Objective
Anti–citrullinated protein antibodies (ACPAs) are disease‐specific biomarkers in rheumatoid arthritis (RA). More than 90% of IgG ACPAs harbor N‐linked glycans in the antibody variable (V) ...domain. The corresponding N‐glycosylation sites in ACPA V‐region sequences result from somatic hypermutation, a T cell–dependent process. As ample evidence indicates that T cells drive the maturation of the ACPA response prior to arthritis onset, we undertook this study to investigate whether the presence of glycans in IgG ACPA V domains predicts the transition from predisease autoimmunity to overt RA.
Methods
We analyzed 2 independent sets of serum samples obtained from 126 ACPA‐positive first‐degree relatives (FDRs) of RA patients. Both sets originated from an Indigenous North American population and comprised cross‐sectional and longitudinal samples of individuals who did or did not develop inflammatory arthritis. Serum IgG ACPAs were affinity‐purified and subjected to ultra high‐performance liquid chromatography–based glycan analysis.
Results
In both data sets, FDR‐derived IgG ACPA displayed markedly lower levels of V domain glycans (<50%) compared to IgG ACPA from RA patients. Notably, FDRs who later developed RA showed extensive V‐domain glycosylation before the onset of arthritis. Moreover, IgG ACPA V‐domain glycosylation was strongly associated with future development of RA (hazard ratio 6.07 95% confidence interval 1.46–25.2; P = 0.013).
Conclusion
Extensive glycosylation of the IgG ACPA V domain is present in a subset of predisposed FDRs of Indigenous North American RA patients. The presence of this feature substantially increases the risk of RA development. Based on these findings, we propose that glycosylation of the IgG ACPA V domain represents a predictive marker for RA development in ACPA‐positive individuals and may serve to better target prevention measures.
This chapter contains a nanoscale liquid chromatography-mass spectrometry method for the glycoform profiling of the conserved Fc N-glycosylation site of monoclonal and polyclonal immunoglobulin G ...(IgG). It describes in detail LaCyTools, a program for automated data (pre-)processing of the obtained LC-MS data. The minimal sample preparation necessary is explained as well as an optional method for affinity purification of (polyclonal) antibodies from serum or plasma.After (optional) affinity purification, the pure IgG is cleaved with trypsin. The tryptic glycopeptides are separated almost exclusively on their peptide backbone. This ensures similar response factors for all glycoforms in the MS detection and allows the collection of separate glycoform profiles for different IgG isoforms or allotypes. LaCyTools automatically performs label-free (relative) quantitation of the obtained data after minimal manual input and additionally calculates several quality criteria which can be used for data curation at the level of both individual analytes and entire LC-MS runs.
Human plasma protein N-glycosylation Clerc, Florent; Reiding, Karli R.; Jansen, Bas C. ...
Glycoconjugate journal,
06/2016, Letnik:
33, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be ...involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the
N
-glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma
N
-glycosylation profile determined at the released glycan level.
Sialylation is a glycosylation feature that occurs in different linkages at the non-reducing end of a glycan moiety, the linkage isomers are often differentially associated with various biological ...processes. Due to very similar physico-chemical properties, the separation of isomeric sialylated glycopeptides remains challenging but of utmost importance in the biomedicine and biotechnology, including biomarker discovery, glyco-engineering and biopharmaceutical characterization. This study presents the implementation of a high-resolution separation platform based on capillary electrophoresis - mass spectrometry (CE-MS) allowing for the selective analysis of α2,3- and α2,6-sialylated glycopeptides. These differentially linked glycopeptides showed an identical fragmentation pattern (collision induced dissociation) but different electrophoretic mobilities, allowing for baseline separation of the different linkages without the need for an extensive sample preparation. The different migration behavior between the two moieties was found to correlate with differences in pK
values. Using a novel methodology adapted from the so-called internal standard CE approach, a relative difference of 3.4·10
in pK
unit was determined. This approach was applied for the analysis of tryptic glycopeptides of prostate specific antigen, which shows highly complex and heterogeneous glycosylation. The developed platform therefore appears attractive for the identification of differentially linked sialic acids that may be related to pathological conditions.
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 t r, 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).
The study of N-linked glycosylation has long been complicated by a lack of bioinformatics tools. In particular, there is still a lack of fast and robust data processing tools for targeted (relative) ...quantitation. We have developed modular, high-throughput data processing software, MassyTools, that is capable of calibrating spectra, extracting data, and performing quality control calculations based on a user-defined list of glycan or glycopeptide compositions. Typical examples of output include relative areas after background subtraction, isotopic pattern-based quality scores, spectral quality scores, and signal-to-noise ratios. We demonstrated MassyTools’ performance on MALDI-TOF-MS glycan and glycopeptide data from different samples. MassyTools yielded better calibration than the commercial software flexAnalysis, generally showing 2-fold better ppm errors after internal calibration. Relative quantitation using MassyTools and flexAnalysis gave similar results, yielding a relative standard deviation (RSD) of the main glycan of ∼6%. However, MassyTools yielded 2- to 5-fold lower RSD values for low-abundant analytes than flexAnalysis. Additionally, feature curation based on the computed quality criteria improved the data quality. In conclusion, we show that MassyTools is a robust automated data processing tool for high-throughput, high-performance glycosylation analysis. The package is released under the Apache 2.0 license and is freely available on GitHub (https://github.com/Tarskin/MassyTools).
It has been reported that glycosylation can influence the proteolytic cleavage of proteins. A thorough investigation of this phenomenon was conducted for the serine protease trypsin, which is ...essential in many proteomics workflows. Monoclonal and polyclonal immunoglobulin G biopharmaceuticals were employed as model substances, which are highly relevant for the bioanalytical applications. Relative quantitation of glycopeptides derived from the conserved Fc-glycosylation site allowed resolution of biases on the level of individual glycan compositions. As a result, a strong preferential digestion of high mannose, hybrid, alpha2-3-sialylated and bisected glycoforms was observed over the most abundant neutral, fucosylated glycoforms. Interestingly, this bias was, to a large extent, dependent on the intact higher order structure of the antibodies and, consequently, was drastically reduced in denatured versus intact antibodies. In addition, a cleavage protocol with acidic denaturation was tested, which featured reduced hands-on time and toxicity while showing highly comparable results to a published denaturation, reduction, and alkylation based protocol.
Neurological complications in COVID-19 patients admitted to an intensive care unit (ICU) have been previously reported. As the pandemic progressed, therapeutic strategies were tailored to new ...insights. This study describes the incidence, outcome, and types of reported neurological complications in invasively mechanically ventilated (IMV) COVID-19 patients in relation to three periods during the pandemic.
IMV COVID-19 ICU patients from the Dutch Maastricht Intensive Care COVID (MaastrICCht) cohort were included in a single-center study (March 2020 – October 2021). Demographic, clinical, and follow-up data were collected. Electronic medical records were screened for neurological complications during hospitalization. Three distinct periods (P1, P2, P3) were defined, corresponding to periods with high hospitalization rates. ICU survivors with and without reported neurological complications were compared in an exploratory analysis.
IMV COVID-19 ICU patients (n=324; median age 64 IQR 57–72 years; 238 males (73.5%)) were stratified into P1 (n=94), P2 (n=138), and P3 (n=92). ICU mortality did not significantly change over time (P1=38.3%; P2=41.3%; P3=37.0%; p=.787). The incidence of reported neurological complications during ICU admission gradually decreased over the periods (P1=29.8%; P2=24.6%; P3=18.5%; p=.028). Encephalopathy/delirium (48/324 (14.8%)) and ICU-acquired weakness (32/324 (9.9%)) were most frequently reported and associated with ICU treatment intensity. ICU survivors with neurological complications (n=53) were older (p=.025), predominantly male (p=.037), and had a longer duration of IMV (p<.001) and ICU stay (p<.001), compared to survivors without neurological complications (n=132). A multivariable analysis revealed that only age was independently associated with the occurrence of neurological complications (ORadj=1.0541; 95% CI=1.0171–1.0925; p=.004). Health-related quality-of-life at follow-up was not significantly different between survivors with and without neurological complications (n = 82, p=.054).
A high but decreasing incidence of neurological complications was reported during three consecutive COVID-19 periods in IMV COVID-19 patients. Neurological complications were related to the intensity of ICU support and treatment, and associated with prolonged ICU stay, but did not lead to significantly worse reported health-related quality-of-life at follow-up.
•Neurological complications are common in mechanically ventilated COVID-19 patients.•The most common neurological complications are delirium and ICU-acquired weakness.•The incidence of neurological complications was highest early in the pandemic.•Neurological complications were related to the intensity of ICU support/treatment.
The objective of our study is to investigate the Fc glycosylation profiles of both antigen-specific IgG targeted against proteinase 3 (PR3-ANCA) and total IgG as prognostic markers of relapse in ...patients with Granulomatosis with Polyangiitis (GPA).
Seventy-five patients with GPA and a PR3-ANCA rise during follow-up were included, of whom 43 patients relapsed within a median period of 8 (2–16) months. The N-glycan at Asn297 of affinity-purified and denatured total IgG and PR3-ANCA was determined by mass spectrometry of glycopeptides in samples obtained at the time of the PR3-ANCA rise and at the time of the relapse or time-matched during remission.
Patients with total IgG1 exhibiting low galactosylation or low sialylation were highly prone to relapse after an ANCA rise (HR 3.46 95%-CI 1.73–6.96, p<0.0001 and HR 3.22 95%-CI 1.52–6.83, p=0.002, respectively).
In relapsing patients, total IgG1 galactosylation, sialylation and bisection significantly decreased and fucosylation significantly increased from the time of the PR3-ANCA rise to the relapse (p<0.0001, p=0.0087, p<0.0001 and p=0.0025), while the glycosylation profile remained similar in non-relapsing patients. PR3-ANCA IgG1 galactosylation, sialylation and fucosylation of PR3-ANCA IgG1 decreased in relapsing patients (p=0.0073, p=0.0049 and p=0.0205), but also in non-relapsing patients (p=0.0007, p=0.0114 and p=0.0002), while bisection increased only in non-relapsing patients (p<0.0001).
While Fc glycosylation profiles have been associated with clinically manifest autoimmune diseases, in the present study we show that low galactosylation and sialyation in total IgG1 but not PR3-ANCA IgG1 predicts disease reactivation in patients with GPA who experience an ANCA rise during follow-up. We postulate that glycosylation profiles may be useful in pre-emptive therapy studies using ANCA rises as guideline.
•Low galactosylation and sialyation in total IgG predicts disease reactivation in GPA patients who experience an ANCA rise•Total IgG changes towards a more inflammation-associated phenotype in relapsing patients but not in non-relapsing patients•Glycosylation profiles may be useful in pre-emptive therapy studies using ANCA rises as guideline
Patients with Granulomatosis with Polyangiitis suffer from (life-threatening) symptoms which can be kept under control using immunosuppressive therapy. However, these symptoms return in some patients, for reasons we do not yet fully understand. The disease is most likely caused by an antibody, a protein that is normally made in response to a foreign substance but can also be targeted against parts of our own body. We questioned whether there are differences in the antibodies that cause symptoms to return in some patients but not in all patients.
To do this, within a group of patients in which the disease was inactive we investigated a glycan, a type of sugar group, that is attached to immunoglobulin G, the most common class of antibody. We found that the composition of this glycan showed variation in the number of certain monosaccharides, such as galactose and sialic acids. Patients in which the antibodies carried less galactose or sialic acid were more likely to suffer from symptoms of disease reactivation in the future. Moreover, the amount of sialic acid and galactose decreased over time in patients that developed symptoms, but not in patients for which the disease remained dormant. These findings may in the future be used to monitor people with inactive disease, so that timely treatment can be started if the disease becomes active.
Deubiquitinating enzymes are key regulators in the ubiquitin system and an emerging class of drug targets. These proteases disassemble polyubiquitin chains and many deubiquitinases show selectivity ...for specific polyubiquitin linkages. However, most biochemical insights originate from studies of single diubiquitin linkages in isolation, whereas in cells all linkages coexist. To better mimick this diubiquitin substrate competition, we develop a multiplexed mass spectrometry-based deubiquitinase assay that can probe all ubiquitin linkage types simultaneously to quantify deubiquitinase activity in the presence of all potential diubiquitin substrates. For this, all eight native diubiquitins are generated and each linkage type is designed with a distinct molecular weight by incorporating neutron-encoded amino acids. Overall, 22 deubiquitinases are profiled, providing a three-dimensional overview of deubiquitinase linkage selectivity over time and enzyme concentration.
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