Characterization of Solitalea canadensis α-mannosidase with specific activity towards α1,3-Mannosidic linkages.

A recombinant exo-α-mannosidase from Solitalea canadensis (Sc3Man) has been characterized to exhibit strict specificity for hydrolyzing α1,3-mannosidic linkages located at the non-reducing end of glycans containing α-mannose. Enzymatic characterization revealed that Sc3Man operates optimally at a pH of 5.0 and at a temperature of 37 °C. The enzymatic activity was notably enhanced twofold in the presence of Ca2+ ions, emphasizing its potential dependency on this metal ion, while Cu2+ and Zn2+ ions notably impaired enzyme function. Sc3Man was able to efficiently cleave the terminal α1,3 mannose residue from various high-mannose N-glycan structures and from the model glycoprotein RNase B. This work not only expands the categorical scope of bacterial α-mannosidases, but also offers new insight into the glycan metabolism of S. canadensis, highlighting the enzyme's utility for glycan analysis and potential biotechnological applications.

N-glycan profiles as a tool in qualitative and quantitative analysis of goat milk adulteration.

Goat milk is closer to human milk in some respects than cow milk, and therefore preferred by many consumers. Because of the short lactation period and consequently less milk production of goats, the price of goat milk is often higher than that of cow milk, so that adulteration of goat milk is common. N-glycans have stability and thus have a good potential for acting as a new biomarker for identifying dairy adulteration. In this study, the N-glycan structures of goat milk and cow milk were analyzed by Ultra-high performance liquid chromatography (UPLC) and MALDI-TOF-MS. Based on the high species specificity of N-glycans, a method for identifying goat milk mixed with cow milk was established. The adulteration content of 5% cow milk in goat milk could be qualitatively and quantitatively detected. A prediction model of adulteration in goat milk was established by using partial least squares (PLS).

ß-1,2-Oligomannan phosphorylase-mediated synthesis of potential oligosaccharide vaccine candidates.

ß-(1,2)-Mannan antigens incorporated into vaccines candidates for immunization studies, showed that antibodies raised against ß-(1,2)-mannotriose antigens can protect against disseminated candidiasis. Until recently, ß-(1,2)- mannans could only be obtained by isolation from microbial cultures, or by lengthy synthetic strategies involving protecting group manipulation. The discovery of two ß-(1,2)-mannoside phosphorylases, Teth514_1788 and Teth514_1789, allowed efficient access to these compounds. In this work, Teth514_1788 was utilised to generate ß-(1,2)-mannan antigens, tri- and tetra-saccharides, decorated with a conjugation tether at the reducing end, suitable to be incorporated on a carrier en-route to novel vaccine candidates, illustrated here by conjugation of the trisaccharide to BSA.

Analysis of Immunogenic Galactose-α-1,3-galactose-Containing N-Glycans in Beef, Mutton, and Pork Tenderloin by Combining Matrix-Assisted Laser Desorption/Ionization-Mass Spectroscopy and Capillary Electrophoresis Hyphenated with Mass Spectrometry via Electrospray Ionization.

Severe allergic reactions to certain types of meat following tick bites have been reported in geographic regions which are endemic with ticks. This immune response is directed to a carbohydrate antigen (galactose-α-1,3-galactose or α-Gal), which is present in glycoproteins of mammalian meats. At the moment, asparagine-linked complex carbohydrates (N-glycans) with α-Gal motifs in meat glycoproteins and in which cell types or tissue morphologies these α-Gal moieties are present in mammalian meats are still unclear. In this study, we analyzed α-Gal-containing N-glycans in beef, mutton, and pork tenderloin and provided for the first time the spatial distribution of these types of N-glycans in various meat samples. Terminal α-Gal-modified N-glycans were found to be highly abundant in all analyzed samples (55, 45, and 36% of N-glycome in beef, mutton, and pork, respectively). Visualizations of the N-glycans with α-Gal modification revealed that this motif was mainly present in the fibroconnective tissue. To conclude, this study contributes to a better understanding of the glycosylation biology of meat samples and provides guidance for processed meat products, in which only meat fibers are required as an ingredient (i.e., sausages or canned meat).

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.

Recent applications of ionic liquid-based tags in glycoscience.

The functionalization of glycosides with ionic compounds such as ionic liquids provides enhanced polarity for the labelled glycans thanks to the presence of a permanent positive charge. The chemical derivatisation of glycans with ionic liquids constitutes an emerging strategy to boost the detection sensitivity in MS applications. This allows the straightforward monitoring and detection of the presence of labelled glycans in complex matrices and in those cases where very limited amounts of material were available such as in biological samples and chemoenzymatic reactions. The use of ionic liquid based derivatisation agents can be further exploited for the labelling of live cells via metabolic oligosaccharide engineering for the detection of cancer biomarkers and for the tuning of live cells-surface properties with implications in cancer prognosis and progression. In this mini-review we summarise the latest development of the ionic liquid based derivatisation agents in glycoscience focussing on their use for sensitive MS applications.

PNGase H + variant from Rudaea cellulosilytica with improved deglycosylation efficiency for rapid analysis of eukaryotic N-glycans and hydrogen deuterium exchange mass spectrometry analysis of glycoproteins.

The analysis of glycoproteins and the comparison of protein N-glycosylation from different eukaryotic origins require unbiased and robust analytical workflows. The structural and functional analysis of vertebrate protein N-glycosylation currently depends extensively on bacterial peptide-N4-(N-acetyl-ß-glucosaminyl) asparagine amidases (PNGases), which are indispensable enzymatic tools in releasing asparagine-linked oligosaccharides (N-glycans) from glycoproteins. So far, only limited PNGase candidates are available for N-glycans analysis, and particularly the analysis of plant and invertebrate N-glycans is hampered by the lack of suitable PNGases. Furthermore, liquid chromatography-mass spectrometry (LC-MS) workflows, such as hydrogen deuterium exchange mass spectrometry (HDX-MS), require a highly efficient enzymatic release of N-glycans at low pH values to facilitate the comprehensive structural analysis of glycoproteins. Herein, we describe a previously unstudied superacidic bacterial N-glycanase (PNGase H+ ) originating from the soil bacterium Rudaea cellulosilytica (Rc), which has significantly improved enzymatic properties compared to previously described PNGase H+ variants. Active and soluble recombinant PNGase Rc was expressed at a higher protein level (3.8-fold) and with higher specific activity (~56% increase) compared to the currently used PNGase H+ variant from Dyella japonicum (Dj). Recombinant PNGase Rc was able to deglycosylate the glycoproteins horseradish peroxidase and bovine lactoferrin significantly faster than PNGase Dj (10 min vs. 6 h). The versatility of PNGase Rc was demonstrated by releasing N-glycans from a diverse array of samples such as peach fruit, king trumpet mushroom, mouse serum, and the soil nematode Caenorhabditis elegans. The presence of only two disulfide bonds shown in the AlphaFold protein model (so far all other superacidic PNGases possess more disulfide bonds) could be corroborated by intact mass- and peptide mapping analysis and provides a possible explanation for the improved recombinant expression yield of PNGase Rc.

A Predominant Role of AtEDEM1 in Catalyzing a Rate-Limiting Demannosylation Step of an Arabidopsis Endoplasmic Reticulum-Associated Degradation Process.

Endoplasmic reticulum-associated degradation (ERAD) is a key cellular process for degrading misfolded proteins. It was well known that an asparagine (N)-linked glycan containing a free α1,6-mannose residue is a critical ERAD signal created by Homologous to α-mannosidase 1 (Htm1) in yeast and ER-Degradation Enhancing α-Mannosidase-like proteins (EDEMs) in mammals. An earlier study suggested that two Arabidopsis homologs of Htm1/EDEMs function redundantly in generating such a conserved N-glycan signal. Here we report that the Arabidopsis irb1 (reversal of bri1) mutants accumulate brassinosteroid-insensitive 1-5 (bri1-5), an ER-retained mutant variant of the brassinosteroid receptor BRI1 and are defective in one of the Arabidopsis Htm1/EDEM homologs, AtEDEM1. We show that the wild-type AtEDEM1, but not its catalytically inactive mutant, rescues irb1-1. Importantly, an insertional mutation of the Arabidopsis Asparagine-Linked Glycosylation 3 (ALG3), which causes N-linked glycosylation with truncated glycans carrying a different free α1,6-mannose residue, completely nullifies the inhibitory effect of irb1-1 on bri1-5 ERAD. Interestingly, an insertional mutation in AtEDEM2, the other Htm1/EDEM homolog, has no detectable effect on bri1-5 ERAD; however, it enhances the inhibitory effect of irb1-1 on bri1-5 degradation. Moreover, AtEDEM2 transgenes rescued the irb1-1 mutation with lower efficacy than AtEDEM1. Simultaneous elimination of AtEDEM1 and AtEDEM2 completely blocks generation of α1,6-mannose-exposed N-glycans on bri1-5, while overexpression of either AtEDEM1 or AtEDEM2 stimulates bri1-5 ERAD and enhances the bri1-5 dwarfism. We concluded that, despite its functional redundancy with AtEDEM2, AtEDEM1 plays a predominant role in promoting bri1-5 degradation.

A Novel PNGase Rc for Improved Protein N-Deglycosylation in Bioanalytics and Hydrogen-Deuterium Exchange Coupled With Mass Spectrometry Epitope Mapping under Challenging Conditions.

N-linked glycosylation is a ubiquitous posttranslational modification of proteins. While it plays an important role in the biological function of proteins, it often poses a major challenge for their analytical characterization. Currently available peptide N-glycanases (PNGases) are often inefficient at deglycosylating proteins due to sterically inaccessible N-glycosylation sites. This usually leads to poor sequence coverage in bottom-up analysis using liquid chromatography with tandem mass spectrometry and makes it impossible to obtain an intact mass signal in top-down MS analysis. In addition, most PNGases operate optimally only in the neutral to slightly acidic pH range and are severely compromised in the presence of reducing and denaturing substances, which limits their use for advanced bioanalysis based on hydrogen-deuterium exchange in combination with mass spectrometry (HDX-MS). Here, we present a novel peptide N-glycanase from Rudaea cellulosilytica (PNGase Rc) for which we demonstrate broad substrate specificity for N-glycan hydrolysis from multiply occupied and natively folded proteins. Our results show that PNGase Rc is functional even under challenging, HDX quenching conditions (pH 2.5, 0 °C) and in the presence of 0.4 M tris(2-carboxyethyl)phosphine, 4 M urea, and 1 M guanidinium chloride. Most importantly, we successfully applied the PNGase Rc in an HDX-MS workflow to determine the epitope of a nanobody targeting the extracellular domain of human signal-regulating protein alpha (SIRPα).

Spatial Mapping of Plant N-Glycosylation Cellular Heterogeneity Inside Soybean Root Nodules Provided Insights Into Legume-Rhizobia Symbiosis.

Although ubiquitously present, information on the function of complex N-glycan posttranslational modification in plants is very limited and is often neglected. In this work, we adopted an enzyme-assisted matrix-assisted laser desorption/ionization mass spectrometry imaging strategy to visualize the distribution and identity of N-glycans in soybean root nodules at a cellular resolution. We additionally performed proteomics analysis to probe the potential correlation to proteome changes during symbiotic rhizobia-legume interactions. Our ion images reveal that intense N-glycosylation occurs in the sclerenchyma layer, and inside the infected cells within the infection zone, while morphological structures such as the cortex, uninfected cells, and cells that form the attachment with the root are fewer N-glycosylated. Notably, we observed different N-glycan profiles between soybean root nodules infected with wild-type rhizobia and those infected with mutant rhizobia incapable of efficiently fixing atmospheric nitrogen. The majority of complex N-glycan structures, particularly those with characteristic Lewis-a epitopes, are more abundant in the mutant nodules. Our proteomic results revealed that these glycans likely originated from proteins that maintain the redox balance crucial for proper nitrogen fixation, but also from enzymes involved in N-glycan and phenylpropanoid biosynthesis. These findings indicate the possible involvement of Lewis-a glycans in these critical pathways during legume-rhizobia symbiosis.

Recombinant Snail Sialic Acid Aldolase is Promiscuous towards Aliphatic Aldehydes.

Aldolases are enzymes that reversibly catalyze the cleavage of carbon-carbon bonds. Here we describe a recombinant sialic acid aldolase originating from the freshwater snail Biomphalaria glabrata (sNPL), and compare its substrate spectrum with a sialic acid aldolase originating from chicken (chNPL). In contrast to vertebrate animals which can synthesize, degrade, and incorporate sialic acids on glycoconjugate ubiquitously, snails (as all mollusks) cannot synthesize sialic acids endogenously, and therefore the biological function and substrate scope of sNPL ought to differ significantly from vertebrate sialic aldolases such as chNPL. sNPL was active towards a series of sialic acid derivatives but was in contrast to chNPL unable to catalyze the cleavage of N-acetylneuraminic acid into N-acetylmannosamine and pyruvate. Interestingly, chNPL and sNPL showed contrasting C4(R)/(S) diastereoselectivity towards the substrates d-mannose and d-galactose in the presence of pyruvate. In addition, sNPL was able to synthesize a series of 4-hydroxy-2-oxoates using the corresponding aliphatic aldehyde substrates in the presence of pyruvate, which could be not achieved by chNPL.

Recombinant human N-acetylneuraminate lyase as a tool to study clinically relevant mutant variants.

N-acetylneuraminic acid (sialic acid) is an abundantly found carbohydrate moiety covering the surface of all vertebrate cells and secreted glycoproteins. The human N-acetylneuraminate pyruvate lyase (NPL) interconverts sialic acid to N-acetylmannosamine and pyruvate, and mutations of the NPL gene were found to cause sialuria and impair the functionality of muscles. Here we report the soluble and functional expression of human NPL in Escherichia coli, which allowed us to study the biochemical properties of two clinically relevant NLP mutations (Asn45Asp and Arg63Cys). The Asn45Asp mutant variant was enzymatically active, but had lower expression levels and showed reduced stability when compared to the wild-type NPL variant. Expression trials of the Arg63Cys mutant did not yield any recombinant protein and consequently, no enzymatic activity was detected. The locations of these clinically relevant amino acid substitutions are also discussed by using a human NPL homology model.

mAbs N-glycosylation: Implications for biotechnology and analytics.

Monoclonal antibodies (mAbs) are amazingly successful in treating diseases such as various types of cancers, immune disorders, and infections. Currently, mAbs are considered the most versatile therapeutic available. Seminal scientific progress over the last decade on elucidating the functional role of antibody N-glycosylation revealed the significance of this post-translational modification on the stability, half-life, and biological activity of antibodies. Therefore, antibody N-glycosylation became a key aspect of biopharmaceutical mAb production in large-scale manufacturing processes. This Mini-Review summarizes the recent scientific attempts of producing mAbs in glycoengineered animal-, plant-, and yeast cell lines. Furthermore, we also describe novel approaches for analyzing mAb N-glycosylation.

The mechanism of probiotic action of human milk N-glycome towards B. infantis ATCC 15697 and identification of the principal functional components.

Human milk N-glycome promotes the growth of Bifidobacterium longum subsp. infantis ATCC 15697. However, the action mode of, and the major functional components for, the bifidogenic function of human milk N-glycome remain unclear. In this study, we demonstrated that milk N-glycome was transferred in an intact form from culture into the bacterial cell and then decomposed intracellularly, evidenced by the following facts: (1) No UHPLC peak shift of N-glycome recovered from culture was observed. (2) No milk N-glycan specific monosugar was detected in culture supernatant. (3) High intracellular exoglycosidase activities were detected. (4) Fluorescently labeled N-glycans were found to be located intracellularly using Laser Scanning Confocal Microscopy (LSCM). Regarding the principal components identification, a novel sequential deglycosylation-based strategy was established. Degalactosylation, defucosylation-desialylation, and defucosylation-desialylation-degalactosylation treatments of human milk N-glycome showed that galactose-containing glycans were the principal components for the probiotic function of human milk N-glycome towards B. infantis ATCC 15697.

Protein Glycosylation and Gut Microbiota Utilization Can Limit the In Vitro and In Vivo Metabolic Cellular Incorporation of Neu5Gc.

SCOPE: Red meat intake is reported to be correlated with chronic diseases. A potential causal factor is N-glycolylneuraminic acid (Neu5Gc) which metabolically incorporates into diverse glycoconjugates in humans. This study aims to investigate the impact of exposure to Neu5Gc-rich red meat on healthy cytidine-5'-monophospho-N-acetylneuraminic acid hydroxylase (Cmah) knock-out mice and the underlying mechanisms. METHODS AND RESULTS: CMAH-/- mice are fed Neu5Gc-rich diet for short-term (4 months) and long-term (10 months). Health status and levels of inflammatory cytokines are assessed. Caco-2 cells are used to investigate the intestinal absorption of Neu5Gc-containing glycoprotein, and in vitro fermentation is used to investigate the Neu5Gc utilization by gut microbiota. Neu5Gc-rich diets show neither measurable abnormality in physio-biochemical and inflammatory indexes nor observable alterations of liver tissue in mice. Glycosylation of lactoferrin limits its intestinal epithelial absorption, and the absorption of Neu5Gc attach onto glycoprotein is thus limited. Neu5Gc is also simultaneously utilized by microorganisms under simulated gut conditions. CONCLUSION: The results indicate that the long-term intake of Neu5Gc-rich red meat has no adverse effect on the health of CMAH-/- mice, which may be related to the limited absorption of Neu5Gc that is regulated by protein glycosylation, and the metabolism of Neu5Gc by gut microorganisms.

Heterologous Expression of a Thermostable Chitinase from Myxococcus xanthus and Its Application for High Yield Production of Glucosamine from Shrimp Shell.

Glucosamine (GlcN) is a widely used food supplement. Hence, enormous attention has been concerned with enzymatic production of GlcN owing to its advantage over a chemical approach. In this study, a previously unstudied chitinase gene (MxChi) in the genome of Myxococcus xanthus was cloned, expressed in recombinant soluble form and purified to homogeneity. TLC-, UPLC-, and microplate-reader- based activity tests confirmed MxChi hydrolyzes colloidal chitin to chitobiose as sole product. The optimal catalytic pH and temperature of MxChi was identified as 7.0 and 55 °C, respectively. MxChi exhibited 80% activity after 72 h incubation at 37 °C. The site-directed mutagenesis revealed that the amino acids D323A, D325A, and E327A of MxChi were in the DXDXE catalytic motif of GH18. When coupled with ß-N-acetylhexosaminidase (SnHex) and deacetylase (CmCBDA), the enzyme allowed one-pot extraction of GlcN from colloidal chitin and shrimp shell. The optimal condition was 37 °C, pH 8.0, and 1/3/16.5 (MxChi/SnHex/CmCBDA), conducted by orthogonal design for the enzymatic cascades. Under this condition, the yield of GlcN was 26.33 mg from 400 mg shrimp shell. Facile recombinant in E. coli, robust thermostability and pure product herein makes newly discovered chitinase a valuable candidate for the green recycling of chitin rich waste.

Hydrogen/Deuterium Exchange Mass Spectrometry with Integrated Electrochemical Reduction and Microchip-Enabled Deglycosylation for Epitope Mapping of Heavily Glycosylated and Disulfide-Bonded Proteins.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a recognized method to study protein conformational dynamics and interactions. Proteins encompassing post-translational modifications (PTMs), such as disulfide bonds and glycosylations, present challenges to HDX-MS, as disulfide bond reduction and deglycosylation is often required to extract HDX information from regions containing these PTMs. In-solution deglycosylation with peptide-N4-(N-acetyl-ß-d-glucosaminyl)-asparagine amidase A (PNGase A) or PNGase H+ combined with chemical reduction using tris-(2-carboxyethyl)phosphine (TCEP) has previously been used for HDX-MS analysis of disulfide-linked glycoproteins. However, this workflow requires extensive manual sample preparation and consumes large amounts of enzyme. Furthermore, large amounts of TCEP and glycosidases often result in suboptimal liquid chromatography-mass spectrometry (LC-MS) performance. Here, we compare the in-solution activity of PNGase A, PNGase H+, and the newly discovered PNGase Dj under quench conditions and immobilize them onto thiol-ene microfluidic chips to create HDX-MS-compatible immobilized microfluidic enzyme reactors (IMERs). The IMERS retain deglycosylation activity, also following repeated use and long-term storage. Furthermore, we combine a PNGase Dj IMER, a pepsin IMER, and an electrochemical cell to develop an HDX-MS setup capable of efficient online disulfide-bond reduction, deglycosylation, and proteolysis. We demonstrate the applicability of this setup by mapping the epitope of a monoclonal antibody (mAb) on the heavily disulfide-bonded and glycosylated sema-domain of the tyrosine-protein kinase Met (SD c-Met). We achieve near-complete sequence coverage and extract HDX data to identify regions of SD c-Met involved in mAb binding. The described methodology thus presents an integrated and online workflow for improved HDX-MS analysis of challenging PTM-rich proteins.

Imidazolium labelling permits the sensitive mass-spectrometric detection of N-glycosides directly from serum.

A novel imidazolium derivative (GITag) shows superior ionisation and consequently allows increased mass spectrometric detection capabilities of oligosaccharides and N-glycans. Here we demonstrate that human serum samples can be directly labelled by GITag on a MALDI target plate, abrogating prevalently required sample pretreatment or clean-up steps.

Construction and Evaluation of Peptide-Linked Lactobacillus brevis ß-Galactosidase Heterodimers.

BACKGROUND: ß-galactosidases are enzymes that are utilized to hydrolyze lactose into galactose and glucose, and are is widely used in the food industry. OBJECTIVE: We describe the recombinant expression of an unstudied, heterodimeric ß-galactosidase originating from Lactobacillus brevis ATCC 367 in Escherichia coli. Furthermore, six different constructs, in which the two protein subunits were fused with different peptide linkers, were also investigated. METHODS: The heterodimeric subunits of the ß-galactosidase were cloned in expressed in various expression constructs, by using either two vectors for the independent expression of each subunit, or using a single Duet vector for the co-expression of the two subunits. RESULTS: The co-expression in two independent expression vectors only resulted in low ß-galactosidase activities, whereas the co-expression in a single Duet vector of the independent and fused subunits increased the ß-galactosidase activity significantly. The recombinant ß-galactosidase showed comparable hydrolyzing properties towards lactose, N-acetyllactosamine, and pNP-ß-D-galactoside. CONCLUSION: The usability of the recombinant L. brevis ß-galactosidase was further demonstrated by the hydrolysis of human, bovine, and goat milk samples. The herein presented fused ß-galactosidase constructs may be of interest for analytical research as well as in food- and biotechnological applications.

Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated protein regions. Here, we have investigated the use of the acidic glycosidase PNGase H+, which has a pH optimum at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our results show that PNGase H+ retains high deglycosylation activity at HDX quench conditions. When used in an HDX-MS workflow, PNGase H+ allowed the extraction of HDX data from all five glycosylated regions of the serpin α1-antichymotrypsin. We demonstrate that PNGase A and PNGase H+ are capable of similar deglycosylation performance during HDX-MS analysis of α1-antichymotrypsin and the IgG1 antibody trastuzumab (TZ). However, PNGase H+ provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while PNGase A offers advantages in terms of commercial availability and purity. Overall, our findings demonstrate the unique features of PNGase H+ for improving conformational analysis of glycoproteins by HDX-MS, in particular, challenging glycoproteins containing both glycosylations and disulfide bonds.