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N-Linked glycans are structurally diverse polysaccharides that represent significant biological relevance due to their involvement in disease progression and cancer. Due to their complex nature, N-linked glycans pose many analytical challenges requiring the continued development of analytical technologies. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a hybrid ionization technique commonly used for mass spectrometry imaging (MSI) applications. Previous work demonstrated IR-MALDESI to significantly preserve sialic acid containing N-linked glycans that otherwise require chemical derivatization prior to detection. Here, we demonstrate the first analysis of N-linked glycans in situ by IR-MALDESI MSI. A formalin-fixed paraffin-embedded human prostate tissue was analyzed in negative ionization mode after tissue washing, antigen retrieval, and pneumatic application of PNGase F for enzymatic digestion of N-linked glycans. Fifty-three N-linked glycans were confidently identified in the prostate sample where more than 60% contained sialic acid residues. This work demonstrates the first steps in N-linked glycan imaging of biological tissues by IR-MALDESI MSI. Raw data files are available in MassIVE (identifier: MSV000088414).

Protein glycosylation is one of the ubiquitous post-translational modifications in eukaryotic cells, which play important roles in plant growth and adverse response. In this study, we performed the first comprehensive wheat plasma membrane N-glycoproteome analysis under drought stress via glycopeptide HILIC enrichment and LC-MS/MS identification. In total, 414 glycosylated sites corresponding to 407 glycopeptides and 312 unique glycoproteins were identified, of which 173 plasma membrane glycoproteins with 215 N-glycosylation sites were significantly regulated by drought stress. Functional enrichment analysis reveals that the significantly regulated N-glycosylation proteins were particularly related to protein kinase activity involved in the reception and transduction of extracellular signal and plant cell wall remolding. The motifs and sequence structures analysis showed that the significantly regulated N-glycosylation sites were concentrated within [NxT] motif, and 79.5% of them were located on the random coil that is always on the protein surface and flexible regions, which could facilitate protein glycosylated modification and enhance protein structural stability via reducing protein flexibility. PNGase F enzyme digestion and glycosylation site mutation further indicated that N-glycosylated modification could increase protein stability. Therefore, N-glycosylated modification is involved in plant adaptation to drought stress by improving the stability of cell wall remodeling related plasma membrane proteins.

Asparagine (Asn, N) -linked glycosylation within Nglyco -X-S/T; X ≠ P motif is a ubiquitously distributed post-translational modification that participates in diverse cellular processes. In this work, N-glycosylation inhibitor was shown to prevent Phytophthora sojae growth, suggesting that N-glycosylation is necessary for oomycete development. We conducted a glycoproteomic analysis of P. sojae to identify and map N-glycosylated proteins and to quantify differentially expressed glycoproteins associated with mycelia, asexual cyst, and sexual oospore developmental stages. A total of 355 N-glycosylated proteins was found, containing 496 glycosites, potentially involved in glycan degradation, carbon metabolism, glycolysis, or other metabolic pathways. Through PNGase F deglycosylation assays and site-directed mutagenesis of a GPI transamidase protein (GPI16) up-regulated in cysts and a heat shock protein 70 (HSP70) up-regulated in oospores, we demonstrated that both proteins were N-glycosylated and that the Nglyco -N motif is a target site for asparagine - oligosaccharide linkage. Glycosite mutations of Asn 94 Nglyco -X-S/T in the GPI16 led to impaired cyst germination and pathogenicity, while mutation of the previously unknown Asn 270 Nglyco -N motif in HSP70 led to decreased oospore production. In addition to providing a map of the oomycete N-glycoproteome, this work confirms that P. sojae has evolved multiple N-glycosylation motifs essential for growth. This article is protected by copyright. All rights reserved.

Recently microdroplet reactions have aroused much interest because the microdroplet provides a unique medium where organic reactions could be accelerated by a factor of 10(3) or more. However, microdroplet reactions of proteins have been rarely studied. We report the occurrence of multiple-step reactions of a large protein, specifically, the digestion, reduction, and deglycosylation of an intact antibody, which can take place in microseconds with high reaction yields in aqueous microdroplets at room temperature. As a result, fast structural characterization of a monoclonal antibody, essential for assessing its quality as a therapeutic drug, can be enabled. We found that the IgG1 antibody can be digested completely by the IdeS protease in aqueous microdroplets in 250 microseconds, a 7.5 million-fold improvement in speed in comparison to traditional digestion in bulk solution (>30 min). Strikingly, inclusion of the reductant tris(2-carboxyethyl)phosphine (TCEP) in the spray solution caused simultaneous antibody digestion and disulfide bond reduction. Digested and reduced antibody fragments were either collected or analyzed online by mass spectrometry. Further addition of PNGase F glycosylase into the spray solution led to antibody deglycosylation, thereby producing reduced and deglycosylated fragments of analytical importance. In addition, glycated fragments of IgG1 derived from a glucose modification were identified quickly with this ultrafast digestion/reduction technique. We suggest that microdroplets can serve as powerful microreactors for both exploring large molecule reactions and speeding their structural analyses.

The elucidation of glycans biological function is essential to understand their role in biological processes, both normal and pathological. Immobilized glycoenzymes are excellent tools for this purpose as they can selectively release glycans from glycoproteins without altering their backbone. They can be easily removed from the reaction mixture avoiding their interference in subsequent experiments. Here, we describe the immobilization of peptide-N-glycosidase F (PNGase F) onto silica magnetic nanoparticles with immobilization yields of 86% and activity yields of 12%. Immobilized PNGase F showed higher thermal stability than its soluble counterpart, and could be reused for at least seven deglycosylation cycles. It was efficient in the deglycosylation of several glycoproteins (ribonuclease B, bovine fetuin, and ovalbumin) and a protein lysate from the parasite Fasciola hepatica under native conditions, with similar performance to that of the soluble enzyme. Successful deglycosylation was evidenced by a decrease in specific lectin recognition of the glycoproteins (40%-80%). Moreover, deglycosylated F. hepatica lysate allowed us to confirm the role of parasite N-glycans in the inhibition of the lipopolysaccharide-induced maturation of dendritic cells. Immobilized PNGase F probed to be a robust biotechnological tool for deglycosylation of glycoproteins and complex biological samples under native conditions.

The detection of glycans and glycoconjugates has gained increasing attention in biological fields. Traditional mass spectrometry (MS)-based methods for glycoconjugate analysis are challenged with poor intensity when dealing with complex biological samples. We developed a desalting paper spray mass spectrometry (DPS-MS) strategy to overcome the issue of signal suppression of carbohydrates in salted buffer. Glycans and glycoconjugates (i.e., glycopeptides, nucleotide sugars, etc.) in non-volatile buffer (e.g., Tris buffer) can be loaded on the paper substrate from which buffers can be removed by washing with ACN/H(2)O (90/10 v/v) solution. Glycans or glycoconjugates can then be eluted and spray ionized by adding ACN/H(2)O/formic acid (FA) (10/90/1 v/v/v) solvent and applying a high voltage (HV) to the paper substrate. This work also showed that DPS-MS is applicable for direct detection of intact glycopeptides and nucleotide sugars as well as determination of glycosylation profiling of antibody, such as NIST monoclonal antibody IgG (NISTmAb). NISTmAb was deglycosylated with PNGase F to release N-linked oligosaccharides. Twenty-six N-linked oligosaccharides were detected by DPS-MS within a 5-minute timeframe without the need for further enrichment or derivatization. This work demonstrates that DPS-MS allows fast and sensitive detection of glycans/oligosaccharides and glycosylated species in complex matrices and has great potential in bioanalysis.

Chitinase 3-like-1 (Chi3l1) and IL-13 are both ligands of IL-13 receptor α2 (IL-13Rα2). The binding of the former activates mitogen-activated protein kinase, AKT, and Wnt/β-catenin signaling, and plays important roles in innate and adaptive immunity, cellular apoptosis, oxidative injury, allergic inflammation, tumor metastasis and wound healing, fibrosis, and repair in the lung. In contrast, the latter binding is largely a decoy event that diminishes the effects of IL-13. Here, we demonstrate that IL-13Rα2 N-glycosylation is a critical determinant of which ligand binds. Structure-function evaluations demonstrated that Chi3l1-IL-13Rα2 binding was increased when sites of N-glycosylation are mutated, and studies with tunicamycin and Peptide:N-glycosidase F (PNGase F) demonstrated that Chi3l1-IL-13Rα2 binding and signaling were increased when N-glycosylation was diminished. In contrast, structure-function experiments demonstrated that IL-13 binding to IL-13Rα2 was dependent on each of the four sites of N-glycosylation in IL-13Rα2, and experiments with tunicamycin and PNGase F demonstrated that IL-13-IL-13Rα2 binding was decreased when IL-13Rα2 N-glycosylation was diminished. Studies with primary lung epithelial cells also demonstrated that Chi3l1 inhibited, whereas IL-13 stimulated, N-glycosylation as evidenced by the ability of Chi3l1 to inhibit and IL-13 to stimulate the subunits of the oligosaccharide complex A and B (STT3A and STT3B). These studies demonstrate that N-glycosylation is a critical determinant of Chi3l1 and IL-13 binding to IL-13Rα2, and highlight the ability of Chi3l1 and IL-13 to alter key elements of the N-glycosylation apparatus in a manner that would augment their respective binding.

Objective To investigate the effects of macromolecular crowding on the folding and aggregation of MUC5AC with different levels of glycosylation during refolding. Methods Part 1:An in vitro catalytic reaction comprising the ppGalNAc T2 enzyme, uridine-5′-diphospho-N-galactosamine (UDP-GalNAc) and an 11-amino acid peptide substrate, was used to assess the enzyme activity of the ppGalNAc T2 enzyme in macromolecular crowding environment respectively with bovine serum albumin (BSA), polyethylene glycol (PEG2000), Dextran70 and Ficoll70 at different concentration and temperature. Part 2: The recombinant MUC5AC was expressed in HEK293 cells and purified by nickel column chromatography. The purified protein was treated with PNGase F, and the degree of glycosylation was analyzed by SDS-PAGE. Macromolecular crowding was simulated using PEG2000 at the concentrations of 50, 100, and 200 g/L. Deglycosylated-MUC5AC (d-MUC5AC) and glycosylated MUC5AC (g-MUC5AC) were denatured by GdnHCl and renatured by dilution in a refolding buffer. Protein aggregation was monitored continuously by absorbance reading at 488 nm using a UV spectrophotometer at 25 °C. The refolded proteins were centrifuged, the protein concentration of the supernatant was measured, and refolding yield in different refolding buffers was determined. Results Enzyme activityof ppGalNAc T2 was observed to increase with increasing crowding agent concentration, with highest enzyme activity at 200 g/L. Compared with the group in the absence of crowding reagent, the refolding yield of g-MUC5AC and d-MUC5AC were reduced significantly in the presence of different concentrations of PEG2000 (200, 100, and 50 g/L). Compared with the dilute solution, aggregation increased significantly in the presence of PEG2000, especially at 200 g/L. Moreover, in the crowded reagent with the same concentration, the refolding yield of d-MUC5AC was higher than that of g-MUC5AC, whereas the degree of aggregation of d-MUC5AC was lower than that of g-MUC5AC. Conclusion The crowded intracellular environment reduces the refolding rate of MUC5AC and strongly induces the misfolding and aggregation of glycosylated MUC5AC.

The analysis of N-linked glycans using liquid chromatography and mass spectrometry (LC-MS) presents significant challenges, particularly owing to their hydrophilic nature. To address these difficulties, a variety of derivatization methods has been developed to facilitate improved ionization and detection sensitivity. One such method, the Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)™ strategy for labeling glycans, has previously been utilized in the analysis of N- and O-linked glycans in biological samples. To assess the maximum sensitivity and separability of the INLIGHT ™ preparation and analysis pipeline, several critical steps were investigated. First, recombinant and nonrecombinant sources of PNGase F were compared to assess variations in the released glycans. Second, modifications in the INLIGHT™ derivatization step were evaluated including temperature optimization, solvent composition changes, and reaction condition length and tag concentration. Optimization of the modified method resulted in 20–100 times greater peak areas for the detected N-linked glycans in fetuin and horseradish peroxidase compared to the standard method. Furthermore, the identification of low abundance glycans, such as (Fuc)(1)(Gal)(2)(GlcNAc)(4)(Man)(3)(NeuAc)(1) and (Gal)(3)(GlcNAc)(5)(Man)(3)(NeuAc)(3,) was possible. Finally, the optimal LC setup for the INLIGHT™ derivatized N-linked glycan analyses was found to be a C18 reverse-phase (RP) column with mobile phases typical of RPLC.

Glycan analysis by mass spectrometry has rapidly progressed due to the interest in understanding the role of glycans in disease and tumor progression. Glycans are complex molecules that pose analytical challenges due to their isomeric compositions, labile character, and ionization preferences. This study sought to demonstrate infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) as a novel approach for the direct analysis of N-linked glycans. The glycoprotein bovine fetuin was chosen for this analysis as its glycome is well-characterized and heavily composed of sialylated glycans. Native N-linked glycans produced by enzymatic cleavage (via PNGase F) of bovine fetuin were analyzed directly by IR-MALDESI in both positive and negative ionization mode. In this study, we detected 12 N-linked glycans in negative mode and 4 N-linked glycans in positive mode, a significant increase in the amount of underivatized glycans detected by other ionization sources. Importantly, all N-linked glycans detected contained at least one sialic acid residue, which are known to be labile. This work represents a critical first step for N-linked glycan analysis by IR-MALDESI with future efforts directed at mass spectrometry imaging.

Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which α-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins. N-glycans were released by PNGase F digestion and O-glycans by β-elimination. Released oligosaccharides were analyzed by liquid chromatography – tandem mass spectrometry. In total, 102 N-glycans and 40 O-glycans were identified in animal heart tissue lysates. The N- and O-glycan patterns were different between species. α-Gal and Neu5Gc were identified on both N- and O-linked glycans, N,N´-diacetyllactosamine (LacdiNAc) on N-glycans only and sulfated O-glycans. The relative amounts of α-Gal-containing N-glycans were higher in bovine compared to equine and porcine pericardia. In contrast to the restricted number of proteins carrying α-Gal and LacdiNAc, the distribution of proteins carrying Neu5Gc-determinants varied between species and between different tissues of the same species. Porcine pericardium carried the highest level of Neu5Gc-sialylated O-glycans, and bovine pericardium the highest level of Neu5Gc-sialylated N-glycans. The identified N- and O-linked glycans, some of which may be immunogenic and remain in BHVs manufactured for clinical use, could direct future genetic engineering to prevent glycan expression rendering the donor tissues less immunogenic in humans.

Proteins on cell membrane are modified by N- and O-glycans. N-Glycans have been extensively characterized using advanced separation and mass spectrometry techniques. However, O-glycans remain a challenge, because of the lack of universal enzymes to release them and the large background abundances of N-glycans. Here, we report a method for in-depth structural analysis and quantitation of O-glycans derived from human cell membrane. O-Glycans were chemically released from isolated cell membrane glycoproteins following N-glycan and lipid/glycolipid removal by PNGase F digestion and Folch extraction, respectively. Released O-glycans were purified by an optimized protocol to eliminate interference from small molecules and degraded proteins. Cell surface O-glycans were then analyzed using a nanoLC-chip-QTOF mass spectrometer with a porous graphitized carbon (PGC) column, while the N-glycans and glycolipids isolated from the same cell membrane fractions were analyzed in parallel using previously reported methods. The monosaccharide compositions and linkages of the detected O-glycans were identified by exoglycosidase digestion facilitated with tandem mass spectrometry (MS/MS). Using this method, we identified 44 cell membrane O-glycan isomers with MS/MS, and, among them, we unambiguously characterized 25 O-glycan structures with exoglycosidase digestion to create a library with their complete structures, accurate masses, and retention times. In this process, we identified and characterized unexpected mannose oligomers that are α(1-2/3) linked. This library enabled the identification and quantification of unique cell surface O-glycans from different cell lines and the study of specific O-glycan changes during cell differentiation.

The human serum N-linked glycoproteome has been determined through LC-MS/MS. The intact glycopeptides were identified through a spectral library search method embedded in the pMatchGlyco software. Four types of known N-glycosylation motifs, prevalent variable modifications and semi-tryptic digestion were considered during searching and the identified intact glycopeptides were validated through target-decoy and motif-specific false discovery rate (FDR) control. The results reveal site-specific glycosylation of serum glycoproteins and provide high-quality tandem mass spectra of 22,677 serum N-linked intact glycopeptides., Graphical Abstract Highlights This study proposed a spectral library search method to accurately identify N-linked glycopeptides in human serum through LC-MS/MS with pMatchGlyco software. The identification depth of serum N-linked intact glycopeptides and glycoproteins was increased by combination of acetonitrile precipitation, HILIC enrichment and high-pH RPLC fractionation. 22,677 unique serum N-linked intact glycopeptides corresponding to 526 N-linked glycoproteins were identified with N-glycosylation motif-specific FDR control. This study revealed the great microheterogeneity of N-linked glycoproteins in serum., Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in human serum is challenging because of the wide dynamic range of serum protein abundances, the lack of a complete serum N-glycan database and the existence of proteoforms. In this regard, a spectral library search method was presented for the identification of N-linked intact glycopeptides from N-linked glycoproteins in human serum with target-decoy and motif-specific false discovery rate (FDR) control. Serum proteins were firstly separated into low-abundance and high-abundance proteins by acetonitrile (ACN) precipitation. After digestion, the N-linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) and a portion of the enriched N-linked intact glycopeptides were processed by Peptide-N-Glycosidase F (PNGase F) to generate N-linked deglycopeptides. Both N-linked intact glycopeptides and deglycopeptides were analyzed by LC-MS/MS. From N-linked deglycopeptides data sets, 764 N-linked glycoproteins, 1699 N-linked glycosites and 3328 unique N-linked deglycopeptides were identified. Four types of N-linked glycosylation motifs (NXS/T/C/V, X≠P) were used to recognize the N-linked deglycopeptides. The spectra of these N-linked deglycopeptides were utilized for N-linked deglycopeptides library construction and identification of N-linked intact glycopeptides. A database containing 739 N-glycan masses was constructed and utilized during spectral library search for the identification of N-linked intact glycopeptides. In total, 526 N-linked glycoproteins, 1036 N-linked glycosites, 22,677 N-linked intact glycopeptides and 738 N-glycan masses were identified under 1% FDR, representing the most in-depth serum N-glycoproteome identified by LC-MS/MS at N-linked intact glycopeptide level.

N-glycoproteins are involved in various biological processes. Certain distinctive glycoforms on specific glycoproteins enhance the specificity and/or sensitivity of cancer diagnosis. Therefore, the characterization of plasma N-glycoproteome is essential for a new biomarker discovery. The absence of suitable analytical methods for in-depth and large-scale analyses of low-abundance plasma glycoproteins makes it challenging to investigate the role of glycosylation. In this study, we developed an integrated method termed Glyco-CPLL, which integrates combinatorial peptide ligand libraries, high-pH reversed-phase prefractionation, hydrophilic interaction chromatography, trypsin and PNGase F digestion, shotgun proteomics, and various analysis software (MaxQuant and pGlyco2.0) for the low-abundance plasma glycoproteomic profiling. Then, we utilized the method to perform a comparative study and to explore papillary thyroid carcinoma-related proteins and glycosylations with reference to healthy controls. Finally, a large and comprehensive human plasma N-glycoproteomic database was established, containing 786 proteins, 369 N-glycoproteins, 862 glycosites, 171 glycan compositions, and 1644 unique intact N-glycopeptides. Additionally, several low-abundance plasma glycoproteins were identified, including SVEP1 (∼0.54 ng/mL), F8 (∼0.83 ng/mL), and ADAMTS13 (∼1.2 ng/mL). These results suggest that this method will be useful for analyzing plasma intact glycopeptides in future studies. Besides, the Glyco-CPLL method has a great potential to be translated to clinical applications. Data are available via ProteomeXchange with identifier PXD016428.

Antibody drug conjugates (ADCs) can undergo in vivo biotransformation (e.g., payload metabolism, deconjugation) leading to reduced or complete loss of activity. The location/site of conjugation of payload-linker can have an effect on ADC stability and hence needs to be carefully optimized. Affinity capture LC-MS of intact ADCs or ADC subfragments has been extensively used to evaluate ADC biotransformation. However, the current methods have certain limitations such as the requirement of specific capture reagents, limited mass resolution of low mass change metabolites, low sensitivity, and use of capillary or nanoflow LC-MS. To address these challenges, we developed a generic affinity capture LC-MS assay that can be utilized to evaluate the biotransformation of any site-specific ADC independent of antibody type and site of conjugation (Fab and Fc) in preclinical studies. The method involves a combination of some or all of these steps: (1) "mono capture" or "dual capture" of ADCs from serum with streptavidin magnetic beads coated with a generic biotinylated antihuman capture reagent, (2) "on-bead" digestion with IdeS and/or PNGase F, and (3) reduction of interchain disulfide bonds to generate ∼25 kDa ADC subfragments, which are finally analyzed by LC-HRMS on a TOF mass spectrometer. The advantages of this method are that it can be performed using commercially available generic reagents and requires sample preparation time of less than 7 h. Furthermore, by reducing the size of intact ADC (∼150 kDa) to subfragments (∼25 kDa), the identification of conjugated payload and its metabolites can be achieved with excellent sensitivity and resolution (hydrolysis and other small mass change metabolites). This method was successfully applied to evaluate the in vitro and in vivo biotransformation of ADCs conjugated at different sites (LC, HC-Fab, and HC-Fc) with various classes of payload-linkers.

The N-glycosylation profile of a monoclonal antibody (mAb) is a critical quality attribute in relation to its therapeutic application. The control of this profile during biomanufacture is difficult because of the multiple parameters that affect the glycosylation metabolism within the cell and the environment in which the cell is grown. One of the approaches that can be used to produce a preferred glycan profile or a single glycoform is through chemoenzymatic remodeling during the isolation of a mAb. Here we describe protocols that can be utilized to produce preferred glycoforms that include galactosylated, agalactosylated, or sialylated glycoforms following isolation of a mAb. Methods for analysis and assignment of structures of the samples following glycoengineering are also described. Chemoenzymatic modeling of mAb glycans has the potential for scale-up and to be introduced into biomanufacturing of mAbs with higher specific therapeutic activities.

The densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22 N-glycosites and site-specific N-glycans in the S protein protomer. Herein, we report the O-glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized through high-resolution mass spectrometry. Following digestion with trypsin and trypsin/Glu-C, and de-N-glycosylation using PNGase F, we determined the GalNAc-type O-glycosylation pattern of S proteins, including O-glycosites and the six most common O-glycans occupying them, via Byonic identification and manual validation. Finally, 255 intact O-glycopeptides composed of 50 peptides sequences and 43 O-glycosites were discovered by higher energy collision-induced dissociation (HCD), and three O-glycosites were confidently identified by electron transfer/higher energy collision-induced dissociation (EThcD) in the insect cell-expressed S protein. Most glycosites were modified by non-sialylated O-glycans such as HexNAc(1) and HexNAc(1)Hex (1). In contrast, in the human cell-expressed S protein S1 subunit, 407 intact O-glycopeptides composed of 34 peptides sequences and 30 O-glycosites were discovered by HCD, and 11 O-glycosites were unambiguously assigned by EThcD. However, the measurement of O-glycosylation occupancy hasn’t been made. Most glycosites were modified by sialylated O-glycans such as HexNAc(1)Hex (1)NeuAc (1) and HexNAc(1)Hex (1)NeuAc (2). Our results reveal that the SARS-CoV-2 S protein is an O-glycoprotein; the O-glycosites and O-glycan compositions vary with the host cell type. These comprehensive O-glycosylation landscapes of the S protein are expected to provide novel insights into the viral binding mechanism and present a strategy for the development of vaccines and targeted drugs.

A new method to probe the conformational changes of glycoproteins on a systems-wide scale, termed limited deglycosylation assay (LDA), is described. The method measures the differential rate of deglycosylation of N-glycans on natively folded proteins by the common peptide:N-glycosidase F (PNGase F) enzyme which in turn informs on their spatial presentation and solvent exposure on the protein surface hence ultimately the glycoprotein conformation. LDA involves 1) protein-level N-deglycosylation under native conditions, 2) trypsin digestion, 3) glycopeptide enrichment, 4) peptide-level N-deglycosylation and 5) quantitative MS-based analysis of formerly N-glycosylated peptides (FNGPs). LDA was initially developed and the experimental conditions optimized using bovine RNase B and fetuin. The method was then applied to glycoprotein extracts from LLC-MK2 epithelial cells upon treatment with dithiothreitol to induce endoplasmic reticulum stress and promote protein misfolding. Data from the LDA and 3D structure analysis showed that glycoproteins predominantly undergo structural changes in loops/turns upon ER stress as exemplified with detailed analysis of ephrin-A5, GALNT10, PVR and BCAM. These results show that LDA accurately reports on systems-wide conformational changes of glycoproteins induced under controlled treatment regimes. Thus, LDA opens avenues to study glycoprotein structural changes in a range of other physiological and pathophysiological conditions relevant to acute and chronic diseases. Significance: We describe a novel method termed limited deglycosylation assay (LDA), to probe conformational changes of glycoproteins on a systems-wide scale. This method improves the current toolbox of structural proteomics by combining site and conformational-specific PNGase F enzymatic activity with large scale quantitative proteomics. X-ray crystallography, nuclear magnetic resonance spectroscopy and cryoEM techniques are the major techniques applied to elucidate macromolecule structures. However, the size and heterogeneity of the oligosaccharide chains poses several challenges to the applications of these techniques to glycoproteins. The LDA method presented here, can be applied to a range of pathophysiological conditions and expanded to investigate PTMs-mediated structural changes in complex proteomes.

Background: The accessory β1 subunits, regulating the pharmacological and biophysical properties of BK channels, always undergo post-translational modifications, especially glycosylation. To date, it remains elusive whether the glycosylation contributes to the regulation of BK channels by β1 subunits. Methods: Herein, we combined the electrophysiological approach with molecular mutations and biochemical manipulation to investigate the function roles of N-glycosylation in β1 subunits. Results: The results show that deglycosylation of β1 subunits through double-site mutations (β1 N80A/N142A or β1 N80Q/N142Q) could significantly increase the inhibitory potency of iberiotoxin, a specific BK channel blocker. The deglycosylated channels also have a different sensitivity to martentoxin, another BK channel modulator with some remarkable effects as reported before. On the contrary to enhancing effects of martentoxin on glycosylated BK channels under the presence of cytoplasmic Ca2+, deglycosylated channels were not affected by the toxin. However, the deglycosylated channels were surprisingly inhibited by martentoxin under the absence of cytoplasmic Ca2+, while the glycosylated channels were not inhibited under this same condition. In addition, wild type BK (α+β1) channels treated with PNGase F also showed the same trend of pharmacological results to the mutants. Similar to this modulation of glycosylation on BK channel pharmacology, the deglycosylated forms of the channels were activated at a faster speed than the glycosylated ones. However, the V1/2 and slope were not changed by the glycosylation. Conclusion: The present study reveals that glycosylation is an indispensable determinant of the modulation of β1-subunit on BK channel pharmacology and its activation. The loss of glycosylation of β1 subunits could lead to the dysfunction of BK channel, resulting in a pathological state.

Background To directly assess the biological role of oligosaccharides in recombinant equine chorionic gonadotropin (rec-eCG) functioning, cDNA encoding the full-length eCGβ-subunit was fused with the mature protein part of the α-subunit, and we examined the expression levels of deglycosylated eCG mutants, the ovulation rate for deglycosylated mutants in C57BL/6 mice. Results The characterizations of heterodimeric and tethered mutants were studied following their respective secretions in culture medium, molecular weight and ovulation in vivo. Rec-eCG variants containing mutations at glycosylation sites at Asn82 of the α-subunit (eCGβ/αΔ82) and Asn13 of the β-subunit (eCGβΔ13/α) were not efficiently secreted into the culture medium from transfected cells. Western blot analysis revealed that the rec-eCGβ/α proteins have an approximate broad range of molecular weights of 40–46 kDa. Three rec-eCG mutants—a deglycosylated site at Asn56 of the α-subunit (eCGβ/αΔ56), a deletion of the C-terminal region of the β-subunit (eCGβ-D/α), and the double mutant (eCGβ-D/αΔ56)—turned out to have clearly lower (approximately 4–23 kDa) molecular weights. Protein N-glycosydase F (PNGase F) treatment markedly decreased the molecular weight to approximately 2–10 kDa. Normal oocytes were significantly more abundant in the natural eCG–treated group than in mutant rec-eCG–treated groups. In particular, numbers of nonfuntional oocytes were remarkably lower in all rec-eCG groups. Conclusions Our results indicate that the ovulation rates of oocytes are not affected by the deglycosylated rec-eCGβ/α mutant proteins. There are around 20% non-functional oocytes with natural eCG and only 2% with the rec-eCGs tested. These results provide insight into the molecular mechanisms underlying the production of rec-eCG hormones with excellent bioactivity in vivo. Electronic supplementary material The online version of this article (10.1186/s12896-019-0550-6) contains supplementary material, which is available to authorized users.

Glycosylation alters the molecular and functional features of glycoproteins, which is closely related with many physiological processes and diseases. During "window of implantation", uterine endometrium transforms into a receptive status to accept the embryo, thereby establishing successful embryo implantation. In this article, we aimed at investigating the role of N-glycosylation, a major modification type of glycoproteins, in the process of endometrial receptivity establishment. Results found that human uterine endometrial tissues at mid-secretory phase exhibited Lectin PHA-E+L (recognizes the branched N-glycans) positive N-glycans as measured by the Lectin fluorescent staining analysis. By utilizing in vitro implantation model, we found that de-N-glycosylation of human endometrial Ishikawa and RL95-2 cells by tunicamycin (inhibitor of N-glycosylation) and peptide-N-glycosidase F (PNGase F) impaired their receptive ability to human trophoblastic JAR cells. Meanwhile, N-glycosylation of integrin αvβ3 and leukemia inhibitory factor receptor (LIFR) are found to play key roles in regulating the ECM-dependent FAK/Paxillin and LIF-induced STAT3 signaling pathways, respectively, thus affecting the receptive potentials of endometrial cells. Furthermore, in vivo experiments and primary mouse endometrial cells-embryos coculture model further verified that N-glycosylation of mouse endometrial cells contributed to the successful implantation. Our results provide new evidence to show that N-glycosylation of uterine endometrium is essential for maintaining the receptive functions, which gives a better understanding of the glycobiology of implantation.

Reliable biomarkers for oral cancer (OC) remain scarce, and routine tests for the detection of precancerous lesions are not routine in the clinical setting. This study addresses a current unmet need for more sensitive and quantitative tools for the management of OC. Whole saliva was used to identify and characterize the nature of glycans present in saliva and determine their potential as OC biomarkers. Proteins obtained from whole saliva were subjected to PNGase F enzymatic digestion. The resulting N-glycans were analyzed with weak anion exchange chromatography, exoglycosidase digestions coupled to ultra-high performance liquid chromatography and/or mass spectrometry. To determine N-glycan changes, 23 individuals with or without cancerous oral lesions were analyzed using Hydrophilic interaction ultra performance liquid chromatography (HILIC–UPLC), and peak-based area relative quantitation was performed. An abundant and complex salivary N-glycomic profile was identified. The main structures present in saliva were neutral oligosaccharides consisting of high mannose, hybrid and complex structures, followed by smaller fractions of mono and di-sialylated structures. To determine if differential N-glycosylation patterns distinguish between OC and control groups, Mann–Whitney testing and principle component analysis (PCA) were used. Eleven peaks were shown to be statistically significant (P ≤ 0.05), while PCA analysis showed segregation of the two groups based on their glycan profile. N-glycosylation changes are active in the oral carcinogenic process and may serve as biomarkers for early detection to reduce morbidity and mortality. Identifying which N-glycans contribute most in the carcinogenic process may lead to their use in the detection, prognosis and treatment of OC.

BACKGROUND Congenital disorders of glycosylation (CDG) represent 1 of the largest groups of metabolic disorders with >130 subtypes identified to date. The majority of CDG subtypes are disorders of N-linked glycosylation, in which carbohydrate residues, namely, N-glycans, are posttranslationally linked to asparagine molecules in peptides. To improve the diagnostic capability for CDG, we developed and validated a plasma N-glycan assay using flow injection–electrospray ionization–quadrupole time-of-flight mass spectrometry. METHODS After PNGase F digestion of plasma glycoproteins, N-glycans were linked to a quinolone using a transient amine group at the reducing end, isolated by a hydrophilic interaction chromatography column, and then identified by accurate mass and quantified using a stable isotope-labeled glycopeptide as the internal standard. RESULTS This assay differed from other N-glycan profiling methods because it was free of any contamination from circulating free glycans and was semiquantitative. The low end of the detection range tested was at 63 nmol/L for disialo-biantennary N-glycan. The majority of N-glycans in normal plasma had CONCLUSIONS The clinical specificity and sensitivity of N-glycan analysis was much improved with this method. Additional CDGs can be diagnosed that would be missed by carbohydrate-deficient transferrin analysis. The assay provides novel biomarkers with diagnostic and potentially therapeutic significance.

Aspergillus glaucus MA0196 produces a highly glycosylated aspartic protease (PepA_MA0196) that shows hydrolytic and decolorization activities toward hemoglobin. Sequence analysis of PepA_MA0196 indicated two potential N-glycosylation consensus sites, at Asn131 and Asn275. To elucidate the role of N-glycosylation in the biochemical properties of PepA_MA0196 and the resulting effects, recombinant PepA_MA0196 and three mutant proteases deglycosylated via site-direct mutagenesis were heterologously expressed in Pichia pastoris. After hydrolysis of the enzymes by PNGase F or Endo H, the molecular masses on SDS-PAGE of the enzymatically deglycosylated wild-type PepA_MA0196 and recombinant and single-mutant proteases were identical to that of the double-mutant protease (42.2 kDa). PepA_MA0196 and its recombinant were stable over a broader pH range and more thermostable than the deglycosylated mutant proteases, especially the double-mutant protease. In addition, PepA_MA0196 retained a higher activity at low water activity than did the double-mutant protease. The commercial applications of the glycosylated protease from strain MA0196 include hydrolysis of the heme proteins in dried tuna meat under low water content. Hemoprotein lysis is also a property of interest to the meat processing and detergent industries.

The disulfide linkages of two etanercept products, Enbrel® (innovator drug) and TuNEX®, were characterized and compared using a multi-fragmentation approach consisting of electron transfer dissociation (ETD) and collision induced dissociation (CID) in combination with multi-enzyme digestion protocols (from Lys-C, trypsin, Glu-C, and PNGase F). Multi-fragmentation approach allowed multi-disulfide linkages contained in a peptide to be un-ambiguously assigned based on the cleavage of both the disulfide and the backbone linkages in a MS3 schedule. New insights gained using this approach were discussed. A total of 29 disulfides, Cys18-Cys31, Cys32-Cys45, Cys35-Cys53, Cys56-Cys71, Cys74-Cys88, Cys78-Cys-96, Cys98-Cys104, Cys112-Cys121, Cys115-Cys139, Cys-142-Cys157, Cys163-Cys178 in TNFR portion and Cys240-Cys240, Cys246-Cys246, Cys249-Cys249, Cys281-Cys341, Cys387-Cys445 in IgG1 Fc domain, were completely assigned with the demonstration of the same disulfide linkages between the Enbrel® and TuNEX® products. The data showed the higher order structure was preserved throughout the recombinant manufacturing processes and consistent between the two products. Keywords: Enbrel®, TuNEX®, LC-MS, CID, ETD

The hepatitis B virus (HBV)-induced chronic liver diseases are serious health threats worldwide. There is evidence to display the alterations of salivary N-linked glycans related to the development of HBV-infected liver diseases. Here, we further investigated the alterations of fucosylated N/O-glycans recognized by LTL in saliva from 120 subjects (30 healthy volunteers (HV), 30 patients with hepatitis B (HB), 30 patients with hepatic cirrhosis (HC), and 30 patients with hepatocellular carcinoma (HCC)) using salivary microarrys and MALDI-TOF/TOF-MS. The results showed that the expression level of fucosylated glycans recognized by LTL was significantly increased in HCC compared with other subjects (P < 0.0001). Besides, the fucosylated glycoproteins were isolated from pooled saliva of HV, HB, HC, and HCC by LTL-magnetic particle conjugates. Then, N/O- glycans were released from the isolated glycoproteins with PNGase F and NaClO, and were identified by MALDI-TOF-MS, respectively. Totally, there were 21/20, 25/18, 29/19, and 28/24 N/O-glycan peaks that were identified and annotated with proposed structures in saliva of HV, HB, HC, and HCC. Among the total, there were 8 N-glycan peaks (e.g., m/z 1905.634, 2158.777 and 2905.036) and 15 O-glycan peaks (e.g., 1177.407, 1308.444 and 1322.444) that only presented in patients with HBV-induced liver diseases. One N-glycan peak (m/z 2205.766) was unique in HC, and 9 O-glycan peaks (e.g., m/z 1157.420, 1163.417 and 1193.402) were unique in HCC. This study could facilitate the discovery of biomarkers for HC and HCC based on precise alterations of fucosylated N/O-glycans in saliva.

[Background]: Colorectal cancer (CRC) is the second most common cause of cancer death worldwide. It is broadly described that cyclooxygenase-2 (COX-2) is mainly overexpressed in CRC but less is known regarding post-translational modifications of this enzyme that may regulate its activity, intracellular localization and stability. Since metabolic and proteomic profile analysis is essential for cancer prognosis and diagnosis, our hypothesis is that the analysis of correlations between these specific parameters and COX-2 state in tumors of a high number of CRC patients could be useful for the understanding of the basis of this cancer in humans., [Aim]: To analyze COX-2 regulation in colorectal cancer and to perform a detailed analysis of their metabolic and proteomic profile., [Methods]: Biopsies from both healthy and pathological colorectal tissues were taken under informed consent from patients during standard colonoscopy procedure in the University Hospital of Bellvitge (Barcelona, Spain) and Germans Trias i Pujol University Hospital (Campus Can Ruti) (Barcelona, Spain). Western blot analysis was used to determine COX-2 levels. Deglycosylation assays were performed in both cells and tumor samples incubating each sample with peptide N-glycosidase F (PNGase F). Prostaglandin E2 (PGE2) levels were determined using a specific ELISA. 1H high resolution magic angle spinning (HRMAS) analysis was performed using a Bruker AVIII 500 MHz spectrometer and proteomic analysis was performed in a nano-liquid chromatography-tandem mass spectrometer (nano LC-MS/MS) using a QExactive HF orbitrap MS., [Results]: Our data show that COX-2 has a differential expression profile in tumor tissue of CRC patients vs the adjacent non-tumor area, which correspond to a glycosylated and less active state of the protein. This fact was associated to a lesser PGE2 production in tumors. These results were corroborated in vitro performing deglycosylation assays in HT29 cell line where COX-2 protein profile was modified after PNGase F incubation, showing higher PGE2 levels. Moreover, HRMAS analysis indicated that tumor tissue has altered metabolic features vs non-tumor counterparts, presenting increased levels of certain metabolites such as taurine and phosphocholine and lower levels of lactate. In proteomic experiments, we detected an enlarged number of proteins in tumors that are mainly implicated in basic biological functions like mitochondrial activity, DNA/RNA processing, vesicular trafficking, metabolism, cytoskeleton and splicing., [Conclusion]: In our colorectal cancer cohort, tumor tissue presents a differential COX-2 expression pattern with lower enzymatic activity that can be related to an altered metabolic and proteomic profile., MINECO, No. SAF2017-82436R, SAF2016-75004R, RTC-2017-6283-1, PRB3 (IPT17/0019-ISCIII-SGEFI/ERDF) and BIO2015-67580P; Comunidad de Madrid, No. S2017/BMD-3686; Fundación Ramón Areces, No. 2016/CIVP18A3864; Instituto de Salud Carlos III, Spain, CIBERCV, No. CB/11/00222 and CB16/11/00277; FEDER, CIBEREHD; the Ministerio de Ciencia, Innovación y Universidades (MCNU); the Pro CNIC Foundation; and Severo Ochoa Center of Excellence, No. SEV-2015-0505.

N-Glycosylation is one of the most common and important post-translational modification methods, and it plays a vital role in controlling many biological processes. Increasing discovery of abnormal alterations in N-linked glycans associated with many diseases leads to greater demands for rapid and efficient N-glycosylation profiling in large-scale clinical samples. In the workflow of global N-glycosylation analysis, enzymatic digestion is the main rate-limiting step, and it includes both protease digestion and peptide-N4–(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase) F deglycosylation. Prolonged incubation time is generally required because of the limited digestion efficiency of the conventional in-solution digestion method. Here, we propose novel thermoresponsive magnetic fluid (TMF)-immobilized enzymes (trypsin or PNGase F) for ultrafast and highly efficient proteome digestion and deglycosylation. Unlike other magnetic material-immobilized enzymes, TMF-immobilized enzymes display a unique temperature-triggered magnetic response behavior. At room temperature, a TMF-immobilized enzyme completely dissolves in an aqueous solution and forms a homogeneous system with a protein/peptide sample for efficient digestion but cannot be separated by magnetic force because of its excellent water dispersity. Above its lower critical solution temperature (LCST), thermoflocculation of a TMF-immobilized enzyme allows it to be easily recovered by increasing the temperature and magnetic force. Taking advantage of the unique homogeneous reaction of a TMF-immobilized enzyme, both protein digestion and glycopeptide deglycosylation can be finished within 3 min, and the whole sample processing time can be reduced by more than 20 times. The application of a TMF-immobilized enzyme in large-scale profiling of protein N-glycosylation in urine samples led to the successful identification of 2,197 N-glycopeptides and further demonstrated the potential of this strategy for fast and high-throughput analysis of N-glycoproteome in clinical samples.

Traditionally the biotransformation of antibody drug conjugates (ADCs) has been evaluated by affinity capture on streptavidin magnetic beads coated with a biotinylated capture reagent. To reduce the complexity of the analyte, the affinity captured ADCs are digested with enzymes ("on-bead" or after elution), and/or interchain disulfides are reduced to generate LC and HC fragments prior to mass spectrometry analysis. The "on-bead" enzymatic digestion with IdeS and PNGase F is not efficient and requires longer incubation times to achieve complete Fc and N-glycan removal. This results in a prolonged sample preparation time (7-18 h) and is not suitable for labile ADCs due to the possibility of assay-induced artifacts. To address these challenges, we developed an affinity capture method, where the ADCs are first captured onto streptavidin cartridges coated with a biotinylated generic capture reagent, followed by a 15 min "on-cartridge" digestion with IdeS or PNGase F. The ADCs are then eluted and directly analyzed by LC-HRMS. This method was successfully applied for the biotransformation assessment of site-specific ADCs with payload conjugated on the Fab or Fc. The reduced complexity of the analyte (Fc and N-glycan removal) combined with HRMS enabled sensitive and accurate identification of minor mass change catabolites and changes in the DAR distribution. This automated cartridge-based affinity capture method is fast with a total sample preparation time of less than 4 h (hands-on time of less than 1 h) and can be utilized for any human mAb/ADC independent of isotype (IgG1, IgG2, and IgG4).

rHuPH20, a neutral pH-active hyaluronidase that degrades glycosaminoglycans under physiologic conditions, has six potential N-glycosylation sites. In this report, the rHuPH20 expressed in Chinese hamster ovary (CHO) cells was analyzed and characterized using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Removal of the N-linked glycans from rHuPH20 with PNGase F shifted the molecular weight from 66 kDa to approximately 52 kDa, its deduced molecular weight based on sequence analysis, suggesting that most, if not all, of the potential N-glycosylation sites are linked to oligosaccharides. Then the N-linked glycans released from the rHuPH20 by PNGase F were characterized by UPLC-FLR-MS, and the six N-glycosylation sites of the rHuPH20 were identified and characterized by UPLC-MS/MS at peptide levels. Subsequently, we found that the rHuPH20 increased the dispersion of locally subcutaneous injected drugs and the in vitro and in vivo bioactivity were decreased significantly after PNGase F treatment. In particular, rHuPH20 significantly augmented the absolute bioavailability of locally subcutaneous injected large protein therapeutics, while the bioavailability decreased after being digested by PNGase F. These results demonstrated that N-glycosylation is important for the bioactivity of the rHuPH20.

The newly identified pyroglutamylated RFamide peptide (QRFP) signaling system has been shown to be implicated in regulating a variety of physiological processes. G-protein-coupled receptors (GPCRs) are preferentially N-glycosylated on extracellular domains. The human QRFP receptor QRFPR (GPR103) possesses three N-glycosylation consensus sites, two located on the N-terminal domain (N5 and N19) and one on the first extracellular loop (ECL1) (N106); however, to date, their role in QRFPR expression and signaling has not been established. Here, we combined mutants with glutamine substitution of the critical asparagines of the consensus sites with glycosidase PNGase F and N-glycosylation inhibitor tunicamycin to study the effect of N-glycosylation in the regulation of QRFPR cell surface expression and signaling. Western blot analysis performed with site-directed mutagenesis revealed that two asparagines at N19 in the N-terminus and N106 in ECL1, but not N5 in the N-terminus, served as sites for N-glycosylation. Treatment with PNGase F and tunicamycin resulted in a reduction in both two-protein species, ~43 kDa and ~85 kDa in size, by 2-4 kDa. Analysis with confocal microscopy and quantitative ELISA showed that N-glycosylation of QRFPR is not essentially required for targeting the cell membrane. However, further binding assay and functional assays demonstrated that removal of N-glycosylation sequons or treatment with tunicamycin led to significant impairments in the interaction of receptor with QRFP26 and downstream signaling. Thus, our findings suggest that for the human QRFP receptor (QRFPR), N-glycosylation is not important for cell surface expression but is a pre-requisite for ligand binding and receptor activation.

Capillary gel electrophoresis-based methods were applied to comprehensively characterize two development phase new modality monoclonal antibodies including a glycoengineered and a bispecific test compound. The samples were subjected to multilevel characterization at the intact (both by SDS-SGE and cIEF) as well as the reduced protein and the released N-glycan levels. SDS capillary gel electrophoresis analysis showed excellent separation of the light and heavy chains of both samples. The bispecific antibody required a special temperature gradient denaturation process and a longer capillary to resolve its two light chain fragments. Separation of PNGase F digested antibodies revealed migration time shifts, suggesting the presence of N-linked glycosylation on the corresponding subunits. For efficient glycan removal, the highly glycosylated glycoengineered monoclonal antibody was trypsin digested prior to the endoglycosidase treatment. The released glycans were profiled by capillary gel electrophoresis after APTS labeling and their oligosaccharide structures were identified by exoglycosidase based carbohydrate sequencing. Finally, capillary isoelectric focusing shed light on the charge heterogeneity of the test compounds, providing important complementary information. A flowchart was established for workflow optimization.

The analysis of N-glycan distributions in formalin-fixed, paraffin-embedded (FFPE) tissues by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is an effective approach for characterization of many disease states. As the workflow has matured and new technology emerged, approaches are needed to more efficiently characterize the isomeric structures of these N-glycans to expand on the specificity of their localization within tissue. Sialic acid chemical derivatization can be used to determine the isomeric linkage (α2,3 or α2,6) of sialic acids attached to N-glycans, while endoglycosidase F3 (Endo F3) can be enzymatically applied to preferentially release α1,6-linked core fucosylated glycans, further describing the linkage of fucose on N-glycans. Here we describe workflows where N-glycans are chemically derivatized to reveal sialic acid isomeric linkages, combined with a dual-enzymatic approach of endoglycosidase F3 and PNGase F to further elucidate fucosylation isomers on the same tissue section.

Glycoproteins, proteins that are co- and posttranslationally modified by sugars (glycans), have significant roles in pathophysiology of many different diseases. One of the main steps in sample preparation for free N-glycan analysis is deglycosylation or glycan removal. The aim of this study was to compare different peptide N-glycosidase F (PNGase F) enzymes (Rapid PNGase F and two recombinant versions) for deglycosylation of total human plasma glycoproteins and different amounts of human immunoglobulin G (IgG). Deglycosylation with different PNGase F enzymes resulted in different IgG and plasma N-glycosylation hydrophilic interaction liquid chromatography ultra-performance liquid chromatography profiles. Additionally, one recombinant version of PNGase F is more efficient in deglycosylation of complex N-glycans compared with Rapid PNGase F and recombinant version of PNGase F from a different manufacturer. In terms of chromatographic peak intensities and coefficient of variation %Area values, all tested versions of PNGase F enzymes were very reproducible and on the similar level when used in optimal conditions. However, care should be taken in terms of which enzyme is used with which protocol, particularly when scaling up.

The use of sequential exoglycosidase digestion of oligosaccharides followed by LC-FLD, LC-MS or CE analysis provides detailed carbohydrate structural information. Highly specific exoglycosidases cleave monosaccharides from the nonreducing end of an oligosaccharide and yield information about the linkage, stereochemistry and configuration of the anomeric carbon. Here we use combinations of exoglycosidases to precisely characterize glycans on the Fc domain of therapeutic antibodies and dimeric fusion proteins. The workflow described includes glycan release with Rapid™ PNGase F (NEB #P0710), direct labeling of released glycans with procainamide (PCA) or 2-aminobenzamide (2AB), cleanup of labeled glycans and a 3 h enzymatic digestion with exoglycosidases. This protocol is designed for completion within an 8 h time frame to allow for subsequent LC-FLD, LC-MS, or CE analysis overnight.

Released N-glycan analysis using the fluorescent label 2-AB (2-aminobenzamide) has been the "gold standard" method for released glycan analysis for several years. The more recent RapiFluor-MS™ labeling technique, however, offers enhanced mass spectrometric detection of released N-glycans, improving the sensitivity and detection limits of the method. The optimized multidimensional detection offers increased confidence in glycan identification which can be further supported by an exoglycosidase digestion array (optional). Here we describe the PNGase F release of N-glycans from a typical IgG1 monoclonal antibody (mAb) with subsequent labeling with RapiFluor-MS™ for detection by HILIC-FLR-MS. The method output quantifies the relative proportion of each glycan species including core afucosylation, sialylation, and high-mannose content, and has a limit of detection (LOD) of 0.01% relative abundance.

Purpose Tear fluid N-Glycome from patients affected with vernal (VKC) and atopic keratoconjunctivitis (AKC) was investigated to identify specific changes in tears and to recognize possible glyco-biomarkers. Methods The analysis of the N-glycans was performed using matrix-assisted laser desorption ionization mass spectrometry on single tear samples. Tears from control normal subjects (CTRL), VKC and AKC patients were processed and treated with peptide N-glycosidase F (PNGase F) to deglycosylate N-glycoproteins. Released N-glycans were purified, permethylated, and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and tandem mass spectrometry (MALDI-TOF MS and MALDI-TOF MS/MS). Results More than 150 complex N-glycans, including highly fucosylated biantennary, triantennary, tetra-antennary, and bisecting species, were observed in our spectra. Three distinct patterns for CTRL, VKC, and AKC patients were identified in terms of relative intensities for some N-glycans structures. Major variations involved bisecting and hyperfucosylated glycoforms. The most intense ions were associated with species at m/z 1907.0 (asialo, agalacto, bisected, biantennary structure-NGA2B) in CTRL MS profiles, at m/z 2605.3 and 2966.5 in VKC, and at m/z 2792.4 in AKC corresponding to a well-known biantennary, disialylated N-glycan. Several peaks were associated with structures bearing one or two Lewis X epitopes. Structures were confirmed by MS/MS analysis. Quantitative differences among the three groups were statistically significant. Conclusions Tear MS profiles are rich in specific glycoforms, particularly those with a high fucosylation degree, indicating both core and peripheral decoration. Tear N-glycome analysis provided important information for a better comprehension of VKC and AKC alterations at the molecular level.

N-glycan imaging mass spectrometry (N-glycan IMS) enables the detection and characterization of N-glycans in thin histological tissue sections. N-glycan IMS is used to study N-glycan regulation and localization in tissue-specific regions, such as tumor and normal adjacent to tumor, or by cell type within a tissue. Once a specific tissue-localized N-glycan signature is found to be associated with by a disease state, it has been challenging to study modulation of the same N-glycan signature by conventional molecular biology techniques. Here we describe a protocol that adapts tissue N-glycan IMS analysis workflows to cells grown on glass slides in an array format. Cells are grown under normal conditions in a cell culture chamber, fixed to maintain normal morphology, and sprayed with a thin coating of PNGase F to release N-glycans for imaging mass spectrometry profiling.

Glycosylation is an important protein post-translational modification in eukaryotic organisms. It is involved in many important life processes, such as cell recognition, differentiation, development, signal transduction and immune response. This study carried out the first N-linked glycosylation proteome analysis of wheat seedling leaves using HILIC glycosylation enrichment, chemical deglycosylation, HPLC separation and tandem mass spectrometric identification. In total, we detected 308 glycosylated peptides and 316 glycosylated sites corresponding to 248 unique glycoproteins. The identified glycoproteins were mainly concentrated in plasma membranes (25.6%), cell wall (16.8%) and extracellular area (16%). In terms of molecular function, 65% glycoproteins belonged to various enzymes with catalytic activity such as kinase, carboxypeptidase, peroxidase and phosphatase, and, particularly, 25% of glycoproteins were related to binding functions. These glycoproteins are involved in cell wall reconstruction, biomacromolecular metabolism, signal transduction, endoplasmic reticulum quality control and stress response. Analysis indicated that 57.66% of glycoproteins were highly conserved in other plant species while 42.34% of glycoproteins went unidentified among the conserved glycosylated homologous proteins in other plant species; these may be the new N-linked glycosylated proteins first identified in wheat. The glycosylation sites generally occurred on the random coil, which could play roles in maintaining the structural stability of proteins. PNGase F digestion and glycosylation site mutations further verified the glycosylation modification and glycosylation sites of LRR receptor-like serine/threonine-protein kinase (LRR-RLK) and Beta-D-glucan exohydrolase (β-D-GEH). Our results indicated that N-linked glycosylated proteins could play important roles in the early seedling growth of wheat.

Glycosylation is one of the most crucial posttranslational modifications of proteins, containing a remarkable amount of biological information. The alteration of glycosylation is closely associated with certain diseases. Exploring glyco-code in the development of diseases is a hot topic in recent years. Esophageal squamous cell carcinoma (ESCC) is the primary pathological histology in developing countries and a severe threat to human health. Although the glycan profiles in the blood samples of ESCC patients were analyzed using glycomic and glycoproteomic methods, the difference of salivary glycopatterns between healthy subjects and ESCC patients is not explicit yet. In the present study, ESCC patients (n = 16) and healthy volunteers (HVs, n = 25) were enrolled. The glycomic strategy combining lectin microarray and lectin blotting was employed to investigate and confirm the altered salivary glycopatterns. Datura stramonium (DSA) was selected to isolate the GlcNAc or Galβ1-4GlcNA-containing glycoproteins due to the distinct difference between ESCC patients and HVs. The N-glycans from DSA-enriched glycoproteins were released by PNGase F and further identified by MALDI-TOF/TOF-MS to obtain the precise structural information of the altered glycans. As a result, the glycopatterns recognized by 13 lectins (e.g., ECA, RCA120, and DSA) showed significant alterations in ESCC patients’ saliva. The ESCC patients showed higher levels of GalNAc and Gal, sialic acid, and GlcNAc expression profiles and lower levels of mannose and fucose expression profiles. The MALDI-TOF/TOF-MS results indicated that the proportion of the GlcNAc or Galβ1-4GlcNAc-containing N-glycans was increased in ESCC patients (79.04%) compared with HV (63.20%), which was consistent with the results of lectin microarrays. Our findings provide comprehensive information to understand the complex physiological changes in ESCC patients. And the altered salivary glycopatterns such as GlcNAc or Galβ1-4GlcNAc-containing N-glycans recognized by DSA might serve as potential biomarkers for the diagnosis of ESCC patients.