Plant N-glycan breakdown by human gut Bacteroides.

The major nutrients available to the human colonic microbiota are complex glycans derived from the diet. To degrade this highly variable mix of sugar structures, gut microbes have acquired a huge array of different carbohydrate-active enzymes (CAZymes), predominantly glycoside hydrolases, many of which have specificities that can be exploited for a range of different applications. Plant N-glycans are prevalent on proteins produced by plants and thus components of the diet, but the breakdown of these complex molecules by the gut microbiota has not been explored. Plant N-glycans are also well characterized allergens in pollen and some plant-based foods, and when plants are used in heterologous protein production for medical applications, the N-glycans present can pose a risk to therapeutic function and stability. Here we use a novel genome association approach for enzyme discovery to identify a breakdown pathway for plant complex N-glycans encoded by a gut Bacteroides species and biochemically characterize five CAZymes involved, including structures of the PNGase and GH92 α-mannosidase. These enzymes provide a toolbox for the modification of plant N-glycans for a range of potential applications. Furthermore, the keystone PNGase also has activity against insect-type N-glycans, which we discuss from the perspective of insects as a nutrient source.

The human gut symbiont Ruminococcus gnavus shows specificity to blood group A antigen during mucin glycan foraging: Implication for niche colonisation in the gastrointestinal tract.

The human gut symbiont Ruminococcus gnavus displays strain-specific repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-ß-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection.

Elevated concentrations of Neu5Ac and Neu5,9Ac2 in human plasma: potential biomarkers of cardiovascular disease.

Cardiovascular disease (CVD) is a group of health conditions affecting the heart and vascular system with very high prevalence and mortality rates. The presence of CVD is characterised by high levels of inflammation which have previously been associated with increased plasma concentrations of N-acetyl neuraminic acid (Neu5Ac). While Neu5Ac has been studied in the context of CVD, Neu5,9Ac2 has not, despite being the second most abundant sialic acid in human plasma. A small-scale pilot study of thirty plasma samples from patients with diagnosed CVD, and thirty age and sex-matched healthy controls, was designed to gain insight into sialic acids as biomarkers for CVD and potential future areas of study. Each sample was assayed for Neu5Ac and Neu5,9Ac2 concentrations. Mean Neu5Ac and Neu5,9Ac2 concentrations were significantly elevated in patients with CVD compared to healthy controls (Neu5Ac: P < 0.001; Neu5,9Ac2: P < 0.04). Receiver operator curve (ROC) analysis indicated that both Neu5Ac and Neu5,9Ac2 have reasonable predictive power for the presence of CVD (Neu5Ac AUC: 0.86; Neu5,9Ac2 AUC: 0.71). However, while Neu5Ac had both good sensitivity (0.82) and specificity (0.81), Neu5,9Ac2 had equivalent specificity (0.81) but very poor sensitivity (0.44). A combination marker of Neu5Ac + Neu5,9Ac2 showed improvement over Neu5Ac alone in terms of predictive power (AUC: 0.93), sensitivity (0.87), and specificity (0.90). Comparison to a known inflammatory marker, high sensitivity c-reactive protein (hs-CRP: P-value: NS, ROC:0.50) was carried out, showing that both Neu5Ac and Neu5,9Ac2 outperformed this marker. Further to this, hs-CRP values were combined with the three different sialic acid markers to determine any effect on the AUC values. A slight improvement in AUC was noted for each of the combinations, with Neu5Ac + Neu5,9Ac2 + hs-CRP giving the best AUC of 0.97 overall. Thus, Neu5Ac would appear to offer good potential as a predictive marker for the presence of CVD, which the addition of Neu5,9Ac2 predictive power improves, with further improvement seen by the addition of hs-CRP.

Structural and functional characterisation of a stable, broad-specificity multimeric sialidase from the oral pathogen Tannerella forsythia.

Sialidases are glycosyl hydrolase enzymes targeting the glycosidic bond between terminal sialic acids and underlying sugars. The NanH sialidase of Tannerella forsythia, one of the bacteria associated with severe periodontal disease plays a role in virulence. Here, we show that this broad-specificity enzyme (but higher affinity for α2,3 over α2,6 linked sialic acids) digests complex glycans but not those containing Neu5,9Ac. Furthermore, we show it to be a highly stable dimeric enzyme and present a thorough structural analysis of the native enzyme in its apo-form and in complex with a sialic acid analogue/ inhibitor (Oseltamivir). We also use non-catalytic (D237A) variant to characterise molecular interactions while in complex with the natural substrates 3- and 6-siallylactose. This dataset also reveals the NanH carbohydrate-binding module (CBM, CAZy CBM 93) has a novel fold made of antiparallel beta-strands. The catalytic domain structure contains novel features that include a non-prolyl cis-peptide and an uncommon arginine sidechain rotamer (R306) proximal to the active site. Via a mutagenesis programme, we identified key active site residues (D237, R212 and Y518) and probed the effects of mutation of residues in proximity to the glycosidic linkage within 2,3 and 2,6-linked substrates. These data revealed that mutagenesis of R306 and residues S235 and V236 adjacent to the acid-base catalyst D237 influence the linkage specificity preference of this bacterial sialidase, opening up possibilities for enzyme engineering for glycotechology applications and providing key structural information that for in silico design of specific inhibitors of this enzyme for the treatment of periodontitis.

Development of an exoglycosidase plate-based assay for detecting α1-3,4 fucosylation biomarker in individuals with HNF1A-MODY.

Maturity-onset diabetes of the young due to hepatocyte nuclear factor-1 alpha variants (HNF1A-MODY) causes monogenic diabetes. Individuals carrying damaging variants in HNF1A show decreased levels of α1-3,4 fucosylation, as demonstrated on antennary fucosylation of blood plasma N-glycans. The excellent diagnostic performance of this glycan biomarker in blood plasma N-glycans of individuals with HNF1A-MODY has been demonstrated using liquid chromatography methods. Here, we have developed a high-throughput exoglycosidase plate-based assay to measure α1-3,4 fucosylation levels in blood plasma samples. The assay has been optimized and its validity tested using 1000 clinical samples from a cohort of individuals with young-adult onset diabetes including cases with HNF1A-MODY. The α1-3,4 fucosylation levels in blood plasma showed a good differentiating power in identifying cases with damaging HNF1A variants, as demonstrated by receiver operating characteristic curve analysis with the AUC values of 0.87 and 0.95. This study supports future development of a simple diagnostic test to measure this glycan biomarker for application in a clinical setting.

Spatial Bayesian GLM on the cortical surface produces reliable task activations in individuals and groups.

The general linear model (GLM) is a widely popular and convenient tool for estimating the functional brain response and identifying areas of significant activation during a task or stimulus. However, the classical GLM is based on a massive univariate approach that does not explicitly leverage the similarity of activation patterns among neighboring brain locations. As a result, it tends to produce noisy estimates and be underpowered to detect significant activations, particularly in individual subjects and small groups. A recently proposed alternative, a cortical surface-based spatial Bayesian GLM, leverages spatial dependencies among neighboring cortical vertices to produce more accurate estimates and areas of functional activation. The spatial Bayesian GLM can be applied to individual and group-level analysis. In this study, we assess the reliability and power of individual and group-average measures of task activation produced via the surface-based spatial Bayesian GLM. We analyze motor task data from 45 subjects in the Human Connectome Project (HCP) and HCP Retest datasets. We also extend the model to multi-run analysis and employ subject-specific cortical surfaces rather than surfaces inflated to a sphere for more accurate distance-based modeling. Results show that the surface-based spatial Bayesian GLM produces highly reliable activations in individual subjects and is powerful enough to detect trait-like functional topologies. Additionally, spatial Bayesian modeling enhances reliability of group-level analysis even in moderately sized samples (n=45). Notably, the power of the spatial Bayesian GLM to detect activations above a scientifically meaningful effect size is nearly invariant to sample size, exhibiting high power even in small samples (n=10). The spatial Bayesian GLM is computationally efficient in individuals and groups and is convenient to implement with the open-source BayesfMRI R package.

Sialic Acid Derivatization of Fluorescently Labeled N-Glycans Allows Linkage Differentiation by Reversed-Phase Liquid Chromatography-Fluorescence Detection-Mass Spectrometry.

Sialic acids have diverse biological roles, ranging from promoting up to preventing protein and cellular recognition in health and disease. The various functions of these monosaccharides are owed, in part, to linkage variants, and as a result, linkage-specific analysis of sialic acids is an important aspect of glycomic studies. This has been addressed by derivatization strategies using matrix-assisted laser desorption/ionization mass spectrometry (MS) or sialidase digestion arrays followed by liquid chromatography (LC)-MS. Despite this, these approaches are unable to simultaneously provide unambiguous assignment of sialic acid linkages and assess further isomeric glycan features within a single measurement. Thus, for the first time, we present the combination of procainamide fluorescent labeling with sialic acid linkage-specific derivatization via ethyl esterification and amidation for the analysis of released plasma N-glycans using reversed-phase (RP)LC-fluorescence detection (FD)-MS. As a result, α2,3- and α2,6-sialylated N-glycans, with the same mass prior to derivatization, are differentiated based on retention time, precursor mass, and fragmentation spectra, and additional sialylated isomers were also separated. Furthermore, improved glycan coverage and protocol precision were found via the novel application using a combined FD-MS quantification approach. Overall, this platform achieved unambiguous assignment of N-glycan sialic acid linkages within a single RPLC-FD-MS measurement, and by improving their retention on RPLC, this technique can be used for future investigations of released N-glycans as an additional or orthogonal method to current analytical approaches.

Quantitative Standards of 4-O-Acetyl- and 9-O-Acetyl-N-Acetylneuraminic Acid for the Analysis of Plasma and Serum.

N-Acetylneuraminic acid (sialic acid, Neu5Ac) is one of a large, diverse family of nine-carbon monosaccharides that play roles in many biological functions such as immune response. Neu5Ac has previously been identified as a potential biomarker for the presence and pathogenesis of cardiovascular disease (CVD), diabetes and cancer. More recent research has highlighted acetylated sialic acid derivatives, specifically Neu5,9Ac2 , as biomarkers for oral and breast cancers, but advances in analysis have been hampered due to a lack of commercially available quantitative standards. We report here the synthesis of 9-O- and 4-O-acetylated sialic acids (Neu5,9Ac2 and Neu4,5Ac2 ) with optimisation of previously reported synthetic routes. Neu5,9Ac2 was synthesised in 1 step in 68 % yield. Neu4,5Ac2 was synthesised in 4 steps in 39 % overall yield. Synthesis was followed by analysis of these standards via quantitative NMR (qNMR) spectroscopy. Their utilisation for the identification and quantification of specific acetylated sialic acid derivatives in biological samples is also demonstrated.

Sialylation on O-linked glycans protects von Willebrand factor from macrophage galactose lectin-mediated clearance.

Terminal sialylation determines the plasma half-life of von Willebrand factor (VWF). A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we showed that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF antigen levels (P<0.05). Using a series of VWF truncations, we further demonstrated that the A1 domain of VWF is predominantly responsible for enabling the MGL interaction. Binding of both full-length and VWF-A1-A2-A3 to MGL was significantly enhanced in the presence of ristocetin (P<0.05), suggesting that the MGL-binding site in A1 is not fully accessible in globular VWF. Additional studies using different VWF glycoforms demonstrated that VWF O-linked glycans, clustered at either end of the A1 domain, play a key role in protecting VWF against MGLmediated clearance. Reduced sialylation has been associated with pathological, increased clearance of VWF in patients with von Willebrand disease. Herein, we demonstrate that specific loss of α2-3 linked sialylation from O-glycans results in markedly increased MGL-binding in vitro, and markedly enhanced MGL-mediated clearance of VWF in vivo. Our data further show that the asialoglycoprotein receptor (ASGPR) does not have a significant role in mediating the increased clearance of VWF following loss of O-sialylation. Conversely however, we observed that loss of N-linked sialylation from VWF drives enhanced circulatory clearance predominantly via the ASGPR. Collectively, our data support the hypothesis that in addition to regulating physiological VWF clearance, the MGL receptor works in tandem with ASGPR to modulate enhanced clearance of aberrantly sialylated VWF in the pathogenesis of von Willebrand disease.

Egg yolk sialylglycopeptide: purification, isolation and characterization of N-glycans from minor glycopeptide species.

Sialylglycopeptide (SGP) is a readily available naturally occurring glycopeptide obtained from hen egg yolk which is now commercially available. During SGP extraction, other minor glycopeptide species are identified, bearing N-glycan structures that might be of interest, such as asymmetrically branched and triantennary glycans. As the scale of SGP production increases, recovery of minor glycopeptides and their N-glycans can become more feasible. In this paper, we aim to provide structural characterization of the N-glycans derived from these minor glycopeptides.

Fucosidases from the human gut symbiont Ruminococcus gnavus.

The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-L-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-L-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1-3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3'-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers.

A capaciflector provides continuous and accurate respiratory rate monitoring for patients at rest and during exercise.

Respiratory rate (RR) is a marker of critical illness, but during hospital care, RR is often inaccurately measured. The capaciflector is a novel sensor that is small, inexpensive, and flexible, thus it has the potential to provide a single-use, real-time RR monitoring device. We evaluated the accuracy of continuous RR measurements by capaciflector hardware both at rest and during exercise. Continuous RR measurements were made with capaciflectors at four chest locations. In healthy subjects (n = 20), RR was compared with strain gauge chest belt recordings during timed breathing and two different body positions at rest. In patients undertaking routine cardiopulmonary exercise testing (CPET, n = 50), RR was compared with pneumotachometer recordings. Comparative RR measurement bias and limits of agreement were calculated and presented in Bland-Altman plots. The capaciflector was shown to provide continuous RR measurements with a bias less than 1 breath per minute (BPM) across four chest locations. Accuracy and continuity of monitoring were upheld even during vigorous CPET exercise, often with narrower limits of agreement than those reported for comparable technologies. We provide a unique clinical demonstration of the capaciflector as an accurate breathing monitor, which may have the potential to become a simple and affordable medical device.Clinical trial number: NCT03832205 https://clinicaltrials.gov/ct2/show/NCT03832205 registered February 6th, 2019.

Generation and Characterization of Native and Sialic Acid-Deficient IgE.

Efficient characterization of IgE antibodies and their glycan structures is required for understanding their function in allergy and in the emerging AllergoOncology field for antibody immunotherapy. We report the generation, glyco-profiling and functional analysis of native and sialic acid-deficient glyco-engineered human IgE. The antibodies produced from human embryonic kidney cells were purified via a human IgE class-specific affinity matrix and structural integrity was confirmed by SDS-PAGE and size-exclusion chromatography (SEC). Purified IgEs specific for the tumor-associated antigens Chondroitin Sulfate Proteoglycan 4 (CSPG4-IgE) and Human Epidermal Growth Factor Receptor 2 (HER2-IgE) were devoid of by-products such as free light chains. Using neuraminidase-A, we generated sialic acid-deficient CSPG4-IgE as example glyco-engineered antibody. Comparative glycan analyses of native and glyco-engineered IgEs by Hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC) indicated loss of sialic acid terminal residues and differential glycan profiles. Native and glyco-engineered CSPG4-IgEs recognized Fc receptors on the surface of human FcεRI-expressing rat basophilic leukemia RBL-SX38 cells, and of CD23/FcεRII-expressing human RPMI-8866 B-lymphocytes and bound to CSPG4-expressing A2058 human melanoma cells, confirming Fab-mediated recognition. When cross-linked on the cell surface, both IgEs triggered RBL-SX38 degranulation. We demonstrate efficient generation and functional competence of recombinant native and sialic acid-deficient IgEs.

Loss of ARNT in skeletal muscle limits muscle regeneration in aging.

The ability of skeletal muscle to regenerate declines significantly with aging. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT), a critical component of the hypoxia signaling pathway, was less abundant in skeletal muscle of old (23-25 months old) mice. This loss of ARNT was associated with decreased levels of Notch1 intracellular domain (N1ICD) and impaired regenerative response to injury in comparison to young (2-3 months old) mice. Knockdown of ARNT in a primary muscle cell line impaired differentiation in vitro. Skeletal muscle-specific ARNT deletion in young mice resulted in decreased levels of whole muscle N1ICD and limited muscle regeneration. Administration of a systemic hypoxia pathway activator (ML228), which simulates the actions of ARNT, rescued skeletal muscle regeneration in both old and ARNT-deleted mice. These results suggest that the loss of ARNT in skeletal muscle is partially responsible for diminished myogenic potential in aging and activation of hypoxia signaling holds promise for rescuing regenerative activity in old muscle.

A semi-automated, high throughput approach for O-glycosylation profiling of in vitro established cancer cell lines by MALDI-FT-ICR MS.

The study of protein O-glycosylation is important in biological research as O-glycans have been reported to regulate a multitude of molecular and cell biology processes occurring in cancer. It is known that alterations in O-glycosylation are involved in the development and progression of cancer. Their easy accessibility makes in vitro established cell lines suitable and useful models for studying biological mechanisms in disease. However, the O-glycosylation analysis of large numbers of samples, as required in systems biology and biomarker discovery studies, is often challenging. In the present study, O-glycans from three human colorectal cancer cell lines and two human pancreatic cancer cell lines were released by semi-automated, high throughput reductive ß-elimination and analysed using ultrahigh resolution MALDI-FT-ICR MS. Automated data integration and processing was performed using MassyTools, where the analyte was automatically included for relative quantitation based on a range of selection criteria including signal-to-noise ratio, mass error and isotopic pattern quality scores. A total of 126 O-glycan compositions, ranging from a single monosaccharide to large oligosaccharides exhibiting complex glycan motifs, were detected. The use of ultrahigh resolution MALDI-FTICR MS enabled glycan identification and quantitation in the matrix region of the spectrum. This approach has the potential to be used for O-glycosylation analysis of large numbers of samples, such as patient sample cohorts.

Interlaboratory evaluation of plasma N-glycan antennary fucosylation as a clinical biomarker for HNF1A-MODY using liquid chromatography methods.

Antennary fucosylation alterations in plasma glycoproteins have been previously proposed and tested as a biomarker for differentiation of maturity onset diabetes of the young (MODY) patients carrying a functional mutation in the HNF1A gene. Here, we developed a novel LC-based workflow to analyze blood plasma N-glycan fucosylation in 320 diabetes cases with clinical features matching those at risk of HNF1A-MODY. Fucosylation levels measured in two independent research centers by using similar LC-based methods were correlated to evaluate the interlaboratory performance of the biomarker. The interlaboratory study showed good correlation between fucosylation levels measured for the 320 cases in the two centers with the correlation coefficient (r) of up to 0.88 for a single trait A3FG3S2. The improved chromatographic separation allowed the identification of six single glycan traits and a derived antennary fucosylation trait that were able to differentiate individuals carrying pathogenic mutations from benign or no HNF1A mutation cases, as determined by the area under the curve (AUC) of up to 0.94. The excellent (r = 0.88) interlaboratory performance of the glycan biomarker for HNF1A-MODY further supports the development of a clinically relevant diagnostic test measuring antennary fucosylation levels.

Assays for the identification and quantification of sialic acids: Challenges, opportunities and future perspectives.

N-Acetyl neuraminic acid (sialic acid) is a monosaccharide generally found as the terminating unit on glycans, which in turn are found on the surface of cells and glycoproteins. These glycans aid in a variety of biological functions such as cell interactions and immune response. Sialic acid has been identified as a biomarker for cardiovascular disease, diabetes and a range of other inflammatory and degenerative conditions. It has also been identified as a marker for different types of cancer. Sialic acid levels vary depending on the level of inflammation present during the course of an inflammatory disease and it is overexpressed by tumours as a shield against the immune system. Since the discovery of sialic acid, numerous assays have been developed for the identification and quantification of different sialic acid derivative monosaccharides and these assays fall into four main groups: colorimetric, fluorometric, enzymatic and chromatographic/mass spectrometric, with much overlap between these. Given the importance of sialic acids in biological pathways, this review article critically appraises assays that are used to detect and quantify sialic acid and its derivatives. Thus it details the method, sensitivity, specificity and wider scope of a range of assays, and concludes by suggesting some future directions for assay development and application. In this way, insight is provided into assays that allow for the accurate quantitation of sialic acid in biological samples, which may facilitate identification of the roles of sialic acid in healthy and disease pathways.

High-throughput Serum N-Glycomics: Method Comparison and Application to Study Rheumatoid Arthritis and Pregnancy-associated Changes.

N-Glycosylation is a fundamentally important protein modification with a major impact on glycoprotein characteristics such as serum half-life and receptor interaction. More than half of the proteins in human serum are glycosylated, and the relative abundances of protein glycoforms often reflect alterations in health and disease. Several analytical methods are currently capable of analyzing the total serum N-glycosylation in a high-throughput manner.Here we evaluate and compare the performance of three high-throughput released N-glycome analysis methods. Included were hydrophilic-interaction ultra-high-performance liquid chromatography with fluorescence detection (HILIC-UHPLC-FLD) with 2-aminobenzamide labeling of the glycans, multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) with 8-aminopyrene-1,3,6-trisulfonic acid labeling, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with linkage-specific sialic acid esterification. All methods assessed the same panel of serum samples, which were obtained at multiple time points during the pregnancies and postpartum periods of healthy women and patients with rheumatoid arthritis (RA). We compared the analytical methods on their technical performance as well as on their ability to describe serum protein N-glycosylation changes throughout pregnancy, with RA, and with RA disease activity.Overall, the methods proved to be similar in their detection and relative quantification of serum protein N-glycosylation. However, the non-MS methods showed superior repeatability over MALDI-TOF-MS and allowed the best structural separation of low-complexity N-glycans. MALDI-TOF-MS achieved the highest throughput and provided compositional information on higher-complexity N-glycans. Consequentially, MALDI-TOF-MS could establish the linkage-specific sialylation differences within pregnancy and RA, whereas HILIC-UHPLC-FLD and xCGE-LIF demonstrated differences in α1,3- and α1,6-branch galactosylation. While the combination of methods proved to be the most beneficial for the analysis of total serum protein N-glycosylation, informed method choices can be made for the glycosylation analysis of single proteins or samples of varying complexity.

rPTMDetermine: A Fully Automated Methodology for Endogenous Tyrosine Nitration Validation, Site-Localization, and Beyond.

We present herein rPTMDetermine, an adaptive and fully automated methodology for validation of the identification of rarely occurring post-translational modifications (PTMs), using a semisupervised approach with a linear discriminant analysis (LDA) algorithm. With this strategy, verification is enhanced through similarity scoring of tandem mass spectrometry (MS/MS) comparisons between modified peptides and their unmodified analogues. We applied rPTMDetermine to (1) perform fully automated validation steps for modified peptides identified from an in silico database and (2) retrieve potential yet-to-be-identified modified peptides from raw data (that had been missed through conventional database searches). In part (1), 99 of 125 3-nitrotyrosyl-containing (nitrated) peptides obtained from a ProteinPilot search were validated and localized. Twenty nitrated peptides were falsely assigned because of incorrect monoisotopic peak assignments, leading to erroneous identification of deamidation and nitration. Five additional nitrated peptides were, however, validated after performing nonmonoisotopic peak correction. In part (2), an additional 236 unique nitrated peptides were retrieved and localized, containing 113 previously unreported nitration sites; 25 endogenous nitrated peptides with novel sites were selected and verified by comparison with synthetic analogues. In summary, we identified and confidently validated 296 unique nitrated peptides-collectively representing the largest number of endogenously identified 3-nitrotyrosyl-containing peptides from the cerebral cortex proteome of a Macaca fascicularis model of stroke. Furthermore, we harnessed the rPTMDetermine strategy to complement conventional database searching and enhance the confidence of assigning rarely occurring PTMs, while recovering many missed peptides. In a final demonstration, we successfully extended the application of rPTMDetermine to peptides featuring tryptophan oxidation.

A novel glycosidase plate-based assay for the quantification of galactosylation and sialylation on human IgG.

Changes in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories. Hence there is a growing demand for simple biochemical assays for analyzing these glycosylation changes. We have addressed this need by introducing a novel glycosidase plate-based assay for the absolute quantification of galactosylation and sialylation on IgG. IgG glycoproteins are treated with specific exoglycosidases to release the galactose and/or sialic acid residues. The released galactose monosaccharides are subsequently used in an enzymatic redox reaction that produces a fluorescence signal that is quantitative for the amount of galactosylation and, in-turn, sialylation on IgG. The glycosidase plate-based assay has the potential to be a simple, initial screening assay or an alternative assay to the usage of high-end analytical platforms such as HILIC-FLD-MSn when considering the analysis of galactosylation and sialylation on IgG. We have demonstrated this by comparing our assay to an industrial established HILIC-FLD-MSn glycomic analysis of 15 patient samples and obtained a Pearson's r correlation coefficient of 0.8208 between the two methods.