N-linked Glycopeptides Research Articles

Microparticle-assisted protein capture method facilitates proteomic and glycoproteomic analysis of urine samples

Serum tests have become a partial alternative to renal biopsy for diagnosing primary membranous nephropathy (pMN). However, urine tests, due to their non-invasive nature and ability to more accurately reflect glomerular diseases, hold great promise for the detection of pMN. However, the low protein concentration and the time-consuming sample preparation procedure of urine samples challenges the proteomic and glycoproteomic analysis to find urine-derived signatures associated with pMN. In this study, we presented a microparticle-assisted protein capture (MAPC) method to efficiently prepare urine samples. It was found that proteins and N-linked intact glycopeptides can be sensitively identified from urine samples of pMN patients and healthy controls by using this method. For comparison, proteins and N-linked intact glycopeptides from serum were also analyzed. Interestingly, discrepancy was found in the changing trends for proteins/intact glycopeptides between serum and urine. Moreover, urine derived proteins, N-linked intact glycopeptides, and glycans exhibited more pronounced changes between pMN and healthy control compared to serum sample. Notably, urine IgG4 not only up-regulated in pMN at global peptide level, its corresponding site-specific glycans were specifically identified in pMN urine with significantly up-regulations, suggested the potential of using glycosylated urine IgG4 for the non-invasive diagnosis of pMN.

Site-Selective Construction of N-Linked Glycopeptides through Photoredox Catalysis.

The glycosylation of peptides and proteins can significantly impact their intrinsic properties, such as conformation, stability, antigenicity, and immunogenicity. Current methods for preparing N-linked glycopeptides typically rely on amide bond formation, which can be limited by the presence of reactive functional groups like acids and amines. Late-stage functionalization of peptides offers a promising approach to obtaining N-linked glycopeptides. In this study, we demonstrate the preparation of N-linked glycopeptides through a photoredox-catalyzed site-selective Giese addition between N-glycosyl oxamic acid and peptides containing dehydroalanine (Dha) under visible light conditions. Unlike traditional methods that rely on the coupling of aspartic acid and glycosylamine, this approach utilizes the conjugation of N-glycosylated carbamoyl radicals with Dha, facilitating the straightforward modification of complex peptides.

Biomarker Discovery via N-Glycoproteomics.

Posttranslational modifications (PTMs) of proteins regulate several biological processes, and investigating their diversity is crucial for understanding the mechanisms of cell regulation. Glycosylation is one of the most complex posttranslational modifications that control fundamental cellular processes such as protein folding, protein trafficking, host-pathogen interactions, cell adhesion, and cytokine receptor signaling networks. N-linked glycosylation denotes the attachment of glycans (oligosaccharides) to a nitrogen atom of asparagine (N) residues in the consensus motif Asn-X-Ser/Thr (NXS/T), where X is any amino acid except proline. Therefore, mutations in this posttranslational modification (i.e., N-glycosylation) site cause many human genetic diseases, including cancer. In the past decade, high-throughput quantitative proteome profiling tools have significantly renewed our interest in discovering novel cancer diagnostic or prognostic biomarkers through the simultaneous examination of the enormous amount of high-quality data of thousands of proteins and genes in complex biological systems. In this chapter, we describe how aberrant N-linked glycopeptides could be selectively identified as novel single tumor markers through the use of mass spectrometry (MS)-based proteomics, also known as Solid-phase extraction of N-glycopeptides (SPEG), and reasonable hypotheses that have the potential capacity to revolutionize biomarker discovery and bring those markers to the clinic as early as possible.

Generalizing a Ligation Site at the N-Glycosylation Sequon for Chemical Synthesis of N-Linked Glycopeptides and Glycoproteins.

Chemical synthesis can generate homogeneous glycoproteins with well-defined and modifiable glycan structures at designated sites. The precision and flexibility of the chemical synthetic approach provide a solution to the heterogeneity problem of glycopeptides/glycoproteins obtained through biological approaches. In this study, we reported that the conserved N-glycosylation sequon (Asn-Xaa-Ser/Thr) of glycoproteins can serve as a general site for performing Ser/Thr ligation to achieve N-linked glycoprotein synthesis. We developed an N + 2 strategy to prepare the corresponding glycopeptide salicylaldehyde esters for Ser/Thr ligation and demonstrated that Ser/Thr ligation at the sequon was not affected by the steric hindrance brought about by the large-sized glycan structures. The effectiveness of this strategy was showcased by the total synthesis of the glycosylated receptor-binding domain (RBD) of the SARS-CoV-2 spike protein.

Enrichment methods of N-linked glycopeptides from human serum or plasma: A mini-review

Human diseases often correlate with changes in protein glycosylation, which can be observed in serum or plasma samples. N-glycosylation, the most common form, can provide potential biomarkers for disease prognosis and diagnosis. However, glycoproteins constitute a relatively small proportion of the total proteins in human serum and plasma compared to the non-glycosylated protein albumin, which constitutes the majority. The detection of microheterogeneity and low glycan abundance presents a challenge. Mass spectrometry facilitates glycoproteomics research, yet it faces challenges due to interference from abundant plasma proteins. Therefore, methods have emerged to enrich N-glycans and N-linked glycopeptides using glycan affinity, chemical properties, stationary phase chemical coupling, bioorthogonal techniques, and other alternatives. This review focuses on N-glycans and N-glycopeptides enrichment in human serum or plasma, emphasizing methods and applications. Although not exhaustive, it aims to elucidate principles and showcase the utility and limitations of glycoproteome characterization.

Detection of Pancreatic Ductal Adenocarcinoma-Associated Proteins in Serum

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancer types, partly because it is frequently identified at an advanced stage, when surgery is no longer feasible. Therefore, early detection using minimally invasive methods such as blood tests may improve outcomes. However, studies to discover molecular signatures for the early detection of PDAC using blood tests have only been marginally successful. In the current study, a quantitative glycoproteomic approach via data-independent acquisition mass spectrometry was utilized to detect glycoproteins in 29 patient-matched PDAC tissues and sera. A total of 892 N-linked glycopeptides originating from 141 glycoproteins had PDAC-associated changes beyond normal variation. We further evaluated the specificity of these serum-detectable glycoproteins by comparing their abundance in 53 independent PDAC patient sera and 65 cancer-free controls. The PDAC tissue-associated glycoproteins we have identified represent an inventory of serum-detectable PDAC-associated glycoproteins as candidate biomarkers that can be potentially used for the detection of PDAC using blood tests.

Click-iG: Simultaneous Enrichment and Profiling of Intact N-linked, O-GalNAc, and O-GlcNAcylated Glycopeptides.

Proteins are ubiquitously modified with glycans of varied chemical structures through distinct glycosidic linkages, making the landscape of protein glycosylation challenging to map. Profiling of intact glycopeptides with mass spectrometry (MS) has recently emerged as a powerful tool for revealing matched information of the glycosylation sites and attached glycans (i.e., intact glycosites), but is largely limited to individual glycosylation types. Herein, we describe Click-iG, which integrates metabolic labeling of glycans with clickable unnatural sugars, an optimized MS method, and a tailored version of pGlyco3 software to enable simultaneous enrichment and profiling of three types of intact glycopeptides: N-linked, mucin-type O-linked, and O-GlcNAcylated glycopeptides. We demonstrate the utility of Click-iG by the identification of thousands of intact glycosites in cell lines and living mice. From the mouse lung, heart, and spleen, a total of 2053 intact N-glycosites, 262 intact O-GalNAc glycosites, and 1947 O-GlcNAcylation sites were identified. Click-iG-enabled comprehensive coverage of the protein glycosylation landscape lays the foundation for interrogating crosstalk between different glycosylation pathways.

Mapping the low abundant plasma glycoproteome using Ranachrome-5 immobilized magnetic terpolymer as improved HILIC sorbent

HILIC (hydrophilic interaction liquid chromatography) materials enrich glycopeptides. The non-specific interactions because of support material and inadequate hydrophilicity render loss of less abundant glycopeptides in SPE-based enrichments. In this work, magnetic terpolymer (Fe3O4@MAA/DVB/1,2-Epoxy-5-hexene) is functionalized with Ranachrome-5 to generate enhanced hydrophilicity. Amine, carboxylic, and amide groups of ranachrome-5 provide zwitterionic chemistry. Material’s magnetic core contributes to ease of operation while higher surface area 97.0711 m2 g−1 immobilizes better quantities of Ranachrome-5. Homogeneous morphology, nano-size, and super hydrophilicity enhance enrichment. Ranachrome-5 functionalized polymeric core-shell beads enrich 25, 18 and 16 N-linked glycopeptides via SPE strategy from tryptic digests of model glycoproteins i.e., immunoglobulin G (IgG), horseradish peroxidase (HRP) and chicken avidin, respectively. Zwitterionic chemistry of ranachrome-5 helps in achieving higher selectivity (1:250, HRP / Bovine Serum Albumin), and lower detection limit (100 attomole, HRP digest) with complete glycosylation profile of each standard digest. High binding capacity (137.1 mg/g) and reuse of affinity material up to seven cycles reduce the cost and amount of affinity material for complex sample analysis. A recovery of 91.76% and relative standard deviation (RSD) values less than 1 define the application of HILIC beads for complex samples like plasma. 508 N-linked intact low abundant glycopeptides corresponding to 50 glycoproteins are identified from depleted human plasma samples via nano-Liquid Chromatography-Tandem Mass Spectrometry (nLC-MS/MS). Using Single Nucleotide Variances (BioMuta) for low abundant plasma glycoproteins, the potential association of proteins to four cancers, i.e., breast, lung, uterine, and melanoma is evaluated. Via the bottom-up approach, HILIC beads can analyze clinically important low-abundant glycoproteins.

Boronic acid and fructose-1,6-diphosphate dual-functionalized highly hydrophilic Zr-MOF for HILIC enrichment of N-linked glycopeptides.

The HILIC enrichment is a greatly compatible strategy for the extraction of glycopeptides in proteomics. Herein, a boric acid and fructose-1,6-diphosphate (FDP) dual-functionalized Zr-based metal-organic framework material UIO-PBA&FDP (UIO is the abbreviation for the University of Oslo, and PBA is the abbreviation for carboxy phenylboronic acid) was synthesized, characterized with the desirable excellent hydrophilicity and thus was explored for the enrichment of N-linked glycopeptides utilizing the HILIC interaction between the glycopeptides and the hydrophilic UIO-PBA&FDP at a high level of ACN concentration. A total of 359 N-linked glycopeptides corresponding to 104 glycoproteins were identified from only 1 μL of digested human serum by the enrichment of UIO-PBA&FDP, which showed a superiorly high coverage of the identified glycopeptides. The dual hydrophilic functionalized UIO-PBA&FDP could be an efficient HILIC material for the enrichment of N-linked glycopeptides from complex biological samples.

Development of an Integrated Platform for the Simultaneous Enrichment and Characterization of N- and O-Linked Intact Glycopeptides.

Both N-linked glycosylation and O-linked glycosylation play essential roles in the onset and progression of various diseases including cancer, and N-/O-linked site-specific glycans have been proven to be promising biomarkers for the discrimination of cancer. However, the micro-heterogeneity and low abundance nature of N-/O-linked glycosylation, as well as the time-consuming and tedious procedures for the enrichment of O-linked intact glycopeptides, pose great challenges for their efficient and accurate characterization. In this study, we developed an integrated platform for the simultaneous enrichment and characterization of N- and O-linked intact glycopeptides from the same serum sample. By fine-tuning the experimental conditions, we demonstrated that this platform allowed the selective separation of N- and O-linked intact glycopeptides into two fractions, with 85.1% O-linked intact glycopeptides presented in the first fraction and 93.4% N-linked intact glycopeptides presented in the second fraction. Determined with high reproducibility, this platform was further applied to the differential analysis of serum samples of gastric cancer and health control, which revealed 17 and 181 significantly changed O-linked and N-linked intact glycopeptides. Interestingly, five glycoproteins containing both significant regulation of N- and O-glycosylation were observed, hinting potential co-regulation of different types of glycosylation during tumor progress. In summary, this integrated platform opened a potentially useful avenue for the global analysis of protein glycosylation and can serve as a useful tool for the characterization of N-/O-linked intact glycopeptides at the proteomics scale.

Analysis of Sialic Acid Linkage in N-Linked Glycopeptides Using Liquid Chromatography-Electron-Activated Dissociation Time-of-Flight Mass Spectrometry.

Herein, we report a novel liquid chromatography coupled with tandem mass spectrometry method to characterize N-acetylneuraminic acid (Neu5Ac, Sa) linkage in N-linked glycans in glycopeptides with no sialic acid derivatization. First, we established a separation in reversed-phase high-performance liquid chromatography (HPLC) using a higher formic acid concentration in the mobile phases, which separated the N-glycopeptides depending on the Sa linkage. We also demonstrated a novel characterization method of Sa linkages in N-glycopeptides using electron-activated dissociation. We found that hot electron capture dissociation using an electron beam energy higher than 5 eV cleaved glycosidic bonds in glycopeptides, resulting in each glycosidic bond in the antennas being broken on both sides of the oxygen atom. Such glycosidic bond cleavage at the reducing end (C-type ion) showed the difference in Sa linkages between Sa-Gal, Gal-GlcNAc, and GlcNAc-Man. We proposed a rule to characterize the Sa linkages using the Sa-Gal products. This method was applied to N-glycopeptides in tryptic fetuin digest separated by an optimized reversed-phase HPLC. We successfully identified a number of isomeric glycoforms in the glycopeptides with different Sa links, whose peptide backbones were also simultaneously sequenced by hot ECD.

Facile fabrication of hydrophilic covalent organic framework composites for highly selective enrichment of N-glycopeptides

The development of facilely synthetic materials acts an essential role in glycoproteome analysis, especially for the highly efficient enrichment of N-linked glycopeptides. In this work, a facile and timesaving route was introduced in which COFTP-TAPT served as a carrier and poly (ethylenimine) (PEI) and carrageenan (Carr) were successively coated on the surface via electrostatic interaction. The resultant COFTP-TAPT@PEI@Carr showed remarkable performance in glycopeptide enrichment with high sensitivity (2 fmol μL−1), high selectivity (1:800, molar ratio of human serum IgG to BSA digests), large loading capacity (300 mg g−1), satisfactory recovery (102.4 ± 6.0%) and reusability (at least eight times). Due to the brilliant hydrophilicity and electrostatic interactions between COFTP-TAPT@PEI@Carr and positively charged glycopeptides, the prepared materials could be applied in the identification and analysis in the human plasma of healthy subjects and patients with nasopharyngeal carcinoma. As a result, 113 N-glycopeptides with 141 glycosylation sites corresponding to 59 proteins and 144 N-glycopeptides with 177 glycosylation sites corresponding to 67 proteins were enriched from 2 μL plasma trypsin digests of the control groups and patients with nasopharyngeal carcinoma, respectively. 22 glycopeptides were identified only from the normal controls and 53 glycopeptides were detected only from the other set. The results demonstrated that this hydrophilic material was promising on a large scale and further N-glycoproteome research.

New hydrophilic material based on hydrogel polymer for the selective enrichment of intact glycopeptides from serum protein digests

The paper describes the preparation and characterization of a new HILIC material for the enrichment of N-linked glycopeptides. The material was prepared using 2-acrylamido-2-methyl-1-propanesulfonic acid as the monomer and ethylene glycol dimethacrylate as the cross-linker. The material was developed by a Box-Behnken experimental design, taking into consideration the amount of monomer-to-crosslinker ratio, the composition, and the amount of porogen mixture. By this approach, the property of the resulting polymer could be fine-tuned to modulate the hydrophilicity and porosity. As HILIC enrichment is mostly dependent on hydrophilic interactions, including H-bonding, the amount of swelling was expected to have an important function, therefore the optimization considered a monomer percent in the range of 20–80%, which implied very different water swelling capacities. After assessing the potential of this new polymer family on fetuin digests, the 17 materials resulting from the Box-Behnken experimental design were used for the enrichment of glycopeptides from serum protein digests. The materials displayed a superior performance over cotton HILIC enrichment, both in terms of the number of enriched N-linked glycopeptides and selectivity, providing up to 762 N-linked glycopeptides with 77% selectivity. The optimization indicated that a high amount of monomer significantly affected the number of enriched glycopeptides, which is also closely connected with the hydrogel nature of the resulting polymers. The results not only provide one additional HILIC material for the enrichment of glycopeptides but also pave the way for the use and development of hydrogel materials for the enrichment of N-linked glycopeptides.

Simultaneous N-Deglycosylation and Digestion of Complex Samples on S-Traps Enables Efficient Glycosite Hypothesis Generation.

N-linked glycosylation is an important post-translational modification that is difficult to identify and quantify in traditional bottom-up proteomics experiments. Enzymatic deglycosylation of proteins by peptide:N-glycosidase F (PNGase F) prior to digestion and subsequent mass spectrometry analysis has been shown to improve coverage of various N-linked glycopeptides, but the inclusion of this step may add up to a day to an already lengthy sample preparation process. An efficient way to integrate deglycosylation with bottom-up proteomics would be a valuable contribution to the glycoproteomics field. Here, we demonstrate a proteomics workflow in which deglycosylation and proteolytic digestion of samples occur simultaneously using suspension trapping (S-Trap). This approach adds no time to standard digestion protocols. Applying this sample preparation strategy to a human serum sample, we demonstrate improved identification of potential N-glycosylated peptides in deglycosylated samples compared with non-deglycosylated samples, identifying 156 unique peptides that contain the N-glycosylation motif (asparagine-X-serine/threonine), the deamidation modification characteristic of PNGase F, and an increase in peptide intensity over a control sample. We expect that this rapid sample preparation strategy will assist in the identification and quantification of both known and potential glycoproteins. Data are available via ProteomeXchange with the identifier PXD037921.

Advances in hydrophilic metal-organic frameworks for N-linked glycopeptide enrichment.

The comprehensive profiling of glycoproteins is of great significance for the timely clinical diagnosis and therapy. However, inherent obstacles hamper their direct analysis from biological samples, and specific enrichment prior to analysis is indispensable. Among the various approaches for glycopeptide enrichment, hydrophilic interaction liquid chromatography (HILIC) has attracted special focus, especially for the development of novel hydrophilic materials, which is the key of HILIC. Metal-organic frameworks (MOFs) are a type of porous materials constructed from the self-assembly of metal and organic linkers. Advantages such as high surface area, flexible pore size, and easy modification render hydrophilic MOFs as ideal candidates for HILIC, which has inspired many studies over the past years. In this review, advances in hydrophilic MOFs for N-linked glycopeptide enrichment are summarized. According to the synthesis strategies, those materials are categorized into three classes, namely pristine MOFs, MOFs with chemical modifications, and MOFs-derived composite. In each categorization, the preparation and the function of different moieties are covered, as well as the enrichment performances of sensitivity, selectivity, and practical application. Finally, a summary and future perspective on the applications of hydrophilic MOFs for N-linked glycopeptide enrichment are briefly discussed. This review is expected to raise awareness of the properties of hydrophilic MOFs and offer some valuable information to further research in glycoproteomics.

Proteome and Glycoproteome Analyses Reveal the Protein N-Linked Glycosylation Specificity of STT3A and STT3B

STT3A and STT3B are the main catalytic subunits of the oligosaccharyltransferase complex (OST-A and OST-B in mammalian cells), which primarily mediate cotranslational and post-translocational N-linked glycosylation, respectively. To determine the specificity of STT3A and STT3B, we performed proteomic and glycoproteomic analyses in the gene knock-out (KO) and wild-type HEK293 cells. In total, 3961 proteins, 4265 unique N-linked intact glycopeptides and 629 glycosites representing 349 glycoproteins were identified from all these cells. Deletion of the STT3A gene had a greater impact on the protein expression than deletion of STT3B, especially on glycoproteins. In addition, total mannosylated N-glycans were reduced and fucosylated N-glycans were increased in STT3A-KO cells, which were caused by the differential expression of glycan-related enzymes. Interestingly, hyperglycosylated proteins were identified in KO cells, and the hyperglycosylation of ENPL was caused by the endoplasmic reticulum (ER) stress due to the STT3A deletion. Furthermore, the increased expression of the ATF6 and PERK indicated that the unfolded protein response also happened in STT3A-KO cells. Overall, the specificity of STT3A and STT3B revealed that defects in the OST subunit not only broadly affect N-linked glycosylation of the protein but also affect protein expression.

Simultaneous enrichment and sequential separation of O-linked glycopeptides and phosphopeptides with immobilized titanium (IV) ion affinity chromatography materials

Protein phosphorylation and O-linked glycosylation are two critical posttranslational modifications (PTMs) and they both target identical amino acid residues, generating “crosstalk”. Analysis of the crosstalk between these PTMs is crucial for deciphering their biological functions. Although several strategies have been established to enrich N-linked glycopeptides and phosphopeptides simultaneously, they are not appropriate for O-linked glycopeptides and phosphopeptides. In this study, we established a method for the simultaneous enrichment and sequential elution of O-linked glycopeptides and phosphopeptides into two fractions using commercially available immobilized titanium (IV) ion affinity chromatography (Ti4+-IMAC) materials. The established method exhibited high selectivity, repeatability, and recovery of the targeted peptides. Particularly, the overlap between the O-linked glycopeptide and phosphopeptide fractions was very low (∼3%). The application of this method to cell lysates of the colon cancer HT29 cell line resulted in a comparable number of enriched phosphopeptides as the coeluted method. However, the number of identified O-linked glycopeptides with our established method was 9.7-fold higher than that with the coelution method. Our study demonstrated that the established strategy was beneficial for the simultaneous analysis of O-linked glycopeptides and phosphopeptides, which might facilitate the study of the biological function of PTM crosstalk.

Site-Specific Intact N-Linked Glycopeptide Characterization of Prostate-Specific Membrane Antigen from Metastatic Prostate Cancer Cells.

The composition of N-linked glycans that are conjugated to the prostate-specific membrane antigen (PSMA) and their functional significance in prostate cancer progression have not been fully characterized. PSMA was isolated from two metastatic prostate cancer cell lines, LNCaP and MDAPCa2b, which have different tissue tropism and localization. Isolated PSMA was trypsin-digested, and intact glycopeptides were subjected to LC-HCD-EThcD-MS/MS analysis on a Tribrid Orbitrap Fusion Lumos mass spectrometer. Differential qualitative and quantitative analysis of site-specific N-glycopeptides was performed using Byonic and Byologic software. Comparative quantitative analysis demonstrates that multiple glycopeptides at asparagine residues 51, 76, 121, 195, 336, 459, 476, and 638 were in significantly different abundance in the two cell lines (p < 0.05). Biochemical analysis using endoglycosidase treatment and lectin capture confirm the MS and site occupancy data. The data demonstrate the effectiveness of the strategy for comprehensive analysis of PSMA glycopeptides. This approach will form the basis of ongoing experiments to identify site-specific glycan changes in PSMA isolated from disease-stratified clinical samples to uncover targets that may be associated with disease progression and metastatic phenotypes.

Terpolymeric platform with enhanced hydrophilicity via cysteic acid for serum intact glycopeptide analysis.

A new polymeric (methyl methacrylate/ethylene glycol dimethacrylate/1,2-epoxy-5-hexene) base/matrix has been fabricated and decorated with zwitterionic hydrophilic cysteic acid (Cya) for the enrichment of intact N-glycopeptides from standards and biological samples. Terpolymer-Cya provides good enrichment efficiency, improved hydrophilicity, and selectivity by virtue of better surface area (2.09 × 102 m2/g) provided by terpolymer and the zwitterionic property offered by cysteic acid. Cysteic acid-functionalized polymeric hydrophilic interaction liquid chromatography (HILIC) sorbent enriches 35 and 24N-linked glycopeptides via SPE (solid phase extraction) mode from tryptic digests of model glycoproteins, i.e., immunoglobulin G (IgG) and horseradish peroxidase (HRP), respectively. Zwitterionic chemistry of cysteine helps in achieving higher selectivity with BSA digest (1:200), and lower detection limit down to 100 attomoles with a complete glycosylation profile of each standard digest. The recovery of 81% and good reproducibility define the application of terpolymer-Cya for complex samples like a serum. Analysis of human serum provides a profile of 807 intact N-linked glycopeptides via nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS). To the best of our knowledge, this is the highest number of glycopeptides enriched by any HILIC sorbent. Selected glycoproteins are evaluated in link to various cancers including the breast, lung, uterine, and melanoma using single-nucleotide variances (BioMuta). This study represents the complete idea of using an in-house developed strategy as a successful tool to help analyze, relate, and answer glycoprotein-based clinical issues regarding cancers.

Tandem Mass Spectrometry for Structural Characterization of Doubly-Charged N-Linked Glycopeptides

Three dissociation methods, including collision-induced dissociation (CID), electron capture dissociation (ECD), and electronic excitation dissociation (EED), were systematically compared for structural characterization of doubly charged glycopeptide. CID produced distinctively different tandem mass spectra for glycopeptide adducted with different charge carriers. Protonated species produced mainly glycosidic cleavages in high abundance. CID of magnesiated glycopeptide formed more cross-ring cleavages, whereas doubly sodiated species produced cleavages at both glycan and peptide moieties. The effect of charge carriers on the fragmentation in ECD and EED was lower than that in CID. ECD produced mainly peptide backbone cleavages but limited cleavages at the glycan moiety, whereas EED of glycopeptide resulted in extensive fragmentation throughout the molecular ion regardless of the charge carriers. Magnesiated species gave, however, more cross-ring cleavages than other charge carriers did. These results demonstrated that EED of magnesiated species could be used as a one-step dissociation method for comprehensive structural analysis of glycopeptides.