Characterization of glycosylation sites for a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein by liquid chromatography-mass spectrometry peptide mapping

Liquid chromatography mass spectrometry (LC-MS) peptide mapping can be a versatile technique for characterizing protein glycosylation sites without the need to remove the attached glycans as in conventional oligosaccharide mapping methods. In this way, both N-linked and O-linked sites of glycosylation can each be directly identified, characterized, and quantified by LC-MS as intact glycopeptides in a single experiment. LC-MS peptide mapping of the individual glycosylation sites avoids many of the limitations of preparing and analyzing an entire pool of released N-linked oligosaccharides from all sites mixed together. In this study, LC interfaced to a linear ion trap mass spectrometer (ESI-LIT-MS) were used to characterize the glycosylation of a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein with multiple sites of N-and O-glycosylation. Samples were reduced, S-carboxyamidomethylated, and cleaved with either trypsin or endoproteinase Asp-N. Enhanced detection for minor IgG1 glycoforms (～0.1 to 1.0 mol% level) was obtained by LC-MS of the longer 32-residue Asp-N glycopeptide (4+ protonated ion) compared to the 9-residue tryptic glycopeptide (2+ ion). LC-MS peptide mapping was run according to a general procedure: (1) Locate N-linked and/or O-linked sites of glycosylation by selected-ion-monitoring of carbohydrate oxonium fragment ions generated by ESI in-source collision-induced dissociation (CID), i.e. 204, 366, and 292 Da marker ions for HexNAc, HexNAc-Hex, and NeuAc, respectively; (2) Characterize oligosaccharides at each site via MS and MSMS. Use selected ion currents (SIC) to estimate relative amounts of each glycoform; and (3) Measure the percentage of site-occupancy by searching for any corresponding nonglycosylated peptide.     

LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins

Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.

Selective glycopeptide mapping of erythropoietin by on-line high-performance liquid chromatography--electrospray ionization mass spectrometry

Selective glycopeptide mapping of recombinant human erythropoietin (rhEPO) used as a model glycoprotein was successfully carried out by on-line high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) using a Vydac C18 column eluted in acetonitrile-1 mM ammonium acetate, pH 6.8. rhEPO expressed in a Chinese hamster ovary clone was exhaustively digested into four glycopeptides and nine peptides with endoproteinase Glu-C. Both glycopeptides and peptides were eluted with trifluoroacetic acid as the eluent, whereas only glycopeptides were eluted selectively with ammonium acetate in the following order: N38, N24, 0126, and N83. Furthermore, many glycoforms included in each glycopeptide were found to be separated by differences in the numbers of sialic acid and N-acetyllactosaminyl repeats. Twenty, 16 and 22 different N-linked oligosaccharides were determined at Asn24, 38, and 83, respectively, and two different O-linked oligosaccharides were observed at Ser126. Our method is simple, rapid, and useful for determining the carbohydrate structures at each glycosylation site and for elucidating the site-specific carbohydrate heterogeneity.     

Characterization of a gel-separated unknown glycoprotein by liquid chromatography/multistage tandem mass spectrometry: analysis of rat brain Thy-1 separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

We developed an efficient and convenient strategy for protein identification and glycosylation analysis of a small amount of unknown glycoprotein in a biological sample. The procedure involves isolation of proteins by electrophoresis and mass spectrometric peptide/glycopeptide mapping by LC/ion trap mass spectrometer. For the complete glycosylation analysis, proteins were extracted in intact form from the gel, and proteinase-digested glycoproteins were then subjected to LC/multistage tandem MS (MSn) incorporating a full mass scan, in-source collision-induced dissociation (CID), and data-dependent MSn. The glycopeptides were localized in the peptide/glycopeptide map by using oxonium ions such as HexNAc+ and NeuAc+, generated by in-source CID, and neutral loss by CID-MS/MS. We conducted the search analysis for the glycopeptide identification using search parameters containing a possible glycosylation at the Asn residue with N-acetylglucosamine (203 Da). We were able to identify the glycopeptides resulting from predictable digestion with proteinase. The glycopeptides caused by irregular cleavages were not identified by the database search analysis, but their elution positions were localized using oxonium ions produced by in-source CID, and neutral loss by the data-dependent MSn. Then, all glycopeptides could be identified based on the product ion spectra which were sorted from data-dependent CID-MSn spectra acquired around localized positions. Using this strategy, we successfully elucidated site-specific glycosylation of Thy-1, glycosylphosphatidylinositol (GPI)-anchored proteins glycosylated at Asn23, 74, and 98, and at Cys111. High-mannose-type, complex-type, and hybrid-type oligosaccharides were all found to be attached to Asn23, 74 and 98, and four GPI structures could be characterized. Our method is simple, rapid and useful for the characterization of unknown glycoproteins in a complex mixture of proteins.     

Post-translational modifications of recombinant B. cinerea EPG 6

The fungus Botrytis cinerea is a ubiquitous plant pathogen that infects more than 200 different plant species and causes substantial economic losses in a wide range of agricultural crops and harvested products. Endopolygalacturonases (EPGs) are among the first array of cell-wall-degrading enzymes secreted by fungi during infection. Up to 13 EPG glycoforms have been described for B. cinerea. The presence of multiple N-linked glycosylation modifications in BcPG1-6 is predicted by their deduced amino acid sequences. In this work, the glycosylation sites and the attached oligosaccharide structures on BcPG6 were analyzed. The molecular mass of the intact glycoprotein was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis. BcPG6 contains seven potential N-linked glycosylation sites. Occupancy of these glycosylation sites and the attached carbohydrate structures were analyzed by tryptic digestion followed by liquid chromatography/mass spectrometry (LC/MS) using a stepped orifice voltage approach. Five out of seven potential N-linked sites present in BcPG6 were determined to be occupied by high-mannose-type oligosaccharides. Four of them were readily determined to be at Asn58 (T3 peptide), Asn198 (T7 peptide), Asn237 (T9 peptide) and Asn256 (T11 peptide), respectively. Another was located on the T8 peptide, which contained two potential N-linked sites, Asn224 and Asn227 (SNNN224VTN227ITFK). LC/MS/MS of a sample treated with N-glycanase placed the glycan in this peptide at Asn224 rather than at Asn227. The potential glycosylation site on Asn146 (T6 peptide) was not glycosylated. In addition, two disulfide bonds were observed, linking the Cys residues within the T13 and T16 peptides.     

Comparison of HPLC/ESI-FTICR MS versus MALDI-TOF/TOF MS for glycopeptide analysis of a highly glycosylated HIV envelope glycoprotein

Defining the structures and locations of the glycans attached on secreted proteins and virus envelope proteins is important in understanding how glycosylation affects their biological properties. Glycopeptide mass spectrometry (MS)-based analysis is a very powerful, emerging approach to characterize glycoproteins, in which glycosylation sites and the corresponding glycan structures are elucidated in a single MS experiment. However, to date there is not a consensus regarding which mass spectrometric platform provides the best glycosylation coverage information. Herein, we employ two of the most widely used MS approaches, online high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS) and offline HPLC followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), to determine which of the two approaches provides the best glycosylation coverage information of a complex glycoprotein, the group M consensus HIV-1 envelope, CON-S gp140DeltaCFI, which has 31 potential glycosylation sites. Our results highlight differences in the informational content obtained between the two methods such as the overall number of glycosylation sites detected, the numbers of N-linked glycans present at each site, and the type of confirmatory information obtained about the glycopeptide using MS/MS experiments. The two approaches are quite complementary, both in their coverage of glycopeptides and in the information they provide in MS/MS experiments. The information in this study contributes to the field of mass spectrometry by demonstrating the strengths and limitations of two widely used MS platforms in glycoprotein analysis.     

Site-specific protein glycosylation analysis with glycan isomer differentiation

Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one N-glycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.     

Global and site-specific detection of human integrin alpha 5 beta 1 glycosylation using tandem mass spectrometry and the StrOligo algorithm

Glycans are oligosaccharides associated with proteins, and are known to confer specific functions and conformations on glycoproteins. As protein tridimensional structures are related to function, the study of glycans and their impact on protein folding can provide important information to the field of proteomics. The subdiscipline of glycomics (or glycoproteomics) is rapidly growing in importance as glycans in proteins have shown to be involved in protein-protein or protein-(drug, virus, antibody) interactions. Glycomics studies most often aim at identifying glycosylation sites, and thus are performed on deglycosylated proteins resulting in loss of site-specific details concerning the glycosylation. In order to obtain such details by mass spectrometry (MS), either whole glycoproteins must be digested and analyzed as mixtures of peptides and glycopeptides, or glycans must be isolated from glycopeptide fractions and analyzed as pools. This article describes parallel experiments involving both approaches, designed to take advantage of the StrOligo algorithm functionalities with the aim of characterizing glycosylation microheterogeneity on a specific site. A hybrid quadrupole-quadrupole-time-of-flight (QqTOF) instrument equipped with a matrix-assisted laser desorption/ionization (MALDI) source was used. Glycosylation of alpha 5 beta 1 subunits of human integrin was studied to test the methodology. The sample was divided in two aliquots, and glycans from the first aliquot were released enzymatically, labelled with 2-aminobenzamide, and identified using tandem mass spectrometry (MS/MS) and the StrOligo program. The other aliquot was digested with trypsin and the resulting peptides separated by reversed-phase high-performance liquid chromatography (HPLC). A specific collected fraction was then analyzed by MS before and after glycan release. These spectra allowed, by comparison, detection of a glycopeptide (several glycoforms) and elucidation of peptide sequence. Compositions of glycans present were proposed, and identification of possible glycan structures was conducted using MS/MS and StrOligo.     

LC-MS/MS biopharmaceutical glycoanalysis: identification of desirable reference material characteristics

Glycosylation, the enzymatic addition of carbohydrates to a protein, is one of the most abundant post-translational modifications found in nature. There is variability in the number, location, and identity of glycans attached. As a result, a glycoprotein consists of a number of glycoforms with different combinations of glycans, potentially resulting in different stability, toxicity, and activity. This is especially important in the biopharmaceutical industry where product consistency and safety are vital. Glycoprotein analysis involves numerous mass spectrometry based techniques, each of which provides various aspects of characterization. The current paper describes two commonly used analytical techniques for glycoprotein characterization. In one experiment, nonspecific proteolysis is combined with a two-tiered mass spectrometry approach (MALDI-TOF and LC-MS/MS) to gain glycosylation site and glycan identity. In a second approach, glycans were enzymatically released, labeled with a fluorescent dye, and analyzed using LC-Fluorescence-MS/MS to give glycan identification and relative quantification. The type and degree of information yielded by each method is assessed in an effort to identify desired reference material characteristics for improving biopharmaceutical glycoanalysis.     

Glycosylation site analysis of human alpha-1-acid glycoprotein (AGP) by capillary liquid chromatography-electrospray mass spectrometry

A new anionic surfactant (RapiGest SF) was successfully used for site-specific analysis of glycosylation in human alpha-1-acid glycoprotein (AGP). By means of this analytical approach combined with capillary HPLC-mass spectrometry (and tandem mass spectrometry), the N-linked glycosylation pattern of AGP was explored. On the basis of mass matching and MS/MS experiments ca 80 different AGP-derived glycopeptides were identified. Glycosylation shows a markedly different pattern for the various glycosylation sites. At sites I and II, triantennary complex-type oligosaccharides predominate and at sites III, IV and V, tetra-antennary complex-type oligosaccharides predominate. Sites IV and V show the presence of additional N-acetyl lactosamine (Gal-GlcNAc) units (even higher degree of branching and/or longer antennae are also present).     

Glycosylation patterns of human chorionic gonadotropin revealed by liquid chromatography-mass spectrometry and bioinformatics

Due to their extensive structural heterogeneity, the elucidation of glycosylation patterns in glycoproteins such as the subunits of human chorionic gonadotropin (hCG), hCG-alpha, and hCG-beta, remains one of the most challenging problems in the proteomic analysis of post-translational modifications. In consequence, glycosylation is usually studied after decomposition of the intact proteins to the proteolytic peptide level. However, by this approach all information about the combination of the different glycopeptides in the intact protein is lost. In this study we have, therefore, attempted to combine the results of glycan identification after tryptic digestion with molecular mass measurements on the native starting material of the new first WHO Reference Reagents (RR) for hCG-alpha (99/720) and hCG-beta (99/650). Despite the extremely high number of possible combinations of the glycans identified in the tryptic peptides by HPLC-MS (>1000 for hCG-alpha and >10 000 for hCG-beta), the mass spectra of intact hCG-alpha and hCG-beta revealed only a limited number of glycoforms present in hCG preparations from pools of pregnancy urines. Peak annotations for hCG-alpha were performed with the help of a bioinformatic algorithm that generated a database containing all possible modifications of the proteins, including modifications possibly introduced during sample preparation such as oxidation or truncation, for subsequent searches for combinations fitting the mass difference between the polypeptide backbone and the measured molecular masses. Fourteen different glycoforms of hCG-alpha, containing biantennary, partly sialylized hybrid-type glycans, including methionine-oxidized and N-terminally truncated forms, were identified. Mass spectra of high quality were also obtained for hCG-beta, however, a database search mass accuracy of +/-5 Da was insufficient to unambiguously assign the possible combinations of post-translational modifications. In summary, mass spectrometric fingerprints of intact molecules were shown to be highly useful for the characterization of glycosylation patterns of different hCG preparations such as the new first WHO RR for immunoassays and could be the first step in establishing biophysical reference methods for hCG and related molecules.     

Identification of N-linked glycosylation sites in human nephrin using mass spectrometry

Nephrin is a type-1 transmembrane glycoprotein and the first identified principal component of the glomerular filtration barrier. Ten potential asparagine (N)-linked glycosylation sites have been predicted within the ectodomain of nephrin. However, it is not known which of these potential sites are indeed glycosylated and what type of glycans are involved. In this work, we have identified the terminal sugar residues on the ectodomain of human nephrin and utilized a straightforward and reliable mass spectrometry-based approach to selectively identify which of the ten predicted sites are glycosylated. Purified recombinant nephrin was subjected to peptide-N-glycosidase F (PNGase F) to enzymatically remove all the N-linked glycans. Since PNGase F is an amidase, the asparagine residues from which the glycans have been removed are deaminated to aspartic acid residues, resulting in an increase in the peptide mass with 1 mass unit. Following trypsin digestion, deglycosylated tryptic peptides were selectively identified by MALDI-TOF MS and their sequence was confirmed by tandem TOF/TOF. The 1 Da increase in peptide mass for each asparagine-to-aspartic acid conversion, along with preferential cleavage of the amide bond carboxyl-terminal to aspartic acid residues in peptides where the charge is immobilized by an arginine residue, was used as a diagnostic signature to identify the glycosylated peptides. Thus, nine of ten potential glycosylation sites in nephrin were experimentally proven to be modified by N-linked glycosylation.     

Mass spectrometry characterization for chemoenzymatic glycoprotein synthesis

The current project describes the chemoenzymatic modification of bovine ribonuclease B (RNase B) to contain a single glycosylation site with a known glycan. A reactive disaccharide oxazoline derivative was synthesized and stereospecifically added to deglycosylated RNase B through endo-β-N-acetylglucosaminidase M catalyzed chemoenzymatic transglycosylation. Oxazoline formation conditions were optimized using mass spectrometry, and the product verified based on its collision-induced dissociation (CID) mass spectrum. Enzymatic removal of native glycans as well as formation of the desired homogeneous product was also monitored using mass spectrometry. LC-MS(n) using four sequential rounds of CID was used to verify that the original glycosylation site had been reorganized to contain the new glycan. The techniques described herein are not limited to this analyte or glycan and should be amenable to the synthesis of numerous homogeneous glycoconjugates with judicious choice of enzyme/substrate combinations. The combined use of chemoenzymatic synthesis and mass spectrometry-based characterization shows promise for the development of homogeneous glycoprotein reference materials. A well-defined glycoprotein standard containing a single glycan of known composition, linkage and stereochemistry would be of great value for the comparison and evaluation of glycoprotein analysis techniques.     

Glycopeptide analysis by mass spectrometry

Glycosylation is one of the most important post-translational modifications found in nature. Identifying and characterizing glycans is an important step in correlating glycosylation structure to the glycan's function, both in normal glycoproteins and those that are modified in a disease state. Glycans on a protein can be characterized by a variety of methods. This review focuses on the mass spectral analysis of glycopeptides, after subjecting the glycoprotein to proteolysis. This analytical approach is useful in characterizing glycan heterogeneity and correlating glycan compositions to their attachment sites on the protein. The information obtained from this approach can serve as the foundation for understanding how glycan compositions affect protein function, in both normal and aberrant glycoproteins.     

Characterization of N-glycans of recombinant human thyrotropin using mass spectrometry

Thyroid-stimulating hormone is a vital component of the regulatory mechanism that maintains the structure and function of the thyroid gland and governs thyroid hormone release. In this paper we report the first detailed structural characterization of the N-linked oligosaccharides of recombinant human thyroid-stimulating hormone (rhTSH). Using a strategy combining mass spectrometric analysis and sequential exoglycosidase digestion, we have defined the structures of the N-glycans released from recombinant human thyrotropin by peptide N-glycosidase F. All glycans are complex-type glycans and are mainly of the bi- and triantennary type with variable degrees of fucosylation and sialylation. The major non-reducing epitope in the complex-type glycans is: NeuAcalpha2-3Galbeta1-4GlcNAc (sialylated LacNAc). The carbohydrate microheterogeneity at the three glycosylation sites was studied using reversed-phase high-performance liquid chromatography (RP-HPLC), concanavalin A affinity chromatography and mass spectrometric techniques, including both matrix-assisted laser desorption/ionization (MALDI) and electrospray. rhTSH was reduced, carboxymethylated and then digested with trypsin. The mixture of peptides and glycopeptides was subjected to RP-HPLC and the structures of the glycopeptides were determined by MALDI in conjunction with on-target exoglycosidase digestions. After PNGase F digestion, the peptide moiety of the glycopeptide was determined by the presence of the b- and y-series ions derived from its amino acid sequence in the quadrupole time-of-flight tandem mass (QTOF-MS/MS) spectrum. Glycosylation sites Asn-alpha52 and Asn-alpha78 contain mainly bi- and triantennary complex-type glycans. Only glycosylation site Asn-alpha52 bears fucosylated N-glycans. Minor tetraantennary complex structures were also observed on both glycosylation sites. Profiling of the carbohydrate moieties of Asn-beta23 indicates a large heterogeneity. Bi-, tri-, and tetraantennary N-glycans were present at this site. These data demonstrate site-specificity of glycosylation in the alpha subunit but not in the beta subunit of rhTSH with Asn-alpha52 bearing essentially di- and triantennary glycans with or without core fucosylation and bi- and triantennary glycans with no core fucosylation being attached to Asn-alpha78.     

Glycoprotein profiling by electrospray mass spectrometry

This work compares several different methods of site-specific analysis of glycoproteins using electrospray mass spectrometry. The glycoprotein, oLHalpha (ovine luteinizing hormone, alpha-subunit) was chosen as an appropriate example protein for these studies because of its biological relevance and extreme microheterogeneity. More than 20 unique glycoforms were detected for this glycoprotein at the Asn(56) site of oLHalpha. The carbohydrates present at this site affect receptor binding affinity, so understanding the great variety in the composition of these carbohydrates is important in studying ligand binding interactions. MS data was acquired on a quadrupole ion trap, a triple quadrupole, and a quadrupole time of flight mass spectrometer, and carbohydrate composition at the Asn(56) site of oLHalpha was determined using these instruments. Additionally, neutral loss and precursor ion scanning modes were also used to identify the glycoforms present, and these techniques were compared to the standard MS data. Of the three instruments compared in the study, the qTOF mass spectrometer achieved the lowest sample consumption, but all three instruments were useful in profiling the glycopeptide composition.     

Glycosylation of extracellular superoxide dismutase studied by high-performance liquid chromatography and mass spectrometry

Extracellular superoxide dismutase, EC-SOD, the main superoxide dismutase in biological fluids, is known from its lectin binding to be a glycoprotein. We have characterized the glycosylation of recombinant EC-SOD. A tryptic digest of the protein contained only one glycosylated peptide. This peptide was specifically bound to lectins and stained by periodic acid-Schiff stain. Although appearing very large on size-exclusion chromatography, it was shown to be glycosylated at only one site, asparagine-89, by specific cleavage with glycanases followed by mass spectrometry of the resulting peptide. Based on the binding properties of the peptide to concanavalin A and lentil lectin and the elution profile of N-glycanase-treated glycopeptide on ion-exchange chromatography, the carbohydrate appears to be the complex biantennary type with a core fucose.     

Glycoform characterization of intact erythropoietin by capillary electrophoresis-electrospray-time of flight-mass spectrometry

Glycosylated proteins often show a large variation in their glycosylation pattern, complicating their structural characterization. In this paper, we present a method for the accurate mass determination of intact isomeric glycoproteins based on capillary electrophoresis-electrospray-time of flight-mass spectrometry. Human recombinant erythropoietin has been chosen as a showcase. The approach enables the on-line removal of nonglycosylated proteins, salts, and neutral and negatively charged species. More important, different glycosylation forms are separated both on the base of differences in the number of negatively charged sialic acid residues and the size of the glycans. Thus, 44 glycoforms and in total about 135 isoforms of recombinant human erythropoietin, taking also acetylation into account, could be distinguished for the reference material from the European Pharmacopeia. Distinct glycosylation differences for samples from different suppliers are clearly observed. Based on the accurate mass an overall composition of each single isoform is proposed, perfectly in agreement with data on glycan and glycopeptide analysis. This method is an ideal complement to the established techniques for glycopeptide and glycan analysis, not differentiating branching or linkage isoforms, but leading to an overall composition of the glycoprotein. The presented strategy is expected to improve significantly the ability to characterize and quantify isomeric glycoforms for a large variety of glycoproteins.     

Rapid characterization of N-linked glycosylation site using non-specific protease digestion of gel-separated glycoproteins and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Rapid identification of glycosylation sites of glycoproteins is urgently needed in glycoproteomics study. In the present work, a rapid and simple method based on non-specific digestion of gel-separated glycoproteins and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry was described, which can efficiently identify the N-linked glycosylation sites. One-step in-gel digestion of Ribonuclease B (RNase B) by proteinase K was employed to generate glycopeptides with short and discrepant peptide composition. When compared with glycopeptides prepared by two-step in gel-digestion using trypsin-proteinase K or trypsin-pronase, the direct proteinase K treatment showed obvious superiority in both glycopeptide recovery and preparation simplicity. Most importantly, it helps to generate greater variety of glycopeptide series with rich information for glycosylation site identification. In addition, binary matrices 5-chloro-2-mercaptobenzothiazole (CMBT) /2,5-dihydroxybenzoic acid (DHB) were found to form homogeneous microcrystal on the target with the purified glycopeptides, leading to improved detection sensitivity. Thus, the present work provides an optimized solution to speed up the characterization of N-linked glycosylation sites in glycoproteins.     

Improving confidence in detection and characterization of protein N-glycosylation sites and microheterogeneity

Protein glycosylation is one of the most common post-translational modifications, estimated to occur in over 50% of human proteins. Mass spectrometry (MS)-based approaches involving different fragmentation mechanisms have been frequently used to detect and characterize protein N-linked glycosylations. In addition to the popular Collision-Induced Dissociation (CID), high-energy C-trap dissociation (HCD) fragmentation, which is a feature of a linear ion trap orbitrap hybrid mass spectrometer (LTQ Orbitrap), has been recently used for the fragmentation of tryptic N-linked glycopeptides in glycoprotein analysis. The oxonium ions observed with high mass accuracy in the HCD spectrum of glycopeptides can be combined with characteristic fragmentation patterns in the CID spectrum resulting from consecutive glycosidic bond cleavages, to improve the detection and characterization of N-linked glycopeptides. As a means of automating this process, we describe here GlypID 2.0, a software tool that implements several algorithmic approaches to utilize MS information including accurate precursor mass and spectral patterns from both HCD and CID spectra, thus allowing for an unequivocal and accurate characterization of N-linked glycosylation sites of proteins.