALF. However, interestingly, information obtained from BALF profiles was still present (but less prominent) in matched serum profiles. By using information from orthogonal partial least squares discriminant analysis (OPLS-DA) differentiating 1) sarcoidosis-BALF and control-BALF and 2) LS-BALF vs. nonLS-BALF, control-serum and sarcoidosis-serum (p = 0.0007) as well as LS-serum and nonLS-serum (p = 0.006) could be distinguished. Noteworthy, many factors prominent in identifying controls and patients were those associated with Fc-regulation, but also features from the IgG-Fab region and novel peptide variants. Differences between phenotypes were mostly IgG-specificity related. The results support the analytical utility of SpotLight proteomics which prospectively have potential to differentiate closely related phenotypes from a simple blood test.

A unique anti-inflammatory property of IgG, independent of antigen specificity, is described. IgG with modification of the heavy-chain glycan on asparagine 297 by the streptococcal enzyme endo-β- N -acetylglucosaminidase (EndoS) induced a dominant suppression of immune complex (IC)-mediated inflammation, such as arthritis, through destabilization of local ICs by fragment crystallizable–fragment crystallizable (Fc-Fc) interactions. Small amounts (250 µg) of EndoS-hydrolyzed IgG were sufficient to inhibit arthritis in mice and most effective during the formation of ICs in the target tissue. The presence of EndoS-hydrolyzed IgG disrupted larger IC lattice formation both in vitro and in vivo, as visualized with anti-C3b staining. Neither complement binding in vitro nor antigen–antibody binding per se was affected.

Background and aims IgM antibodies against phosphorylcholine (anti-PC) are negatively associated with atherosclerosis, cardiovascular disease (CVD) and systemic lupus erythematosus (SLE), where the risk of CVD and atherosclerosis is high. We here study the effects of IgM anti-PC immune regulation. Methods Mononuclear leukocytes were isolated from peripheral blood (PBMC) obtained from healthy blood donors, six SLE patients with age- and sex-matched controls, and symptom-giving human atherosclerotic plaques. The proportion of Th17 (CD4+CCR6+) and Treg (CD4+CD25+CD127dim/-) cells was determined by flow cytometry in CD4+T cells after 6 days of culture with Th17 or Treg-polarizing cytokines, with PMA and Ionomycin stimulation. IgM anti-PC were extracted from total IgM, with flow-through IgM as controls. Dendritic cells (DC) were differentiated from PBMC. Antibody peptide/protein characterization was done by a proteomics de novo sequencing approach. Results IgM anti-PC increased significantly the proportion of Tregs from healthy donors, SLE patients and atherosclerotic plaque cells while control antibodies did not. T cells from SLE patients had a significantly lower proportion of Tregs and a higher proportion of Th17 cells as compared to matched controls. IgM anti-PC, but not control antibodies, significantly reduced the production of IL-17 and TNF-α in cell cultures from SLE patients and atherosclerotic plaque cells. IgM anti-PC interacted with CD40 and kept DCs in an immature stage, potentially being tolerogenic. We observed differences in the IgM peptide expression levels in anti-PC compared to control antibodies. Conclusions IgM anti-PC promote polarization of Tregs, which could represent a novel protective mechanism in atherosclerosis and autoimmune conditions as SLE.

The human blood proteome is frequently assessed by protein abundance profiling using a combination of liquid chromatography and tandem mass spectrometry (LC-MS/MS). In traditional sequence database search, many good-quality MS/MS data remain unassigned. Here we uncover the hidden part of the blood proteome via novel SpotLight approach. This method combines de novo MS/MS sequencing of enriched antibodies and co-extracted proteins with subsequent label-free quantification of new and known peptides in both enriched and unfractionated samples. In a pilot study on differentiating early stages of Alzheimer’s disease (AD) from Dementia with Lewy Bodies (DLB), on peptide level the hidden proteome contributed almost as much information to patient stratification as the apparent proteome. Intriguingly, many of the new peptide sequences are attributable to antibody variable regions, and are potentially indicative of disease etiology. When the hidden and apparent proteomes are combined, the accuracy of differentiating AD (n = 97) and DLB (n = 47) increased from ≈85% to ≈95%. The low added burden of SpotLight proteome analysis makes it attractive for use in clinical settings.

We here report the lipopolysaccharide (LPS) structures expressed by nontypeable Haemophilus influenzae R2846, a strain whose complete genome sequence has recently been obtained. Results were obtained by using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MS (n) on permethylated dephosphorylated OS. A beta- d-Glc p-(1-->4)- d-alpha- d-Hep p-(1-->6)-beta- d-Glc p-(1-->4) unit was found linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety, l-alpha- d-Hep p-(1-->2)-[ PEtn-->6]- l-alpha- d-Hep p-(1-->3)- l-alpha- d-Hep p-(1-->5)-[ PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A. The beta- d-Glc p (GlcI) linked to HepI was also branched with oligosaccharide extensions from O-4 and O-6. O-4 of GlcI was substituted with sialyllacto- N-neotetraose [alpha-Neu5Ac-(2-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc pNAc-(1-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc p-(1-->] and the related structure [( PEtn-->6)-alpha- d-Gal pNAc-(1-->6)-beta- d-Gal p-(1-->4)-beta- d-Glc pNAc-(1-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc p-(1-->]. The distal heptose (HepIII) was substituted at O-2 by beta- d-Gal. Phosphate, phosphoethanolamine, phosphocholine, acetate, and glycine were found to substitute the core oligosaccharide. Two heptosyltransferase genes, losB1 and losB2, have been identified from the R2846 genome sequence and are candidates to add the noncore heptose to the LPS. Mutant strain R2846 losB1 did not show dd-heptose in the extension from HepI but still contained minor quantities of ld-heptose at the same position, indicating that the losB1 gene is required to add dd-heptose to GlcI. The LPS from strain R2846 losB1/ losB2 expressed no noncore heptose, consistent with losB2 directing the addition of ld-heptose.

Lipopolysaccharide is the major glycolipid of the cell wall of the bacterium Haemophilus influenzae, a Gram-negative commensal and pathogen of humans. Lipopolysaccharide is both a virulence determinant and a target for host immune responses. Glycosyltransferases have high donor and acceptor substrate specificities that are generally limited to catalysis of one unique glycosidic linkage. The H. influenzae glycosyltransferase LpsA is responsible for the addition of a hexose to the distal heptose of the inner core of the lipopolysaccharide molecule and belongs to the glycosyltransferase family 25. The hexose added can be either glucose or galactose and linkage to the heptose can be either beta1-2 or beta1-3. Each H. influenzae strain uniquely produces only one of the four possible combinations of linked sugar in its lipopolysaccharide. We show that, in any given strain, a specific allelic variant of LpsA directs the anomeric linkage and the added hexose, glucose, or galactose. Site-directed mutagenesis of a single key amino acid at position 151 changed the hexose added in vivo from glucose to galactose or vice versa. By constructing chimeric lpsA gene sequences, it was shown that the 3' end of the gene directs the anomeric linkage (beta1-2 or beta1-3) of the added hexose. The lpsA gene is the first known example where interstrain variation in lipopolysaccharide core structure is directed by the specific sequence of a genetic locus encoding enzymes directing one of four alternative possible sugar additions from the inner core.

Eicosanoids are oxygenated, endogenous, unsaturated fatty acids derived from arachidonic acid. Detection and quantification of these compounds are of great interest because they play important roles in a number of significant diseases, including asthma, chronic obstructive pulmonary disease (COPD), cardiovascular disease, and cancer. Because the endogenous levels of eicosanoids are quite low, sensitive and specific analytical methods are required to reliably quantify these compounds. High-performance liquid chromatography mass spectrometry (HPLC/MS) has emerged as one of the main techniques used in eicosanoid profiling. Herein, we describe the main LC/MS techniques and principles as well as their application in eicosanoid analysis. In addition, a protocol is given for extracting eicosanoids from biological samples, using bronchoalveolar lavage fluid (BALF) as an example. The method and instrument optimization procedures are presented, followed by the analysis of eicosanoid standards using reverse phase HPLC interfaced with an ion trap mass spectrometer (LC/MS/MS). This protocol is intended to provide a broad description of the field for readers looking for an introduction to the methodologies involved in eicosanoid quantification.

Otitis media caused by nontypeable Haemophilus influenzae (NTHi) is a common and recurrent bacterial infection of childhood. The structural variability and diversity of H. influenzae lipopolysaccharide (LPS) glycoforms are known to play a significant role in the commensal and disease-causing behavior of this pathogen. In this study, we determined LPS glycoform populations from NTHi strain 1003 during the course of experimental otitis media in the chinchilla model of infection by mass spectrometric techniques. Building on an established structural model of the major LPS glycoforms expressed by this NTHi strain in vitro (M. Månsson, W. Hood, J. Li, J. C. Richards, E. R. Moxon, and E. K. Schweda, Eur. J. Biochem. 269:808-818, 2002), minor isomeric glycoform populations were determined by liquid chromatography multiple-step tandem electrospray mass spectrometry (LC-ESI-MS n ). Using capillary electrophoresis ESI-MS (CE-ESI-MS), we determined glycoform profiles for bacteria from direct middle ear fluid (MEF) samples. The LPS glycan profiles were essentially the same when the MEF samples of 7 of 10 animals were passaged on solid medium (chocolate agar). LC-ESI-MS n provided a sensitive method for determining the isomeric distribution of LPS glycoforms in MEF and passaged specimens. To investigate changes in LPS glycoform distribution during the course of infection, MEF samples were analyzed at 2, 5, and 9 days postinfection by CE-ESI-MS following minimal passage on chocolate agar. As previously observed, sialic acid-containing glycoforms were detected during the early stages of infection, but a trend toward more-truncated and less-complex LPS glycoforms that lacked sialic acid was found as disease progressed.

Hereditary cystatin C amyloid angiopathy (HCCAA) is a severe neurodegenerative disorder related to the point mutation in cystatin C gene resulting in human cystatin C (hCC) L68Q variant. One of the potential immunotherapeutic approaches to HCCAA treatment is based on naturally occurring antibodies against cystatin C. A recent growing interest in autoantibodies, especially in the context of neurodegenerative diseases, emerges from their potential use as valuable diagnostic markers and for controlling protein aggregation. In this work, we present characteristics of natural anti-hCC antibodies isolated from the IgG fraction of human serum by affinity chromatography. The electrophoresis (1-D and 2-D) results demonstrated that the isolated NAbs are a polyclonal mixture, but their electrophoretic properties did not allow to classify the new autoantibodies to any particular type of IgG. The Fc-glycan status of the studied autoantibodies was assessed using mass spectrometry analysis. For the isolated NAbs, the epitopic fragments in hCC sequence were identified by MS-assisted proteolytic excision of the immune complex and compared with the ones predicted theoretically. The knowledge of hCC fragments binding to NAbs and other ligands may contribute to the search for new diagnostic methods for amyloidosis of different types and the search for their treatment.

In our previous work, we showed that electrospray ionization of intact polyclonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift. To determine the origin of the high molecular mass clones, we performed de novo bottom-up mass spectrometry on a purified IgM monoclonal light chain that had a calculated molecular mass of 24 275.03 Da. The entire sequence of the monoclonal light chain was determined using multienzyme digestion and de novo sequence-alignment software and was found to belong to the germline allele IGKV2-30. The alignment of kappa germline sequences revealed ten IGKV2 and one IGKV4 sequences that contained additional amino acids in their CDR1 region, creating the high-molecular-mass phenotype. We also performed an alignment of lambda germline sequences, which showed additional amino acids in the CDR2 region, and the FR3 region of functional germline sequences that result in a high-molecular-mass phenotype. The work presented here illustrates the ability of mass spectrometry to provide information on the diversity of light-chain molecular mass phenotypes in circulation, which reflects the germline sequences selected by the immunoglobulin-secreting B-cell population.

Oxylipins are oxidised fatty acids that can exert lipid mediator functions in inflammation, and several oxylipins derived from arachidonic acid are linked to asthma. This study quantified oxylipin profiles in different regions of the lung to obtain a broad-scale characterisation of the allergic asthmatic inflammation in relation to healthy individuals. Bronchoalveolar lavage fluid (BALF), bronchial wash fluid and endobronchial mucosal biopsies were collected from 16 healthy and 16 mildly allergic asthmatic individuals. Inflammatory cell counts, immunohistochemical staining and oxylipin profiling were performed. Univariate and multivariate statistics were employed to evaluate compartment-dependent and diagnosis-dependent oxylipin profiles in relation to other measured parameters. Multivariate modelling showed significantly different bronchial wash fluid and BALF oxylipin profiles in both groups (R(2)Y[cum]=0.822 and Q(2)[cum]=0.759). Total oxylipin concentrations and five individual oxylipins, primarily from the lipoxygenase (LOX) pathway of arachidonic and linoleic acid, were elevated in bronchial wash fluid from asthmatics compared to that from healthy controls, supported by immunohistochemical staining of 15-LOX-1 in the bronchial epithelium. No difference between the groups was found among BALF oxylipins. In conclusion, bronchial wash fluid and BALF contain distinct oxylipin profiles, which may have ramifications for the study of respiratory diseases. Specific protocols for sampling proximal and distal airways separately should be employed for lipid mediator studies.

Blood-based anti-amyloid-β (Aβ) immunoglobulins (IgGs) and peripheral inflammation are factors correlating with development of Alzheimer's disease (AD). IgG functionality can drastically change from anti- to pro-inflammatory via alterations in the IgG-Fc N-glycan structure. Herein, we tested if IgG-Fc glycosylation in plasma is indeed altered during the development of AD. Samples from age-matched subjects of 23 controls, 58 patients with stable mild cognitive impairment (SMCI), 34 patients with progressive (P)MCI, and 31 patients with AD were investigated. Label-free shotgun proteomics was applied without glycoprotein enrichment. Glycans on peptides EEQYNSTYR (IgG1) and EEQFNSTFR (IgG2) were quantified, and their abundances were normalized to total IgGn glycoform abundance. Univariate and multivariate statistics were employed to investigate the correlations between the patients groups and the abundances of the IgG glycoforms as well as those of inflammatory mediating proteins. Significant differences (p ≤ 0.05) were found, with a lower abundance of complex galactosylated and sialylated forms in AD. For females, a decline in glycoform complexity correlated with disease progress but an inverse change was found in males prior to the onset of AD. Principal component analysis (PCA; Males: R(2)X(cum) = 0.65, Q(2)(cum) = 0.34; Females: R(2)X(cum) = 0.62, Q(2)(cum) = 0.36), confirmed the gender similarities (for controls, SMCI and AD) as well as differences (for PMCI), and showed a close correlation between pro-inflammatory protein markers, AD, female PMCI, and truncated IgG-Fc glycans. The differences observed between genders prior to the onset of AD may indicate a lower ability in females to suppress peripheral inflammation, which may lead to exacerbated disease progression.

SCOPE: In contrast to well-characterized PUFA levels in serum, little is known regarding their downstream metabolic products. However, many of these compounds are lipid mediators with prominent roles during pro- and antiinflammatory processes. METHODS AND RESULTS: In this double blind crossover study on asthmatics, shifts in serum levels of ?-3 and ?-6 PUFA-derived oxidized fatty acids (e.g. eicosanoids, oxylipins) were quantified following dietary fish oil supplementation. Serum was obtained from subjects following fasting at three occasions; (i) prior to supplementation, (ii) following a 3-week supplement intake of either placebo or fish oil, and (iii) following a 3-week washout period with a subsequent 3-week period of either fish oil or placebo supplement. A total of 87 oxylipins representing cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) metabolic pathways were screened via LC-MS/MS. The primary alterations observed were in CYP- and 15-LOX-derived EPA- and CYP-derived DHA oxylipins. CONCLUSION: The results indicate that intake of an ?-3 rich diet alters not only the PUFA ratio, but also the ratio of downstream oxylipins. These data further support that dietary manipulation with ?-3 PUFAs affects not only PUFA levels, but importantly also the downstream metabolic profile.

Mast cells are inflammatory cells that play key roles in health and disease. They are distributed in all tissues and appear in two main phenotypes, connective tissue and mucosal mast cells, with differing capacities to release inflammatory mediators. A metabolic profiling approach was used to obtain a more comprehensive understanding of the ability of mast cell phenotypes to produce eicosanoids and other lipid mediators. A total of 90 lipid mediators (oxylipins) were characterized using liquid chromatography-tandem mass spectrometry (LC-MS/MS), representing the cyclooxygenase (COX), lipoxygenase (LO), and cytochrome P450 (CYP) metabolic pathways. In vitro-derived murine mucosal-like mast cells (MLMC) and connective tissue-like mast cells (CTLMC) exhibited distinct mRNA expression patterns of enzymes involved in oxylipin biosynthesis. Oxylipins produced by 5-LO and COX pathways were the predominant species in both phenotypes, with 5-LO products constituting 90 ± 2% of the CTLMCs compared with 58 ± 8% in the MLMCs. Multivariate analyses demonstrated that CTLMCs and MLMCs secrete differing oxylipin profiles at baseline and following calcium ionophore stimulation, evidencing specificity in both a time- and biosynthetic pathway-dependent manner. In addition to the COX-regulated prostaglandin PGD(2) and 5-LO-regulated cysteinyl-leukotrienes (e.g., LTC(4)), several other mediators evidenced phenotype-specificity, which may have biological implications in mast cell-mediated regulation of inflammatory responses.