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Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC-UHPLC-FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC-UHPLC-FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies.

BPM1 interacts with components of the DDR complex and stimulates DNA methylation at CHH sites, suggesting its involvement in the RdDM methylation pathway. The best-known function of MATH-BTB proteins, including Arabidopsis BPM proteins, is their role as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin-proteasome pathway. This paper reports a new CUL3-independent role of BPM1 in RNA-directed DNA methylation (RdDM). Using quantitative and qualitative Y2H, pull down, microscale thermophoresis and FRET-FLIM, we demonstrate that BPM1 interacts with DMS3 and RDM1, components of the chromatin remodeling DDR complex involved in the recruitment of the RdDM methylation machinery. All three proteins colocalized predominantly in the nucleus. The MATH domain, which specifically binds proteins destined for degradation, was not essential for interactions with DMS3 and RDM1. In plants overexpressing BPM1, endogenous DMS3 protein levels were stable, indicating that BPM1 does not induce proteasomal degradation. In RDM1-overexpressing plants, RDM1 was not ubiquitinated. Together, these results suggest that BPM1 does not mediate the degradation of DMS3 and RDM1. Additionally, overexpression of BPM1 caused increased global methylation levels as well as CHH methylation in promoters of two RdDM-regulated genes, FWA and CML41. Overall, BPM1 seems to have a stimulating effect on RdDM activity, and this role appears to be unrelated to its known function as a Cul3-based E3 ligase adaptor.

Role of protein N-glycosylation and especially changes in IgG Nglycosylation pattern has repeatedly been confirmed as crucial in different physiological and pathological processes. Expanding knowledge requires more affordable, reliable, and higher throughput methods. New approaches to glycosylation analysis would allow its optimization and application in the biopharmaceutical industry, epidemiology as well as advance clinical diagnostics that would rely on glycan biomarkers. Though there were successful attempts to develop new automated strategies for glycan research, many laboratories still leverage manual sample preparation protocols that limit their throughput. To help address this issue, automated methods for glycosylation analysis should be developed further. This research proposes one possible approach to automation of IgG glycan sample preparation using the Tecan Freedom Evo liquid handling automated platform. To improve throughput and robustness, the manual method was substituted by the automated system that was equipped with a liquid handling arm (LiHa) and a robotic manipulator arm (RoMa), while the vacuum manifold operations were replaced with A200, a positive pressure unit which allows for higher pressure and flow-through. The adapted automated protocol features IgG isolation on a protein G plate, IgG deglycosylation, and glycan 2-AB labeling. This automated protocol allows the samples to be analyzed using ultra-high- performance liquid chromatography (UPLC). UPLC analysis showed that peaks' number, retention time, and peak area corresponded with the manual method. Peaks with the largest area showed small variation, while the smaller peaks exhibited larger variation. Method has yielded promising results and could encourage additional automation efforts in other protocols with development.

Role of protein N-glycosylation and especially changes in IgG N-glycosylation pattern has repeatedly been confirmed as crucial in different physiological and pathological processes. Expanding knowledge requires more affordable, reliable, and higher throughput methods. New approaches to glycosylation analysis would allow its optimization and application in the biopharmaceutical industry, epidemiology as well as advance clinical diagnostics that would rely on glycan biomarkers. Though there were successful attempts to develop new automated strategies for glycan research, many laboratories still leverage manual sample preparation protocols that limit their throughput. In order to help address this issue, automated methods for glycosylation analysis should be developed further. This research proposes one possible approach to automation of IgG glycan sample preparation using the Tecan Freedom Evo liquid handling automated platform. To improve throughput and robustness, the manual method was substituted by the automated system that was equipped with a liquid handling arm (LiHa) and a robotic manipulator arm (RoMa), while the vacuum manifold operations were replaced with A200, a positive pressure unit which allows for higher pressure and flow-through. The adapted automated protocol features IgG isolation on a protein G plate, IgG deglycosylation, and glycan 2-AB labeling. This automated protocol allows the samples to be analyzed using ultra-high performance liquid chromatography (UPLC). UPLC analysis showed that peaks’ number, retention time and peak area corresponded with the manual method. Peaks with the largest area showed small variation, while the smaller peaks exhibited larger variation. Method has yielded promising results and with development could encourage additional automation efforts in other protocols.

Aberrant immunoglobulin G (IgG) N-glycosylation offers new prospects to detect changes in cell metabolism and by extension, for biomarker discovery in type 2 diabetes mellitus (T2DM). However, past studies did not analyze the individual IgG subclasses in relation to T2DM pathophysiology. We report here original findings through a comparison of the IgG subclass-specific fragment crystallizable (Fc) glycan biosignatures in 115 T2DM patients with 122 healthy controls within the Uyghur population in China. IgG Fc glycosylation profiles were analyzed using nano- liquid chromatography–mass spectrometry to exclude changes attributed to fragment antigen binding N- glycosylation. After correction for clinical covariates, 27 directly measured and 4 derived glycan traits of the IgG subclass-specific N- glycopeptides were significantly associated with T2DM. Furthermore, we observed in T2DM a decrease in bisecting N-acetylglucosamine of IgG2 and agalactosylation of IgG4, and an increase in sialylation of IgG4 and digalactosylation of IgG2. Classification model based on IgG subclass- specific N-glycan traits was able to distinguish patients with T2DM from controls with an area under the receiver operating characteristic curve of 0.927 (95% confidence interval 0.894–0.960, p < 0.001). In conclusion, a robust association between the IgG subclass-specific Fc N-glycomes and T2DM was observed in the Uyghur population sample in China, suggesting a potential for the IgG Fc glycosylation as a biomarker candidate for type 2 diabetes. The integration of glycomics with other system science biomarkers might offer further hope for innovation in diagnosis and treatment of T2DM in the future. Finally, it is noteworthy that “Population Glycomics” is an emerging approach to biomarker discovery for common complex diseases.

Mass spectrometry and its hyphenated techniques enabled by the improvements in liquid chromatography, capillary electrophoresis, novel ionization, and fragmentation modes are truly a cornerstone of robust and reliable protein glycosylation analysis. Boost in immunoglobulin G (IgG) glycan and glycopeptide profiling demands for both applied biomedical and research applications has brought many new advances in the field in terms of technical innovations, sample preparation, improved throughput, and confidence in glycan structural characterization. This chapter summarizes mass spectrometry basics, focusing on IgG and monoclonal antibody N-glycosylation analysis on several complexity levels. Different approaches, including antibody enrichment, glycan release, labeling, and glycopeptide preparation and purification, are covered and illustrated with recent breakthroughs and examples from the literature omitting excessive theoretical frameworks. Finally, selected highly popular methodologies in IgG glycoanalytics such as liquid chromatography-mass spectrometry and matrix-assisted laser desorption ionization are discussed more thoroughly yet in simple terms making this text a practical starting point either for the beginner in the field or an experienced clinician trying to make sense out of the IgG glycomic or glycoproteomic dataset.

Mass spectrometry and its hyphenated techniques enabled by the improvements in liquid chromatography, capillary electrophoresis, novel ionization, and fragmentation modes are truly a cornerstone of robust and reliable protein glycosylation analysis. Boost in immunoglobulin G (IgG) glycan and glycopeptide profiling demands for both applied biomedical and research applications has brought many new advances in the field in terms of technical innovations, sample preparation, improved throughput, and confidence in glycan structural characterization. This chapter summarizes mass spectrometry basics, focusing on IgG and monoclonal antibody N-glycosylation analysis on several complexity levels. Different approaches, including antibody enrichment, glycan release, labeling, and glycopeptide preparation and purification, are covered and illustrated with recent breakthroughs and examples from the literature omitting excessive theoretical frameworks. Finally, selected highly popular methodologies in IgG glycoanalytics such as liquid chromatography–mass spectrometry and matrix-assisted laser desorption ionization are discussed more thoroughly yet in simple terms making this text a practical starting point either for the beginner in the field or an experienced clinician trying to make sense out of the IgG glycomic or glycoproteomic dataset.

MATH-BTB proteins are known to act as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin proteasome pathway. Their BTB domain binds to CUL3 scaffold proteins and the less conserved MATH domain targets a highly diverse collection of substrate proteins to promote their ubiquitination and subsequent degradation. In plants, a significant expansion of the MATH-BTB family occurred in the grasses. Here, we report analysis of TaMAB2, a MATH-BTB protein transiently expressed at the onset of embryogenesis in wheat. Due to difficulties in studying its role in zygotes and early embryos, we have overexpressed TaMAB2 in Arabidopsis to generate gain-of-function mutants and to elucidate interaction partners and substrates. Overexpression plants showed severe growth defects as well as disorganization of microtubule bundles indicating that TaMAB2 interacts with substrates in Arabidopsis. In tobacco BY-2 cells, TaMAB2 showed a microtubule and ubiquitin-associated cytoplasmic localization pattern in form of foci. Its direct interaction with CUL3 suggests functions in targeting specific substrates for ubiquitin- dependent degradation. Although direct interactions with tubulin could not be confimed, tandem affinity purification of TaMAB2 interactors point towards cytoskeletal proteins including tubulin and actin as well as the translation initiation machinery. The idenification of various subunits of eucaryotic translation initiation factors eIF3 and eIF4 as TaMAB2 interactors indicate regulation of translation initiation as a major function during onset of embryogenesis in plants.

Immunoglobulin G (IgG) is the most abundant serum antibody and is a key determinant of the humoral immune response. Its structural characteristics and effector functions are modulated through the attachment of various sugar moieties called glycans. IgG N-glycome patterns change with the age of individual and in different diseases. Variability of IgG glycosylation within a population is well studied and is affected by a combination of genetic and environmental factors. However, global inter-population differences in IgG glycosylation have never been properly addressed. Here we present population-specific N-glycosylation patterns of whole IgG, analysed in 5 different populations totalling 10,482 IgG glycomes, and of IgG’s fragment crystallisable region (Fc), analysed in 2,530 samples from 27 populations sampled across the world. We observed that country of residence associates with many N-glycan features and is a strong predictor of monogalactosylation variability. IgG galactosylation also strongly correlated with the development level of a country, defined by United Nations health and socioeconomic development indicators. We found that subjects from developing countries had low IgG galactosylation levels, characteristic for inflammation and ageing. Our results suggest that citizens of developing countries may be exposed to country-specific environmental factors that can cause low-grade chronic inflammation and the apparent increase in biological age.

Background: Many genome- and epigenome-wide association studies (GWAS and EWAS) and studies of promoter methylation of candidate genes for inflammatory bowel disease (IBD) have demonstrated significant associations between genetic and epigenetic changes and IBD. Independent GWA studies have identified genetic variants in the BACH2, IL6ST, LAMB1, IKZF1, and MGAT3 loci to be associated with both IBD and immunoglobulin G (IgG) glycosylation. Methods: Using bisulfite pyrosequencing, we analyzed CpG methylation in promoter regions of these five genes from peripheral blood of several hundred IBD patients and healthy controls (HCs) from two independent cohorts, respectively. Results: We found significant differences in the methylation levels in the MGAT3 and BACH2 genes between both Crohn's disease and ulcerative colitis when compared to HC. The same pattern of methylation changes was identified for both genes in CD19 + B cells isolated from the whole blood of a subset of the IBD patients. A correlation analysis was performed between the MGAT3 and BACH2 promoter methylation and individual IgG glycans, measured in the same individuals of the two large cohorts. MGAT3 promoter methylation correlated significantly with galactosylation, sialylation, and bisecting GlcNAc on IgG of the same patients, suggesting that activity of the GnT-III enzyme, encoded by this gene, might be altered in IBD. The correlations between the BACH2 promoter methylation and IgG glycans were less obvious, since BACH2 is not a glycosyltransferase and therefore may affect IgG glycosylation only indirectly. Conclusions: Our results suggest that epigenetic deregulation of key glycosylation genes might lead to an increase in pro-inflammatory properties of IgG in IBD through a decrease in galactosylation and sialylation and an increase of bisecting GlcNAc on digalactosylated glycan structures. Finally, we showed that CpG methylation in the promoter of the MGAT3 gene is altered in CD3 + T cells isolated from inflamed mucosa of patients with ulcerative colitis from a third smaller cohort, for which biopsies were available, suggesting a functional role of this glyco-gene in IBD pathogenesis. The authors would like to thank Stephanie Scott for her organizational and administrational contribution. The study has been funded by the EU FP7 grant European Commission IBD-BIOM (contract # 305479), EU FP7 Regional Potential Grant INTEGRA-Life (contract # 315997), European Structural and Investment Funds grant for the Croatian National Centre of Research Excellence in Personalized Healthcare (contract # KK.01.1.1.01.0010), and Croatian Science Foundation grant EpiGlycoIgG (contract # 3361). Financial support from Portugal (PI: SSP): FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Inovação in the framework of the project (POCI-01/0145-FEDER-016601; PTDC/DTP-PIC/0560/2014) was received. SSP also acknowledges the European Crohn’s and Colitis Organization (ECCO) and the “Broad Medical Research program at Crohn’s and Colitis Foundation of America-CCFA” for funding. SSP acknowledges the Portuguese Group of Study on IBD (GEDII) for funding. A.M.D. [PD/BD/105982/2014] also acknowledges FCT for funding. IBD-BIOM consortium: Daniel Kolarich (Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany), Manfred Wuhrer (Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands; Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, the Netherlands), Dermot P. B. McGovern (F. Widjaja Family Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles), Iain K. Pemberton (IP Research Consulting SAS, Paris, France), Daniel IR Spencer (Ludger Ltd., Culham Science Centre, Oxford, UK, Daryl L. Fernandes (Ludger Ltd., Culham Science Centre, Oxford, UK), Rahul Kalla, Kate O’Leary, Alex T Adams, Hazel Drummond, Elaine Nimmo, Ray Boyapati, David C Wilson (Centre for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK), Ray Doran (Ludger Ltd., Culham Science Centre, Oxford, UK), Igor Rudan (all, Centre for Population Health Sciences, University of Edinburgh, Edinburgh, UK), Paolo Lionetti (Paediatric Gastroenterology Unit, AOU Meyer, Viale Pieraccini, Florence, Italy), Natalia Manetti (Department of Medical and Surgical Sciences, Division of Gastroenterology, University Hospital Careggi, Florence, Italy), Fabrizio Bossa (Department of Medical Sciences, Division of Gastroenterology, IRCCS-CSS Hospital, Viale Cappuccini, Rotondo, Italy), Paola Cantoro, Anna Kohn (Division of Gastroenterology, S. Camillo Hospital, Rome, Italy), Giancarlo Sturniolo (Gastrointestinal Unit, University of Padua, Padua, Italy), Silvio Danese (IBD Unit, Humanitas Research Institute, Rozzano, Milan, Italy), Mariek Pierik (Maastricht University Medical Centre (MUMC), Maastricht, the Netherlands), and David C. Wilson (Centre for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK). This independent research was generously supported by the following grants: EU FP7 research grant IBD-BIOM (contract # 305479) to JS, VA, GL, and VZ; EU FP7 Regional Potential Grant INTEGRA-Life (contract # 315997) to GL and VZ; European Structural and Investment Funds grant for the Croatian National Centre of Research Excellence in Personalized Healthcare (contract # KK.01.1.1.01.0010) to GL and VZ; Croatian Science Foundation grant EpiGlycoIgG (contract # 3361) to VZ; FEDER COMPETE 2020 POCI, Portugal 2020, and Portuguese funds through FCT (contracts # POCI-01/0145-FEDER-016601 and PTDC/DTP-PIC/0560/2014) to SP; and FTC (contract # PD/BD/105982/2014) to AMD.

While N-linked glycosylation has been extensively studied in the context of inflammatory and metabolic disorders, its relationship with major depressive disorder (MDD) and antidepressant treatment response has not been investigated. In our exploratory study, we analysed N-glycan profiles in blood plasma samples collected from MDD patients (n = 18) and found gender-dependent correlations with severity of depressive symptoms prior to initiating antidepressant treatment. In addition, several N-glycosylation traits showed gender-dependent associations with clinical antidepressant response. Follow up proteomics analysis in peripheral blood mononuclear cells (PBMCs) collected from MDD patients (n = 20) identified baseline and post-antidepressant treatment pathway differences between responder and non-responder patients. Reactome data analysis further delineated potential biological reaction differences between responder and non-responder patients. Our preliminary results suggest that specific glycosylation traits are associated with depressive symptom severity and antidepressant response and may be of use as biomarkers.

Immunoglobulin G (IgG) N-glycosylation is crucial for its effector functions. It is a complex trait, and large sample sets are needed to discover multiple genetic factors that underlie it. While in humans such high-throughput studies of IgG N-glycans became usual, only one has been carried out in mice. Here we describe and validate a method for the relative quantification of IgG Fc-linked N-glycans in a subclass-specific manner using nano-reverse phase liquid chromatography coupled with mass-spectrometry (nanoRP-LC-MS) applied to murine IgG. High-throughput data processing is ensured by the LaCyTools software. We have shown that IgG isolation procedure is the main source of technical variation in the current protocol. The major glycoforms were quantified reliably with coefficients of variation below 6% for all the analytes with relative abundances above 5%. We have applied our method to a sample set of 3 inbred strains: BALB/c, C57BL/6 and C3H and observed differences in subclass-specific and strain-specific N-glycosylation of IgG, suggesting a significant genetic component in the regulation of Fc-linked IgG N-glycosylation.

Hypertension results from the interaction of genetic and acquired factors. IgG occurs in the form of different subclasses, of which the effector functions show significant variation. The detailed differences between the glycosylation profiles of the individual IgG subclasses may be lost in a profiling method for total IgG N-glycosylation. In this study, subclass-specific IgG Fc glycosylation profile was investigated in the four northwestern Chinese minority populations, namely, Uygur (UIG), Kazak (KZK), Kirgiz (KGZ), and Tajik (TJK), composed of 274 hypertensive patients and 356 healthy controls. The results showed that ten directly measured IgG N-glycan traits (i.e., IgG1G0F, IgG2G0F, IgG2G1FN, IgG2G1FS, IgG2G2S, IgG4G0F, IgG4G1FS, IgG4G1S, IgG4G2FS, and IgG4G2N) representing galactosylation and sialylation are significantly associated with hypertension, with IgG4 consistently showing weaker associations of its sialylation, across the four ethnic groups. We observed a modest improvement on the AUC of ROC curve when the IgG Fc N-glycan traits are added into the glycan-based model (difference between AUCs, 0.044, 95% CI: 0.016–0.072, P = 0.002). The AUC of the diagnostic model indicated that the subclass-specific IgG Fc N-glycan profiles provide more information reinforcing current models utilizing age, gender, BMI, and ethnicity, and demonstrate the potential of subclass-specific IgG Fc N-glycosylation profiles to serve as a biomarker for hypertension. Further research is however required to determine the additive value of subclass-specific IgG Fc N-glycosylation on top of biomarkers, which are currently used.

Background & Aims Biomarkers are needed for early detection of Crohn’s disease (CD) and ulcerative colitis (UC) or to predict patient outcomes. Glycosylation is a common and complex posttranslational modification of proteins that affects their structure and activity. We compared plasma N-glycosylation profiles between patients with CD or UC and healthy individuals (controls). Methods We analyzed the total plasma N-glycomes of 2635 patients with inflammatory bowel diseases and 996 controls by mass spectrometry with a linkage-specific sialic acid derivatization technique. Plasma samples were acquired from 2 hospitals in Italy (discovery cohort, 1989 patients with inflammatory bowel disease [IBD] and 570 controls) and 1 medical center in the United States (validation cohort, 646 cases of IBD and 426 controls). Sixty-three glycoforms met our criteria for relative quantification and were extracted from the raw data with the software MassyTools. Common features shared by the glycan compositions were combined in 78 derived traits, including the number of antennae of complex-type glycans and levels of fucosylation, bisection, galactosylation, and sialylation. Associations of plasma N-glycomes with age, sex, CD, UC, and IBD-related parameters such as disease location, surgery and medication, level of C-reactive protein, and sedimentation rate were tested by linear and logistic regression. Results Plasma samples from patients with IBD had a higher abundance of large-size glycans compared with controls, a decreased relative abundance of hybrid and high-mannose structures, lower fucosylation, lower galactosylation, and higher sialylation (α2,3- and α2,6-linked). We could discriminate plasma from patients with CD from that of patients with UC based on higher bisection, lower galactosylation, and higher sialylation (α2,3-linked). Glycosylation patterns were associated with disease location and progression, the need for a more potent medication, and surgery. These results were replicated in a large independent cohort. Conclusions We performed high-throughput analysis to compare total plasma N-glycomes of individuals with vs without IBD and to identify patterns associated with disease features and the need for treatment. These profiles might be used in diagnosis and for predicting patients’ responses to treatment.

It is speculated that immunoglobulin G (IgG) plays a regulatory role in allergic reactions. The glycans on the Fc region are known to affect IgG effector functions, thereby possibly having a role in IgG modulation of allergic response. This is the first study investigating patients’ IgG glycosylation profile in allergic diseases. Subclass specific IgG glycosylation profile was analyzed in two cohorts of allergen sensitized and non-sensitized 3- to 11-year-old children (conducted at University of Aberdeen, UK and Children’s Hospital Srebrnjak, Zagreb, Croatia) with 893 subjects in total. IgG was isolated from serum/plasma by affinity chromatography on Protein G. IgG tryptic glycopeptides were analyzed by liquid chromatography electrospray ionization mass spectrometry. In the Zagreb cohort IgG glycome composition changed with age across all IgG subclasses. In both cohorts, IgG glycome composition did not differ in allergen sensitized subjects, nor children sensitized to individual allergens, single allergen mean wheal diameter or positive wheal sum values. In the Zagreb study the results were also replicated for high total serum IgE and in children with self-reported manifest allergic disease. In conclusion, our findings demonstrate no association between serum IgG glycome composition and allergic diseases in children.

Bottom-up glycoproteomics by liquid chromatography-mass spectrometry (LC-MS) is an established approach for assessing glycosylation in a protein- and site-specific manner. Consequently, tools are needed to automatically align, calibrate, and integrate LC-MS glycoproteomics data. We developed a modular software package designed to tackle the individual aspects of an LC-MS experiment, called LaCyTools. Targeted alignment is performed using user defined m/z and retention time (t(r)) combinations. Subsequently, sum spectra are created for each user defined analyte group. Quantitation is performed on the sum spectra, where each user defined analyte can have its own tr, minimum, and maximum charge states. Consequently, LaCyTobls deals with multiple charge states, which gives an output per charge state if desired, and offers various analyte and spectra quality criteria. We compared throughput and performance of LaCyTools to combinations of available tools that deal with individual processing steps. LaCyTools yielded relative quantitation of equal precision (relative standard deviation

Aim Regenerative periodontal therapy is often unpredictable and limited. Cementum regeneration is necessary for the proper repair of a periodontal ligament. The precise mechanism how bone morphogenetic protein-7 (BMP7) induces differentiation and mineralization of cementoblasts remains undetermined. The purpose of this study was to evaluate the effect of BMP7 on early proteome and gene expression profile of cementoblastic OCCM.30 cells in vitro. Materials and methods Immortalized murine cementoblasts (OCCM.30) were exposed to BMP7 and evaluated for: (1) proliferation; (2) mineralization; (3) early proteome profile using liquid chromatography-mass spectrometry (LC-MS); and (4) gene expression by quantitative RT-PCR. Results Bone morphogenetic protein-7 increased the cell proliferation at 24 h and 48 h, while higher doses suppressed the cell proliferation at 48 h. BMP7 induced the mineralization of cementoblasts following 8 days of therapy. Using LC-MS we identified 1117 proteins from the cell lysate. Many belonged to extracellular matrix formation such as PCPE1, collagens, annexins and integrin receptors. RT-PCR analyses revealed a BMP7 dose-dependent upregulation of BMP1, TGFβ1, osterix, osteoprotegerin, procollagen I and II, PCPE1, and noggin, while BMP6 and chordin expression were decreased. The high BMP7 dose down regulated most of the genes 24 h following therapy. Conclusion Bone morphogenetic protein-7 promotes differentiation and mineralization of cementoblasts via inducing PCPE1 and BMP1 responsible for processing of type I collagen.

Immunoglobulin G (IgG) is a major effector molecule of the human immune response, and aberrations in IgG glycosylation are linked to various diseases. However, the molecular mechanisms underlying protein glycosylation are still poorly understood. We present a data-driven approach to infer reactions in the IgG glycosylation pathway using large-scale mass-spectrometry measurements. Gaussian graphical models are used to construct association networks from four cohorts. We find that glycan pairs with high partial correlations represent enzymatic reactions in the known glycosylation pathway, and then predict new biochemical reactions using a rule-based approach. Validation is performed using data from a GWAS and results from three in vitro experiments. We show that one predicted reaction is enzymatically feasible and that one rejected reaction does not occur in vitro. Moreover, in contrast to previous knowledge, enzymes involved in our predictions colocalize in the Golgi of two cell lines, further confirming the in silico predictions., IgG glycosylation is an important factor in immune function, yet the molecular details of protein glycosylation remain poorly understood. The data-driven approach presented here uses large-scale plasma IgG mass spectrometry measurements to infer new biochemical reactions in the glycosylation pathway.

PURPOSE: Iron overload accelerates bone loss in mice lacking the bone morphogenetic protein 6 (Bmp6) gene, which is the key endogenous regulator of hepcidin, iron homeostasis gene. We investigated involvement of other BMPs in preventing haemochromatosis and subsequent osteopenia in Bmp6-/- mice. ----- METHODS: Iron-treated wild-type (WT) and Bmp6-/- mice were analysed for hepcidin messenger RNA (mRNA) and tissue and blood BMP levels by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry, Western blot, enzyme-linked immunosorbent assay (ELISA) and proximity extension assay. BMPs labeled with technetium-99m were used in pharmacokinetic studies. ----- RESULTS: In WT mice, 4 h following iron challenge, liver Bmp6 and hepcidin expression were increased, while expression of other Bmps was not affected. In parallel, we provided the first evidence that BMP6 circulates in WT mice and that iron increased the BMP6 serum level and the specific liver uptake of (99m)Tc-BMP6. In Bmp6-/- mice, iron challenge led to blunted activation of liver Smad signaling and hepcidin expression with a delay of 24 h, associated with increased Bmp5 and Bmp7 expression and increased Bmp2, 4, 5 and 9 expression in the duodenum. Liver Bmp7 expression and increased circulating BMP9 eventually contributed to the late hepcidin response. This was further supported by exogenous BMP7 therapy resulting in an effective hepcidin expression followed by a rapid normalisation of plasma iron values and restored osteopenia in Bmp6-/- mice. ----- CONCLUSION: In Bmp6-/- mice, iron activated endogenous compensatory mechanisms of other BMPs that were not sufficient for preventing hemochromatosis and bone loss. Administration of exogenous BMP7 was effective in correcting the plasma iron level and bone loss, indicating that BMP6 is an essential but not exclusive in vivo regulator of iron homeostasis.

The biological and clinical relevance of glycosylation is becoming increasingly recognized, leading to a growing interest in large-scale clinical and population-based studies. In the past few years, several methods for high-throughput analysis of glycans have been developed, but thorough validation and standardization of these methods is required before significant resources are invested in large-scale studies. In this study, we compared liquid chromatography, capillary gel electrophoresis, and two MS methods for quantitative profiling of N-glycosylation of IgG in the same data set of 1201 individuals. To evaluate the accuracy of the four methods we then performed analysis of association with genetic polymorphisms and age. Chromatographic methods with either fluorescent or MS-detection yielded slightly stronger associations than MS-only and multiplexed capillary gel electrophoresis, but at the expense of lower levels of throughput. Advantages and disadvantages of each method were identified, which should inform the selection of the most appropriate method in future studies. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Svrha: Željelo se pronaći optimalne uvjete i metode za identifikaciju maksimalnog broja proteina u uzorcima tkiva gingive pacijentice s agresivnim parodontitisom. Pritom smo se koristili tehnologijom Label-free kvantifikacije, tekućinske kromatografije i spektrometrije masa (LC –MS/MS), te usporedili proteom zdravih i bolesnih uzoraka tkiva gingive. Materijali i metode: Četiri uzorka gingivnog tkiva (dva zdrava i dva bolesna) uzeta su od pacijentice, inače nepušačice, koja boluje od teškog oblika generaliziranog agresivnog parodontitisa. Proteini tkivnog lizata razdvojeni su 1D gel-elektroforezom nakon koje je slijedila digestija u gelu te mjerenje proteina nanoljestvicom HPLC-sistema preko nanoelektro spreja, ionizacijskog izvora i spektrometra mase. Dobiveni podaci obrađeni su u programu MaxQuant. Rezultati: Label-free kvantifikacija i LC-MS/MS analiza, zajedno s pripremom uzoraka, 1D gel-elektroforezom i digestijom u gelu pokazali su se kao učinkovita metoda za analizu proteoma gingivnog tkiva. Ukupan broj identificiranih i kvantificiranih proteina iznosio je 2429. Proteini koji su pokazali pojačanu ekspresiju u tkivu zahvaćenom agresivnim parodontitisom bili su: imunoglobulinski gama-1 lanac regije C, imunoglobulinski kapa lanac regije C i imunoglobulinski gama-3 lanac regije C (Ig gamma-1 chain C region, Ig kappa chain C region i Ig gamma-3 chain C region), a desmoplakin, aneksini, proteini S100-A8/A9 i keratini pokazali su sniženu ekspresiju. Zaključak: Ovo eksperimentalno istraživanje omogućuje novi uvid u proteomski profil zdravog i bolesnog gingivnog tkiva, što bi moglo pridonijeti napretku u dijagnostici i prognozi te boljem razumijevanju patogeneze agresivnog parodontitisa. Tehnologija LCMS/ MS pokazala se učinkovitom kad je riječ o proteomskoj analizi gingivnog tkiva., Aim: The aim of this study was to find an optimal method to identify maximal number of proteins from human gingival tissue samples in a patient with aggressive periodontitis using label-free quantitative liquid chromatography mass spectrometry technology (LC-MS/MS), and to compare proteome of healthy and diseased gingival tissue samples. Materials and methods: Four gingival tissue samples (2 healthy, 2 diseased) were obtained from a nonsmoking female patient suffering from severe generalized aggressive periodontitis. Proteins from the tissue lysate were separated by 1D gel electrophoresis, in gel digested, measured by nano-scale HPLC system coupled to a mass spectrometer through a nano-electrospray ionization source. Raw data were processed by MaxQuant software. Results: Label-free quantitative LC-MS/MS analysis together with sample preparation, 1D gel electrophoresis and in-gel digestion showed to be an effective method for the proteome analysis of the gingival periodontal tissues. A total of 2429 proteins were identified and quantified. Among proteins upregulated in disease were: Ig gamma-1 chain C region, Ig kappa chain C region, Ig gamma-3 chain C region, and among downregulated proteins in the disease were: Desmoplakin, annexins, S100-A8/A9 proteins, and keratins. Conclusions: This pilot study is providing a novel insight into the protein profiles of gingival healthy and diseased tissues, which may contribute to the improvements in diagnosis, prognosis and better understanding of aggressive periodontitis pathogenesis. LC-MS/MS technology proved to be an effective method for the proteome analysis of the gingival tissues.

Using proteomic approach in this study, we sought to identify proteins with heparin affinity associated with rheumatoid arthritis (RA), psoriatic arthritis (PsA) and non-inflammatory arthritis (NIA).Plasma samples from adult RA, PsA and NIA patients, 20 of each, were collected. After enrichment of proteins with heparin affinity, SDS-PAGE and in-gel digestion with trypsin were performed. Peptides were concentrated, micro-purified, separated and measured by nano-scale HPLC system coupled to a mass spectrometer. Peak lists were generated from raw spectra and searched against human complete proteome set by MaxQuant software. Statistical analysis of protein relative expression levels was done in IPython interactive Python shell using NumPy and Matplotlib libraries. Individual protein impact on the whole dataset correlation was done by excluding one protein at a time and calculating the correlation coefficient of remaining data points.Three hundred and eighty-four different proteins were identified keeping false discovery rate to 1%, from which 163 were identified in all three conditions. The plasma proteome showed a good correlation between rheumatoid (RA) and psoriatic arthritis (PsA). Out of 10 proteins whose impact on the correlation coefficient fell outside of two standard deviations from the mean, four were up-regulated (complement factor I, complement component C8 beta, glyceraldehyde-3-phosphate dehydrogenase and inter-alpha-trypsin inhibitor heavy chain H1), and two were down-regulated (immunoglobulin heavy chain V-III region BRO, and immunoglobulin J chain), both in PsA and RA by a similar ratio when compared to NIA. The remaining four proteins (Serpin A11, complement factor H-related protein 5, cartilage acidic protein 1 and coagulation factor IX) were down-regulated in PsA and up-regulated in RA when compared to NIA.We found differently expressed proteins in patients with inflammatory and non-inflammatory rheumatic conditions. Out of 384 proteins with heparin affinity four proteins should be further validated as potential diagnostic biomarkers in patients with RA and PsA.

1.1 Bone morphogenetic proteins Bone morphogenetic proteins (BMPs) are family of growth factors. (Chen et al., 2004; Vukicevic & Sampath, 2008) Discovered in context of bone biology, today they are recognized as important signaling molecules in wide range of biological processes, such as vertebrate embryonic development (Hogan, 1996), mesenchymal stem cell differentiation (Vukicevic & Grgurevic, 2009), kidney fibrosis, and more. For the last years BMP-2 and BMP-7 are used as therapeutics in orthopedics, harnessing their regenerative potential as growth factors. From the onset of medicine scholars have been aware of the bone regenerative potential. In 1965. Urist was first to show that demineralized bone matrix (DBM) can induce bone growth if implanted into extraskeletal site. Active component from DBM was named bone morphogenetic protein by Urist & Strates (1971). Purification, cloning and sequencing of BMP was done almost 20 years later by Wozney et al. (1988). They showed that BMP is not a single protein but a family of growth factors. From introduction of the BMP term through cloning and sequencing of individual BMPs in late 1980s, scientific output in the field has constantly grown and has exceeded 1500 papers in 2010. (Figure 1) BMPs are part of transforming growth factor (TGF) superfamily of proteins. In humans TGFsuperfamily constitutes of 37 proteins. (Figure 2) Beside BMPs, TGFsuperfamily includes TGFproteins, inhibins (INH), growth/differentiation factors (GDF) and few others: artemin (ARTN), glial cell line-derived neurotrophic factor (GDNF), left-right determination factor 1 (LFTY1), LFTY2, muellerian-inhibiting factor (MIS), nodal homolog (NODAL), neurturin (NRTN) and persephin (PSPN). BMPs are functionally and structurally very conserved throughout animal kingdom. Their biological importance is reflected through functional and structural redundancy of different BMPs in single species. BMPs are translated as pre-propeptides. Signal peptide targets them for secretion out of cell. Prodomain is two thirds to four fifths of total peptide length and The Use of Mass Spectrometry in Characterization of Bone Morphogenetic Proteins from Biological Samples

Regenerative periodontal therapy is unpredictable and limited. Cementum regeneration is necessary for the proper development of a periodontal ligament. The precise mechanism how BMPs induce differentiation and mineralization of cementoblasts remains undetermined. The purpose of this study was to evaluate the effect of BMP7 on an early proteome and gene expression profile of cementoblastic OCCM-30 cells in vitro. Immortalized murine OCCM.30 cells were treated with BMP7 and evaluated for proliferative activity, mineralization by Alizarin red-S, early proteome profile using liquid chromatography-mass spectrometry (LC-MS), and gene expression using quantitative RT-PCR. BMP7 increased cell proliferation at 24 and 48 hours with higher doses decreasing the cell proliferation at 48 hours. BMP7 induced mineralization of cementoblasts after 8 days. Using LC-MS we identified 1117 proteins out of which many belonged to extracellular matrix proteins. We further analyzed by qPCR the gene expression of several relevant genes and found that in a dose of 150 ng/ml, BMP7 upregulated BMP1, TGF-β1, osterix, osteoprotegerin, procollagen I and II, PCPE1, noggin ; and downregulated BMP6 and chordin. The dose of 300 ng/ml of BMP7 downregulated most of the genes at 24 hours following therapy. BMP7 directly affects differentiation, mineralization and expression of genes involved in the function of cementoblasts.

BACKGROUND: Glycosylation is one of the most common post-translation modifications with large influences on protein structure and function. The effector function of immunoglobulin G (IgG) alters between pro- and anti-inflammatory, based on its glycosylation. IgG glycan synthesis is highly complex and dynamic.METHODS: With the use of two different analytical methods for assessing IgG glycosylation, we aim to elucidate the link between DNA methylation and glycosylation of IgG by means of epigenome-wide association studies. In total, 3000 individuals from 4 cohorts were analyzed.RESULTS: The overlap of the results from the two glycan measurement panels yielded DNA methylation of 7 CpG-sites on 5 genomic locations to be associated with IgG glycosylation: cg25189904 (chr.1, GNG12); cg05951221, cg21566642 and cg01940273 (chr.2, ALPPL2); cg05575921 (chr.5, AHRR); cg06126421 (6p21.33); and cg03636183 (chr.19, F2RL3). Mediation analyses with respect to smoking revealed that the effect of smoking on IgG glycosylation may be at least partially mediated via DNA methylation levels at these 7 CpG-sites.CONCLUSION: Our results suggest the presence of an indirect link between DNA methylation and IgG glycosylation that may in part capture environmental exposures.GENERAL SIGNIFICANCE: An epigenome-wide analysis conducted in four population-based cohorts revealed an association between DNA methylation and IgG glycosylation patterns. Presumably, DNA methylation mediates the effect of smoking on IgG glycosylation.

BACKGROUND AND AIMS: Causes of inflammatory bowel diseases are not well understood and the most prominent forms, Crohn's disease (CD) and ulcerative colitis (UC), are sometimes hard to distinguish. Glycosylation of IgG has been associated with CD and UC. IgG Fc-glycosylation affects IgG effector functions. We evaluated changes in IgG Fc- glycosylation associated with UC and CD, as well as with disease characteristics in different patient groups. METHODS: We analyzed 3441 plasma samples obtained from 2 independent cohorts of patients with CD (874 patients from Italy and 391 from the United States) or UC (1056 from Italy and 253 from the US and healthy individuals [controls] ; 427 in Italy and 440 from the United States). IgG Fc-glycosylation (tryptic glycopeptides) was analyzed by liquid chromatography coupled to mass spectrometry. We analyzed associations between disease status (UC vs controls, CD vs controls, and UC vs CD) and glycopeptide traits, and associations between clinical characteristics and glycopeptide traits, using a logistic regression model with age and sex included as covariates. RESULTS: Patients with CD or UC had lower levels of IgG galactosylation than controls. For example, the odds ratio (OR) for IgG1 galactosylation in patients with CD was 0.59 (95% confidence interval [CI], 0.51-0.69) and for patients with UC was 0.81 (95% CI, 0.71-0.92). Fucosylation of IgG was increased in patients with CD vs controls (for IgG1: OR, 1.27 ; 95% CI, 1.12-1.44), but decreased in patients with UC vs controls (for IgG23: OR, 0.72 ; 95% CI, 0.63-0.82). Decreased galactosylation associated with more severe CD or UC, including the need for surgery in patients with UC vs controls (for IgG1: OR, 0.69 ; 95% CI, 0.54-0.89) and in patients with CD vs controls (for IgG23: OR, 0.78 ; 95% CI, 0.66-0.91). CONCLUSIONS: In a retrospective analysis of plasma samples from patients with CD or UC, we associated levels of IgG Fc-glycosylation with disease (compared to controls) and its clinical features. These findings could increase our understanding of mechanisms of CD and UC pathogenesis and be used to develop diagnostics or guide treatment.