Automated high throughput IgG N-glycosylation sample preparation method development on the Tecan Freedom EVO platform.

Introduction: Glycomics, focusing on the role of glycans in biological processes, particularly their influence on the folding, stability and receptor interactions of glycoconjugates like antibodies, is vital for our understanding of biology. Changes in immunoglobulin G (IgG) N-glycosylation have been associated with various physiological and pathophysiological conditions. Nevertheless, time-consuming manual sample preparation is one of the limitations in the glycomics diagnostic implementation. The study aimed to develop an automated method for sample preparation on the Tecan Freedom Evo 200 platform and compare its efficiency and precision with the manual counterpart. Materials and methods: The initial method development included 32 pooled blood plasma technical replicates. An additional 24 pooled samples were used in the method comparison along with 78 random duplicates of plasma samples collected from 10,001 Dalmatians biobank to compare the manual and automated methods. Results: The development resulted in a new automated method. For the automated method, glycan peaks comprising 91% of the total sample glycan showed a variation of less than 5% while 92% of the total sample showed a variation of less than 5% for the manual method. The results of the Passing-Bablok regression indicated no differences between the automated and manual methods for 12 glycan peaks (GPs). However, for 8 GPs systematic difference was present, while both systematic and proportional differences were present for four GPs. Conclusions: The developed automated sample preparation method for IgG glycan analysis reduced exposure to hazardous chemicals and offered a simplified workflow. Despite slight differences between the methods, the new automated method showed high precision and proved to be highly comparable to its manual counterpart.

High-throughput immunoaffinity enrichment and N-glycan analysis of human plasma haptoglobin.

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 regulates RdDM-mediated DNA methylation via a cullin 3 independent mechanism.

KEY MESSAGE: 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.

Developments and perspectives in high-throughput protein glycomics: enabling the analysis of thousands of samples.

Glycans expand the structural complexity of proteins by several orders of magnitude, resulting in a tremendous analytical challenge when including them in biomedical research. Recent glycobiological research is painting a picture in which glycans represent a crucial structural and functional component of the majority of proteins, with alternative glycosylation of proteins and lipids being an important regulatory mechanism in many biological and pathological processes. Since interindividual differences in glycosylation are extensive, large studies are needed to map the structures and to understand the role of glycosylation in human (patho)physiology. Driven by these challenges, methods have emerged, which can tackle the complexity of glycosylation in thousands of samples, also known as high-throughput (HT) glycomics. For facile dissemination and implementation of HT glycomics technology, the sample preparation, analysis, as well as data mining, need to be stable over a long period of time (months/years), amenable to automation, and available to non-specialized laboratories. Current HT glycomics methods mainly focus on protein N-glycosylation and allow to extensively characterize this subset of the human glycome in large numbers of various biological samples. The ultimate goal in HT glycomics is to gain better knowledge and understanding of the complete human glycome using methods that are easy to adapt and implement in (basic) biomedical research. Aiming to promote wider use and development of HT glycomics, here, we present currently available, emerging, and prospective methods and some of their applications, revealing a largely unexplored molecular layer of the complexity of life.

Mass Spectrometry-Based Methods for Immunoglobulin G N-Glycosylation Analysis.

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.

Global variability of the human IgG glycome.

Immunoglobulin G (IgG) is the most abundant serum antibody which structural characteristics and effector functions are modulated through the attachment of various sugar moieties called glycans. Composition of the IgG N-glycome changes with age of an individual and in different diseases. Variability of IgG glycosylation within a population is well studied and is known to be affected by both 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 IgG, analyzed in 5 different populations totaling 10,482 IgG glycomes, and of IgG's fragment crystallizable region (Fc), analyzed in 2,579 samples from 27 populations sampled across the world. Country of residence associated with many N-glycan features and the strongest association was with monogalactosylation where it explained 38% of variability. IgG monogalactosylation strongly correlated with the development level of a country, defined by United Nations health and socioeconomic development indicators, and with the expected lifespan. Subjects from developing countries had low levels of IgG galactosylation, characteristic for inflammation and ageing. Our results suggest that citizens of developing countries may be exposed to environmental factors that can cause low-grade chronic inflammation and the apparent increase in biological age.

The MATH-BTB Protein TaMAB2 Accumulates in Ubiquitin-Containing Foci and Interacts With the Translation Initiation Machinery in Arabidopsis.

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.

Glycomics for Type 2 Diabetes Biomarker Discovery: Promise of Immunoglobulin G Subclass-Specific Fragment Crystallizable N-glycosylation in the Uyghur Population.

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.

MIgGGly (mouse IgG glycosylation analysis) - a high-throughput method for studying Fc-linked IgG N-glycosylation in mice with nanoUPLC-ESI-MS.

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.

Promoter methylation of the MGAT3 and BACH2 genes correlates with the composition of the immunoglobulin G glycome in inflammatory bowel disease.

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.

Genome-Wide Association Study on Immunoglobulin G Glycosylation Patterns.

Immunoglobulin G (IgG), a glycoprotein secreted by plasma B-cells, plays a major role in the human adaptive immune response and are associated with a wide range of diseases. Glycosylation of the Fc binding region of IgGs, responsible for the antibody's effector function, is essential for prompting a proper immune response. This study focuses on the general genetic impact on IgG glycosylation as well as corresponding subclass specificities. To identify genetic loci involved in IgG glycosylation, we performed a genome-wide association study (GWAS) on liquid chromatography electrospray mass spectrometry (LC-ESI-MS)-measured IgG glycopeptides of 1,823 individuals in the Cooperative Health Research in the Augsburg Region (KORA F4) study cohort. In addition, we performed GWAS on subclass-specific ratios of IgG glycans to gain power in identifying genetic factors underlying single enzymatic steps in the glycosylation pathways. We replicated our findings in 1,836 individuals from the Leiden Longevity Study (LLS). We were able to show subclass-specific genetic influences on single IgG glycan structures. The replicated results indicate that, in addition to genes encoding for glycosyltransferases (i.e., ST6GAL1, B4GALT1, FUT8, and MGAT3), other genetic loci have strong influences on the IgG glycosylation patterns. A novel locus on chromosome 1, harboring RUNX3, which encodes for a transcription factor of the runt domain-containing family, is associated with decreased galactosylation. Interestingly, members of the RUNX family are cross-regulated, and RUNX3 is involved in both IgA class switching and B-cell maturation as well as T-cell differentiation and apoptosis. Besides the involvement of glycosyltransferases in IgG glycosylation, we suggest that, due to the impact of variants within RUNX3, potentially mechanisms involved in B-cell activation and T-cell differentiation during the immune response as well as cell migration and invasion involve IgG glycosylation.

Publisher Correction: Network inference from glycoproteomics data reveals new reactions in the IgG glycosylation pathway.

Correction to: Nature Communications (2017) 8:1231. doi:10.1038/s41467-017-01525-0.

Glycosylation of Immunoglobulin G Associates With Clinical Features of Inflammatory Bowel Diseases.

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.

Blood plasma/IgG N-glycome biosignatures associated with major depressive disorder symptom severity and the antidepressant response.

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.

IgG glycosylation and DNA methylation are interconnected with smoking.

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.

Network inference from glycoproteomics data reveals new reactions in the IgG glycosylation pathway.

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.

Effects of allergic diseases and age on the composition of serum IgG glycome in children.

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.

LaCyTools: A Targeted Liquid Chromatography-Mass Spectrometry Data Processing Package for Relative Quantitation of Glycopeptides.

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 (tr) 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, LaCyTools 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 <0.5%) and higher trueness due to the use of MS peak area instead of MS peak intensity. In conclusion, LaCyTools is an accurate automated data processing tool for high-throughput analysis of LC-MS glycoproteomics data. Released under the Apache 2.0 license, it is freely available on GitHub ( https://github.com/Tarskin/LaCyTools ).

Exogenous BMP7 corrects plasma iron overload and bone loss in Bmp6-/- mice.

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.

Comparative performance of four methods for high-throughput glycosylation analysis of immunoglobulin G in genetic and epidemiological research.

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.