The potential of polyaniline-coated stationary phase in hydrophilic interaction liquid chromatography-based solid-phase extraction for glycopeptide enrichment.

Glycopeptide enrichment is a crucial step in glycoproteomic analysis, often achieved through solid-phase extraction (SPE) on polar stationary phases in hydrophilic interaction liquid chromatography (HILIC). This study explores the potential of polyaniline (PANI)-coated silica gel for enriching human immunoglobulin G (IgG). Experimental conditions were varied to assess their impact on glycopeptide enrichment efficiency, comparing PANI-cotton wool SPE with conventional cotton wool as SPE sorbents. Two formic acid concentrations (0.1% and 1%) in elution solvent were tested, revealing that higher concentrations led to earlier elution of studied glycopeptides, especially for sialylated glycopeptides. Substituting formic acid with acetic acid increased the interaction of neutral glycopeptides with the PANI-modified sorbent, while sialylated glycopeptides showed no significant change in enrichment efficiency. Acetonitrile concentration in the elution solvent (5%, 10%, and 20%) affected the enrichment efficiency with most glycopeptides eluting at the lowest acetonitrile concentration. The acetonitrile concentration in conditioning and washing solutions (65%, 75%, and 85%) played a crucial role; at 65% acetonitrile, glycopeptides were least retained on the stationary phase, and neutral glycopeptides were even detected in the flow-through fraction. This study shows the potential of in-house-prepared PANI-modified sorbents for SPE-HILIC glycopeptide enrichment, highlighting the crucial role of tuning experimental conditions in sample preparation to enhance enrichment efficiency and selectivity.

IgG glycopeptide enrichment using hydrophilic interaction chromatography-based solid-phase extraction on an aminopropyl column.

The sample preparation step is pivotal in glycoproteomic analysis. An effective approach in glycoprotein sample preparation involves enriching glycopeptides by solid-phase extraction (SPE) using polar stationary phases in hydrophilic interaction liquid chromatography (HILIC) mode. The aim of this work is to show how different experimental conditions influence the enrichment efficiency of glycopeptides from human immunoglobulin G (IgG) on an aminopropyl-modified SPE column. Different compositions of the elution solvent (acetonitrile, methanol, and isopropanol), along with varying concentrations of elution solvent acidifiers (formic and acetic acid), and different concentrations of acetonitrile for the conditioning and washing solvents (65%, 75%, and 85% acetonitrile) were tested to observe their effects on the glycopeptide enrichment process. Isopropanol proved less effective in enriching glycopeptides, while acetonitrile was the most efficient, with methanol in between. Higher formic acid concentrations in the elution solvent weakened the ionic interactions, particularly with sialylated glycopeptides. Substituting formic acid with acetic acid led to earlier elution of more glycopeptides. The acetonitrile concentration in conditioning and washing solutions played a key role; at 65% acetonitrile, glycopeptides were not retained on the SPE column and were detected in the flow-through fraction. Ultimately, it was proven that the enrichment method was applicable to human plasma samples, resulting in a significant decrease in the abundances of non-glycosylated peptides. To the best of our knowledge, this study represents the first systematic investigation into the impact of the mobile phase on glycopeptide enrichment using an aminopropyl-modified SPE column in HILIC mode. This study demonstrates the substantial impact of even minor variations in experimental conditions, which have not yet been considered in the literature, on SPE-HILIC glycopeptide enrichment. Consequently, meticulous optimization of these conditions is imperative to enhance the specificity and selectivity of glycoproteomic analysis, ensuring accurate and reliable quantification.

Magnetic Bead-Based Workflow for Sensitive and Streamlined Cell Surface Proteomics.

Cell surface proteins represent an important class of molecules for therapeutic targeting and cellular phenotyping. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, post-translational modifications, hydrophobic regions, and processing requirements. To improve their identification, we optimized a Cell-Surface Capture (CSC) workflow that incorporates magnetic bead-based processing. Using this approach, we evaluated labeling conditions (biotin tags and catalysts), enrichment specificity (streptavidin beads), missed cleavages (lysis buffers), nonenzymatic deamidation (digestion and deglycosylation buffers), and data acquisition methods (DDA, DIA, and TMT). Our findings support the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid, SDS/urea-based lysis buffers, single-pot solid-phased-enhanced sample-preparation (SP3), and streptavidin magnetic beads for maximal surfaceome coverage. Notably, with semiautomated processing, sample handling was simplified and between ∼600 and 900 cell surface N-glycoproteins were identified from only 25-200 µg of HeLa protein. CSC also revealed significant differences between in vitro monolayer cultures and in vivo tumor xenografts of murine CT26 colon adenocarcinoma samples that may aid in target identification for drug development. Overall, the improved efficiency of the magnetic-based CSC workflow identified both previously reported and novel N-glycosites with less material and high reproducibility that should help advance the field of surfaceomics by providing insight in cellular phenotypes not previously documented.

An integrated method for IgG N-glycans enrichment and analysis: Understanding the role of IgG glycosylation in diabetic foot ulcer.

Diabetic foot ulcer (DFU) is the most common and serious complication of diabetes, and its incidence, disability, and mortality rates are increasing worldwide. The pathogenesis of DFU is associated with dysregulated inflammation mediated by abnormal immunoglobulin G (IgG) glycosylation. In this study, we developed a comprehensive method for IgG N-linked glycosylation in the serum of DFU patients. Through analysis, we identified 31 IgG1 glycans, 32 IgG2 glycans, and 30 IgG4 glycans in the DFU serum. Furthermore, 13 IgG1 glycans, 12 IgG2 glycans, and 5 IgG4 glycans in the DFU groups were found to be significantly different from those of the control groups (p < 0.05). Of these, compared with the control group, one glycan was unique to DFU patients, and seven glycans were not detected in the DFU group. In terms of glycan characteristics, we observed a substantial decrease in galactosylation, sialylation and bisecting GlcNAcylation, and a significant increase in agalactosylation. Abnormal IgG N-glycosylation modifications were significantly associated with the chronic inflammation that is characteristic of DFU. Further, this is the first comprehensive analysis of subclass-specific IgG N-glycosylation in DFU patients, which not only fills the gap of DFU in terms of the pathological mechanisms related to IgG glycosylation but also may provide valuable clues for the immunotherapeutic pathway of DFU.

Dual-functionalized two-dimensional metal-organic framework composite with highly hydrophilicity for effective enrichment of glycopeptides.

Protein glycosylation research is currently focused on the development of various functionalized materials that can effectively enrich the levels of glycopeptides in samples. However, most of these materials possess limited glycopeptide-specific recognition sites because of large steric hindrance, unsuitable mass transfer kinetics, and relatively low surface areas. Herein, a highly hydrophilic two-dimensional (2-D) metal-organic framework (MOF) nanosheet modified with glutathione (GSH) and l-cysteine (l-Cys) (denoted as Zr-Fc MOF@Au@GC) has been synthesized for efficient glycopeptide enrichment. Using this composite material, 39 and 44 glycopeptides from horseradish peroxidase (HRP) and human serum immunoglobulin G (IgG) digests were detected, respectively, which represents a higher efficiency for glycopeptide enrichment from model glycoprotein digests than has been previously reported. The material Zr-Fc MOF@Au@GC exhibited ultra-high sensitivity (0.1 fmol/µL), excellent selectivity (weight ratio of HRP tryptic digest to bovine serum albumin (BSA) tryptic digest = 1:2000), good binding capacity (200 mg/g), satisfactory reusability, and long-term storage capacity. In addition, 655 glycopeptides corresponding to 366 glycoproteins were identified from human serum samples. To the best of our knowledge, this is the largest number of glycoproteins detected in human serum samples to date. These results indicated that Zr-Fc MOF@Au@GC has the potential to be used for the enrichment of glycopeptides in biological samples and the analysis of protein glycosylation.

Construction of dual-hydrophilic metal-organic framework with hierarchical porous structure for efficient glycopeptide enrichment.

As an important role in life activities, it is necessary and important to study protein glycosylation. The pre-enrichment of N-glycopeptides is a significant step in glycoproteomics research. According to the inherent size, hydrophilicity and other properties of N-glycopeptides, affinity materials designed to match them will be able to separate N-glycopeptides from complex samples. In this work, we designed and prepared dual-hydrophilic hierarchical porous metal-organic frameworks (MOFs) nanospheres by metal-organic assembly (MOA) based template method and post-synthesis modification strategy. The hierarchical porous structure significantly improved the diffusion rate and binding sites for N-glycopeptide enrichment. Furthermore, the combination of hydrophilic MOFs and small molecules endowed the as-prepared MOFs nanospheres excellent hydrophilicity, which is conducive to the enrichment of N-glycopeptides based on hydrophilic interaction liquid chromatography (HILIC). Therefore, the nanospheres showed surprising enrichment ability for N-glycopeptides such as excellent selectivity (1/500, human serum immunoglobulin G/bovine serum albumin, m/m) and extremely low detective limitation (0.5 fmol). Meanwhile, 550 N-glycopeptides were identified from rat liver samples, proving its application potential in glycoproteomics research and providing design idea for porous affinity materials.

Surface functionalization modification of ultra-hydrophilic magnetic spheres with mesoporous silica for specific identification of glycopeptides in serum exosomes.

Protein glycosylation of human serum exosomes can reveal significant physiological information, and the development of large-scale identification strategies is crucial for the in-depth investigation of the serum exosome glycoproteome. In this study, using surface functionalization techniques, an ultra-hydrophilic mesoporous silica magnetic nanosphere (denoted as Fe3O4-CG@mSiO2) was synthesized for the quick and accurate detection of glycopeptides from HRP digests. The Fe3O4-CG@mSiO2 nanospheres demonstrated outstanding enrichment capability, high sensitivity (5 amol/µL), good size exclusion effect (HRP digests/BSA proteins, 1:10,000), stable reusability (at least 10 times), and an excellent recovery rate (108.6 ± 5.5%). Additionally, after enrichment by Fe3O4-CG@mSiO2, 156 glycopeptides assigned to 64 proteins derived from human serum exosomes were successfully identified, which demonstrates that the nanospheres have great potential for the research of the large-scale serum exosome glycoproteome.

A combination of surface-initiated atom transfer radical polymerization and photo-initiated "thiol-ene" click chemistry: Fabrication of functionalized macroporous adsorption resins for enrichment of glycopeptides.

A hydrophilic adsorbent (Cys@poly(AMA)@MAR) was successfully prepared for the enrichment of N-glycopeptides via surface-initiated atom transfer radical polymerization (SI-ATRP) and photo-initiated "thiol-ene" reaction using monodisperse macroporous adsorbent resin (MAR) as adsorption matrix. Due to the presence of electron-deficient acrylic groups and electron-rich vinyl groups in allyl methacrylate (AMA), both of them can participate in free radical reaction. Therefore, the polymerization time of SI-ATRP was optimized. The resulting poly(AMA)@MAR was modified with l-cysteine (L-Cys) via photo-initiated "thiol-ene" reaction, and the amount of vinyl retained was determined by measuring the adsorption of Cu2+. The Cys@poly(AMA)@MAR pendant brushes with high density of amine and carboxyl groups could capture N-glycopeptides from IgG digest and human serum digest by hydrophilic interaction. The 22 N-glycopeptides were identified from IgG digest and the limit of detection reached 10 fmol. The 319 N-glycosylation sites and 583 N-glycopeptides were identified from 2 µL human serum digest and mapped to 147 glycoproteins. It demonstrates great potential and commercialization prospects for the enrichment of N-glycopeptides.

Thiol-ene-based microfluidic chips for glycopeptide enrichment and online digestion of inflammation-related proteins osteopontin and immunoglobulin G.

Proteins, and more specifically glycoproteins, have been widely used as biomarkers, e.g., to monitor disease states. Bottom-up approaches based on mass spectrometry (MS) are techniques commonly utilized in glycoproteomics, involving protein digestion and glycopeptide enrichment. Here, a dual function polymeric thiol-ene-based microfluidic chip (TE microchip) was applied for the analysis of the proteins osteopontin (OPN) and immunoglobulin G (IgG), which have important roles in autoimmune diseases, in inflammatory diseases, and in coronavirus disease 2019 (COVID-19). TE microchips with larger internal surface features immobilized with trypsin were successfully utilized for OPN digestion, providing rapid and efficient digestion with a residence time of a few seconds. Furthermore, TE microchips surface-modified with ascorbic acid linker (TEA microchip) have been successfully utilized for IgG glycopeptide enrichment. To illustrate the use of the chips for more complex samples, they were applied to enrich IgG glycopeptides from human serum samples with antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The dual functional TE microchips could provide high throughput for online protein digestion and glycopeptide enrichment, showing great promise for future extended applications in proteomics and the study of related diseases.

Mesoporous covalent organic framework microspheres with dual-phase separation strategy for high-purity glycopeptide enrichment.

High-performance porous materials and rational enrichment strategies are crucial during sample pretreatment process in glycoproteomics analysis. Herein, we report a dual-phase separation strategy based on hydrophobic and hydrophilic mesoporous covalent-organic framework (COF) microspheres for high-purity glycopeptide enrichment for the first time. The COF microspheres (about 1.8±0.5 µm) with hydrophobic mesopores (2.5 nm) were prepared by a facile method at room temperature. Through the post-synthesis modification strategy, hydrophilic mesopores were obtained by modifying the vinyl ligands with glutathione (GSH), and the hydrophilic properties of COF microspheres were further enhanced by the introduction of Au nanoparticles and GSH to obtain the hydrophilic COF microspheres (denoted as COF@Au-GSH). The low-abundance hydrophilic glycopeptides could be efficiently enriched by the hydrophilic COF@Au-GSH microspheres in low polar solutions after the high-abundance hydrophobic non-glycopeptides were removed with the hydrophobic COF microspheres in high polar solutions. With the help of dual-phase separation strategy and inherent properties of the COF structure, the as-prepared COF microspheres showed splendid enrichment performance for glycopeptides, including ultrahigh sensitivity (2 fmol, IgG digests), extremely high specificity (1:10000, IgG digests/BSA digests), excellent size selectivity (1:500:500, IgG digests/BSA/IgG), and large binding capacity (200 mg g-1, IgG digests). In addition, a total of 1993 glycopeptides could be enriched and identified from the rat liver digests after enrichment by the COF microspheres. As a proof of concept application, the proposed strategy was successfully used in sample pretreatment process for plasma glycoproteomic analysis.

Design and Preparation of Novel Nitro-Oxide-Grafted Nanospheres with Enhanced Hydrogen Bonding Interaction for O-GlcNAc Analysis.

As an essential modification, O-linked ß-N-acetylglucosamine (O-GlcNAc) modulates the functions of many proteins. However, site-specific characterization of O-GlcNAcylated proteins remains challenging. Herein, an innovative material grafted with nitro-oxide (N→O) groups was designed for high affinity enrichment for O-GlcNAc peptides from native proteins. By testing with synthetic O-GlcNAc peptides and standard proteins, the synthesized material exhibited high affinity and selectivity. Based on the material prepared, we developed a workflow for site-specific analysis of O-GlcNAcylated proteins in complex samples. We performed O-GlcNAc proteomics with the PANC-1 cell line, a representative model for pancreatic ductal adenocarcinoma. In total 364 O-GlcNAc peptides from 267 proteins were identified from PANC-1 cells. Among them, 183 proteins were newly found to be O-GlcNAcylated in humans (with 197 O-GlcNAc sites newly reported). The materials and methods can be facilely applied for site-specific O-GlcNAc proteomics in other complex samples.

Advances in glycopeptide enrichment methods for the analysis of protein glycosylation over the past decade.

Advances in bioanalytical technology have accelerated the analysis of complex protein glycosylation, which is beneficial to understand glycosylation in drug discovery and disease diagnosis. Due to its biological uniqueness in the course of disease occurrence and development, disease-specific glycosylation requires quantitative characterization of protein glycosylation. We provide a comprehensive review of recent advances in glycosylation analysis, including workflows for glycoprotein digestion, glycopeptide separation and enrichment, and mass spectrometry sequencing. We specifically focus on different strategies for glycopeptide enrichment through physical interaction, chemical oxidation, or metabolic labeling of intact glycopeptides. Recent advances and challenges of O-glycosylation analysis are presented, and the development of improved enrichment methods combining different proteases to analyze O-glycosylation is also proposed.

Glycopeptide-Centric Approaches for the Characterization of Microbial Glycoproteomes.

Protein glycosylation is increasingly recognized as a common class of modifications within microbial species that can shape protein functions and the proteome at large. Due to this, there is an increasing need for robust analytical methods, which allow for the identification and characterization of microbial glycopeptides from proteome samples in a high-throughput manner. Using affinity-based enrichment (either hydrophilicity or antibody-based approaches) glycopeptides can easily be separated from non-glycosylated peptides and analyzed using mass spectrometry. By utilizing multiple mass spectrometry fragmentation approaches and open searching-based bioinformatic techniques, novel glycopeptides can be identified and characterized without prior knowledge of the glycans used for glycosylation. Using these approaches, glycopeptides within samples can rapidly be identified as well as quantified to understand how glycosylation changes in response to stimuli or how changes in glycosylation systems impact the glycoproteome. This chapter outlines a set of robust protocols for the initial preparation, enrichment, and analysis of microbial glycopeptides for both qualitative and quantitative glycoproteomic studies. Using these approaches, glycosylation events can be easily identified by researchers without the need for extensive manual analysis of proteomic datasets.

Endo-M Mediated Chemoenzymatic Approach Enables Reversible Glycopeptide Labeling for O-GlcNAcylation Analysis.

To selectively enrich O-linked ß-N-acetylglucosamine (O-GlcNAc) peptides in their original form from complex samples, we report the first reversible chemoenzymatic labeling approach for proteomic analysis. In this strategy, the O-GlcNAc moieties are ligated with long N-glycans using an Endo-M mutant, which enables the enrichment of the labeled glycopeptides by hydrophilic interaction liquid chromatography (HILIC). The attached glycans on the enriched glycopeptides are removed by wild-type Endo-M/S to restore the O-GlcNAc moiety. Compared with classic chemoenzymatic labeling, this approach enables the tag-free identification, and eliminates the interference of bulky tags in glycopeptide detection. This approach presents a unique avenue for the proteome-wide analysis of protein O-GlcNAcylation to promote its mechanism research.

One-step preparation of magnetic zwitterionic-hydrophilic dual functional nanospheres for in-depth glycopeptides analysis in Alzheimer's disease patients' serum.

A novel ultra-hydrophilic zwitterionic-HILIC (ZIC-HILIC) nanosphere (Fe3O4-CG) was synthesized via a one-step hydrothermal strategy, which significantly simplified the conventional multi-step procedures for the preparation of ZIC-HILIC materials. The dual-functional Fe3O4-CG nanosphere exhibited excellent selectivity (molar ratio BSA:HRP = 5000:1), low detection limit (0.05 fmol/µL), satisfactory reusability (at least 5 times) and recovery rate (93.7 ± 2.1%). The binding constant of Fe3O4-CG for HRP is 2.45 ± 0.32 × 10-6 M and the theoretical binding capacity is 330 mg g-1. In addition, the Fe3O4-CG microsphere showed excellent performance in the detection of glycopeptides from real biological samples. Furthermore, 131 glycopeptides related to 71 glycoproteins were selectively enriched from healthy human serum and 180 glycopeptides related to 82 glycoproteins were captured from Alzheimer's disease patients' serum analyzed by Nano-LC-MS/MS. Gene ontology analysis of the biological process and molecular function showed that 21 primitive glycoproteins in glycopeptides captured from Alzheimer's disease patients' serum were meaningfully involved in a variety of neurodegenerative disease-related events, including serine-type endopeptidase inhibitor activity, receptor binding, positive regulation of B cell activation, and platelet activation.

Glutathione functionalized magnetic covalent organic frameworks with dual-hydrophilicity for highly efficient and selective enrichment of glycopeptides.

Glycoproteins can be used as biomarkers to detect many diseases. Reliable and efficient sample materials are essential to separate and enrich glycopeptides before detection and analysis. In this report, glutathione (GSH)-modified magnetic covalent organic framework (TpBD) composite Fe3O4@TpBD@Au@GSH was synthesized by a two-step, post-synthesis modification strategy. The native hydrophilic TpBD and the highly hydrophilic GSH furnished the composite with dual-hydrophilic performance that was superior to covalent organic framework-based materials reported previously. The composite material showed excellent performance in enriching glycopeptides from protein standards because of its superior hydrophilicity, with 21 and 36 glycopeptides enriched from horseradish peroxidase (HRP) and immunoglobulin G from human serum (IgG) tryptic digests, respectively. The prepared composite exhibited ultra-high sensitivity (0.1 fmol/µL), excellent selectivity (HRP tryptic digest/bovine serum albumin (BSA) tryptic digest = 1:2000) and macromolecular protein anti-interference ability (HRP tryptic digest/BSA = 1:2000). Moreover, Fe3O4@TpBD@Au@GSH exhibited outstanding binding capacity (160 mg/g), excellent long-term storage capacity and good recycling ability (at least six times). Glycopeptide enrichment of biological samples by Fe3O4@TpBD@Au@GSH was successful, with 492 and 160 glycopeptides, corresponding to 134 and 64 glycoproteins, detected in 5 µL human serum and human saliva samples, respectively. The results showed that Fe3O4@TpBD@Au@GSH provides more information to facilitate in-depth analysis of glycopeptides in biological samples and has broad potential in cancer monitoring and diagnosis.

Dual-function monolithic enzyme reactor based on dopamine/graphene oxide coating for simultaneous protein enzymatic hydrolysis and glycopeptide enrichment.

A new dual-function enzyme reactor was prepared based on a dopamine/graphene oxide coated boron affinity monolithic column, which can be used for simultaneous protein enzymatic hydrolysis and glycopeptide enrichment. Firstly, a boron affinity monolithic column was prepared as the carrier for enzyme reactor. Secondly, the monolithic column was coated with dopamine/graphene oxide to provide higher specific surface area for the increase in the amount of trypsin bound. Then, dopamine can self-polymerize under alkaline conditions to produce multiple reaction sites. By the Schiff base reaction and Michael addition reaction with amino, sulfhydryl groups to trypsin, enzyme were immobilized on the boron affinity monolithic carrier. The enzyme activity was characterized by kinetic parameters maximum rate (Vmax) of the enzyme reaction and Michaelis constant (Km). Km of the dual-function enzyme reactors doped with PDA/GO and without PDA/GO were 34.37 and 120.93 mM, Vmax were 1.35 and 3.35 mM/min, respectively. The performance of the dual-function enzyme reactor was evaluated by protein extraction of mouse liver. After digested by the dual-function enzyme reactor, the number of peptides was 4,801, which was 960 more than the number of peptides in the solution digestion. At the same time, the dual-function enzyme reactor displayed the ability to capture cis-dihydroxy compounds specificly. A total of 55 glycopeptides were enriched in the dual-functional enzyme reactor, corresponding to 33 glycoproteins. The dual-function enzyme reactor provided repeatable performance and robust with long-term storage.

Bi-amino acid functionalized biomimetic honeycomb chitosan membrane as a multifunctional hydrophilic probe for specific capture of N-linked glycopeptides in nasopharyngeal carcinoma's disease patient's serum.

In this work, a novel porous bifunctionalized composite material was synthesized via a simple method. Gold nanoparticles are uniformly dispersed on the surface of the biomimetic honeycomb chitosan membrane through the interaction between amino and Au, and then cysteine and glutathione are successfully grafted onto the surface of the Au by the Au-S bond. The modification of cysteine and glutathione makes this bifunctionalized composite material have significant advantages of superhydrophilicity and small steric hindrance simultaneously. This material manifests excellent property in glycopeptides enrichment, with high selectivity (1:5000), low detection limit (0.1 fmol·µL-1 ), high recovery rate (99.4 ± 0.5%), and good repeatability. In addition, with the help of nano-flow liquid chromatography tandem mass spectrometry, this composite achieved excellent performance in efficiently enriching glycopeptides in the serum of healthy people and nasopharyngeal carcinoma's disease patient. More excitingly, further gene ontology analysis of molecular function and biological process indicated that 41 original glycoproteins of the identified glycopeptides from serum of nasopharyngeal carcinoma's disease patient significantly partake in numerous cancer-associated events, including protease binding, calcium ion binding, enzyme binding, extracellular matrix organization, cellular response to tumor necrosis factor, and inflammatory response.

Efficient HCD-pd-EThcD approach for N-glycan mapping of therapeutic antibodies at intact glycopeptide level.

N-glycosylation is a critical quality attribute for monoclonal antibody (mAb)-based therapeutics due to its significant impact on drug efficacy and safety. Extensive glycosylation mapping is therefore necessary for mAb drug development and quality control. We utilized a higher-energy dissociation product ions-triggered electron-transfer/higher-energy collision dissociation (HCD-pd-EThcD) approach to mapping N-glycosylation in therapeutic mAbs. Due to the improved duty cycle and targeted ability, HCD-pd-EThcD could provide extensive N-glycan identifications as well as higher quality spectra than EThcD mode. On average, ten types of N-glycan were uncovered in two different lots of trastuzumab, demonstrating a significant increment in N-glycan species compared to only four types identified by EThcD. After integrating pre-enrichment of glycopeptides, up to 16 N-glycans were recognized. Significantly, this strategy facilitated the identification of glycopeptides containing fucosylated and sialylated glycans, meanwhile enabled the recognition of different N-glycan classes (high mannose, hybrid, and complex). Further application in the glycosylation analysis of adalimumab and bevacizumab resulted in 19 and 8 N-glycans species, providing a more comprehensive insight into their glycosylation modification status. We demonstrated the benefits of an integrated strategy in characterizing various N-glycans of mAb therapeutics and offer an alternative approach for their quality control at the intact glycopeptides level.

Hydrophilic arginine-functionalized mesoporous polydopamine-graphene oxide composites for glycopeptides analysis.

Considering the importance of glycopeptides in the clinical diagnosis of cancer and some serious diseases, the identification of glycopeptides from complex biological samples has attracted considerable attention. Effective pre-enrichment before mass spectrometry analysis plays an important role. In this work, a kind of hydrophilic two-dimensional composites (denoted as GO@MPDA@Arg) based on mesoporous polydopamine-graphene oxide were used to selectively enrich glycopeptides in biological samples. The mesoporous polydopamine (MPDA) layer self-assembled with template Pluronic F127 provided more binding sites to load arginine, and bound arginine enhanced the hydrophilicity of the material. As a result, GO@MPDA@Arg composites exhibited excellent enrichment performance for glycopeptides, containing good selectivity (IgG digests : BSA digests = 1:50, molar ratio), low detection limit for IgG digests (10 fmol µL-1), high loading capacity for IgG digests (200 µg mg-1), and good size exclusion (IgG digests : IgG : BSA = 1:100:100, mass ratio). In addition, mouse brain tissue was selected as the actual biological sample to further study the enrichment effect of GO@MPDA@Arg composites. In three parallel experiments, a total of 401 glycopeptides belonging to 233 glycoproteins were enriched from 200 µg digestion of mouse brain extract. The enrichment results demonstrate that GO@MPDA@Arg composites have application potential for glycopeptides enrichment in protein post-translational modification research.