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.     

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.     

Ionisation and fragmentation of complex glycans with a quadrupole time-of-flight mass spectrometer fitted with a matrix-assisted laser desorption/ionisation ion source

This paper reports the use of an experimental matrix-assisted laser desorption/ionisation (MALDI) ion source fitted to a quadrupole time-of-flight (Q-Tof) mass spectrometer for the analysis of carbohydrates, particularly the N-linked glycans from glycoproteins. Earlier work on the Q-Tof instrument, using electrospray ionisation, gave excellent MS/MS spectra, particularly from the [M + Na]+ ions, but suffered from the major disadvantages that the signal was often split between singly and multiply charged ions and that sensitivity fell dramatically as the molecular weight of the carbohydrate rose. The MALDI ion source did not suffer from these problems and the instrument produced excellent MS and MS/MS spectra from small amounts of complex, underivatised glycans as well as those derivatised at the reducing terminus. Positive ion MS spectra of sialylated glycans recorded on the new instrument were much less complex than those recorded with a conventional MALDI-TOF instrument because of the absence of ions resulting from metastable (post-source decay, (PSD)) fragmentations occurring in the flight tube. However, considerable fragmentation by loss of sialic acid still occurred. MS/MS spectra of the [M + Na]+ ions from all compounds were almost identical to those recorded earlier with the electrospray-Q-Tof combination and far superior to MALDI-PSD spectra recorded with reflectron-TOF instruments. Spectra are shown for neutral and sialylated N-linked glycans from chicken ovalbumin, riboflavin binding protein, alpha1-acid glycoprotein, bovine fetuin and ribonuclease B, both as free glycans and as those derivatised at their reducing termini. The technique was applied to the structural determination of N-linked glycans from human secretory IgA and Apo-B 100 from human low-density lipoprotein.     

Production and fragmentation of negative ions from neutral N-linked carbohydrates ionized by matrix-assisted laser desorption/ionization

Although negative ion fragmentation mass spectra of neutral N-linked carbohydrates (those attached to Asn in glycoproteins) provide much more structural information than spectra recorded in positive ion mode, neutral carbohydrates are reluctant to form negative ions by matrix-assisted laser desorption/ionization (MALDI) unless ionized from specific matrices such as nor-harmane or adducted with anions such as chloride. This paper reports the results of experiments to optimize negative ion formation from adducts of N-linked glycans with respect to ion abundance and fragment ion production. The best results were obtained with 2,4,6-trihydroxyacetophenone (THAP) as the matrix with added ammonium nitrate as the salt providing the anion. This approach is demonstrated to be applicable for a wide range of N-linked glycan structures. Phosphate adducts, analogous to those that are usually encountered in electrospray spectra from N-glycans released by protein N-glycosidase F, were produced by addition of ammonium phosphate to the matrix but in relatively low yield allowing competitive ionization of endogenous anionic compounds leading to complex spectra. Fragmentation of the nitrate adducts, which were formed in higher yield, generally paralleled that seen by collision-induced dissociation following ionization by electrospray, with the first stage of the dissociation being the elimination of the nitrate with a proton from one of the hydroxyl groups of the sugar. The spectra of the resulting [M-H](-) species displayed very specific fragment ions, mainly cross-ring and C-type glycosidic cleavage products, that revealed more structural (linkage and branching) information of the compounds than the mainly glycosidic cleavage products that dominated the positive ion spectra.     

Detection and Characterization of Low Abundance Glycopeptides Via Higher-Energy C-Trap Dissociation and Orbitrap Mass Analysis

Broad-scale mass spectrometric analyses of glycopeptides are constrained by the considerable complexity inherent to glycoproteomics, and techniques are still being actively developed to address the associated analytical difficulties. Here we apply Orbitrap mass analysis and higher-energy C-trap dissociation (HCD) to facilitate detailed insights into the compositions and heterogeneity of complex mixtures of low abundance glycopeptides. By generating diagnostic oxonium product ions at mass measurement errors of <5 ppm, highly selective glycopeptide precursor ion detections are made at sub-fmol limits of detection: analyses of proteolytic digests of a hen egg glycoprotein mixture detect 88 previously uncharacterized glycopeptides from 666 precursor ions selected for MS/MS, with only one false positive due to co-fragmentation of a non-glycosylated peptide with a glycopeptide. We also demonstrate that by (1) identifying multiple series of glycoforms using high mass accuracy single stage MS spectra, and (2) performing product ion scans at optimized HCD collision energies, the identification of peptide + N-acetylhexosamine (HexNAc) ions (Y1 ions) can be readily achieved at <5 ppm mass measurement errors. These data allow base peptide sequences and glycan compositional information to be attained with high confidence, even for glycopeptides that produce weak precursor ion signals and/or low quality MS/MS spectra. The glycopeptides characterized from low fmol abundances using these methods allow two previously unreported glycosylation sites on the Gallus gallus protein ovoglycoprotein (amino acids 82 and 90) to be confirmed; considerable glycan heterogeneities at amino acid 90 of ovoglycoprotein, and amino acids 34 and 77 of Gallus gallus ovomucoid are also revealed.     

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.     

Structural analysis of a glycoprotein by liquid chromatography-mass spectrometry and liquid chromatography with tandem mass spectrometry. Application to recombinant human thrombomodulin

Using recombinant human thrombomodulin (rhTM) expressed in Chinese hamster ovary (CHO) cells, we studied the structural analysis of a glycoprotein by liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS-MS). First, we analyzed the structure of both the O- and N-linked glycans in rhTM by oligosaccharide mapping using LC-MS equipped with a graphitized carbon column (GCC-LC-MS). Major O- and N-linked glycans were determined to be core 1 structure and fucosyl biantennary containing NeuAc(0-2) respectively. Next, the post-translational modifications and their heterogeneities, including the site-specific glycosylation, were analyzed by mass spectrometric peptide/glycopeptide mapping of trypsin-digested rhTM and precursor-ion scanning. Precursor-ion scanning was successful in the detection of five glycopeptides. Four N-glycosylation sites and their site-specific carbohydrate heterogeneity were determined by their mass spectra. O-Glycosylation could be estimated on the basis of its mass spectrum. We were able to identify partial beta-hydroxylation on Asn324 and Asn439, and O-linked glucose on Ser287 from the peptide/glycopeptide map and their mass spectra. We demonstrated that a sequential analysis of LC-MS and LC-MS-MS are very useful for the structural analysis of O- and N-linked glycans, polypeptides, and post-translational modifications and their heterogeneities, including site-specific glycosylation in a glycoprotein. Our method can be applied to a glycoprotein in biological samples.     

The determination of glycopeptides by liquid chromatography/mass spectrometry with collision-induced dissociation

Glycopeptides derived from ribonuclease B and ovomucoid have been subjected to collision-induced dissociation (CID) in the second quadrupole of a triple quadrupole mass spectrometer. Doubly charged parent ions gave predictable fragmentation that yielded partial sequence information of the attached oligosaccharide as Hex and HexNAc units. Common oxonium ions are observed in the product ion mass spectra of the glycopeptides that correspond to HexNAc⁺ (m/z 204) and HexHexNAc⁺ (m/z 366). A strategy for locating the glycopeptides in the proteolytic digest mixtures of glycoproteins by ions spray liquid chromatography mass spectrometry (LC/MS) is described by utilizing CID in the declustering region of the atmospheric pressure ionization mass spectrometer to produce these characteristic oxonium ions. This LC/CID/MS approach is used to identify glycopeptides in proteolytic digest mixtures of ovomucoid, asialofetuin, and fetuin. LC/CID/MS in the selected ion monitoring mode may be used to identify putative glycopeptides from the proteolytic digest of fetuin.     

A novel precursor ion discovery method on a hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer for studying protein phosphorylation

A tandem quadrupole time-of-flight (Q-TOF) mass spectrometer has been programmed such that phosphorylated peptides can automatically be discovered and identified in a way similar to that of the use of precursor ion or neutral loss scanning, but without the need to scan the quadrupole mass filter. Instead, the method capitalizes on the innate capability of the Q-TOF to record mass spectra and product ion spectra quickly, with good sensitivity and with good mass accuracy. Alternate mass spectra, with and without fragmentation, are recorded at high and low collision energy with the quadrupole operating in wideband mode. The method of analysis is both compatible with and dependant on liquid chromatography for separation of complex mixtures. The method has been demonstrated by searching for the neutral loss of 98 Da (H3PO4) from phosphoserine and phosphothreonine residues, or for the phosphorylated immonium ion at m/z 216 from phosphotyrosine. The method also incorporates acquisition of the product ion spectrum from any candidate precursor ions, thereby allowing confirmation of the neutral loss or product ion and providing additional sequence information to assist identification of the protein and assign the site of phosphorylation.     

Glycosylation analysis of interleukin-23 receptor: elucidation of glycosylation sites and characterization of attached glycan structures

Interleukin-23 (IL-23) is a heterodimeric cytokine, a central factor in chronic/autoimmune inflammation. It signals through a heterodimeric receptor consisting of IL-23r, which is heavily glycosylated. The structural characterization of IL-23r has not been reported. In this work, glycosylation profiles of soluble recombinant human IL-23r (rhIL-23r) were established using mass spectrometry (MS), which included defining glycosylation sites, degree of glycosylation occupancy of each site and structure of attached oligosaccharides. Specifically, precursor ion scan of oxonium ion protonated N-acetylglucosamine (GlcNAc(+)) (m/z 204) was performed using a triple quadrupole MS instrument to locate the retention time of glycopeptides. Both the glycopeptides and their corresponding deglycosylated forms in each collected HPLC fraction were studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (LTQ-Orbitrap) for glycosylation site profiling. The attached glycan structures were elucidated by collision-induced dissociation (CID) fragmentation of target glycopeptides in combination with accurate mass measurement. Eight glycosylation sites were identified on IL-23r (Asn24, Asn209, Asn239, Asn157, Asn118, Asn250, Asn58 and Asn6). Most of the glycosylation sites were > 95% occupied except Asn250 and Asn6. Those two sites were 88% and 45% occupied by estimation from trypsin digestion and were 55% and 42% occupied from LysC digestion. Multiple glycoforms were observed in IL-23r. Most of them were bi-, tri- or tetra-antennary complex type structures with fucose and sialic acid. High mannose and hybrid type glycans were only observed on Asn157. The structural characterization on IL-23r glycosylation provides useful information for better understanding of the biological function of IL-23r.     

The influence of sialylation on glycan negative ion dissociation and energetics

For the analysis of native glycans using tandem mass spectrometry (MS), it is desirable to choose conditions whereby abundances of cross-ring cleavages indicative of branch positions are maximized. Recently, negative ion tandem mass spectrometry has been shown to produce significantly higher abundances of such ions in glycans compared to the positive ion mode. Much of this prior work has concerned fragmentation patterns in asialo glycans. The present work compares the abundances of critical cross-ring cleavage ions using negative mode tandem mass spectrometry for milk oligosaccharides and N-linked glycans. For comparison, product ion formation was studied for deprotonated and nitrated ions formed from asialo glycans and deprotonated ions from sialylated glycans. Breakdown profiles demonstrate clearly that more energy was required to fragment sialylated compounds to the same extent as either their asialo or nitrate adducted counterparts. The extraction of a proton from a ring hydroxyl group during the ionization process may be viewed, qualitatively, as imparting significantly more energy to the ion than would that from a molecule bearing an acidic group, so that acidic glycans are more stable in the gas phase, as the negative charge resides on the carboxyl group. These results have strong practical implications because a major portion of glycans released from mammalian proteins will be sialylated.     

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.     

Automated structural assignment of derivatized complex N-linked oligosaccharides from tandem mass spectra

Glycoprotein function is controlled by several biological factors, one of them being the structure of carbohydrate chains (glycans) attached to specific amino acids of the protein backbone. Changes in glycan structures have been shown to modify the secondary and tertiary conformation of glycoproteins, thus their function. Powerful analytical tools are available for the characterization of sugar structures, and recently mass spectrometry (MS) has been increasingly useful for this purpose. Manual interpretation of tandem mass spectrum is possible but tedious. Automated interpretation should speed the analysis and enhance the results obtained. A new computer program for automated interpretation of tandem MS spectra of complex N-linked glycans oligosaccharides from mammals will be described. N-Linked oligosaccharides standards were derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) and analyzed by matrix-assisted laser desorption/ionization (MALDI)-tandem MS. Simulated tandem mass spectra of other common glycans were also generated to test the algorithm. The MALDI-MS/MS spectra featured resolved isotopic distributions for the [M + H](+) and fragment ions of oligosaccharides. These isotopic distributions complicated the automated analysis of the spectra and were removed to leave only monoisotopic peaks. An algorithm was written for this purpose, yielding simplified tandem mass spectra. Another algorithm is then used to determine the structure of the oligosaccharide. A score is then given to each structure, depending on agreement with experimental results. The program successfully assigned the true structure in 24 out of the 28 cases (86%) and the true structure was among the three top scoring structures in all cases.     

Combining gel and capillary electrophoresis, nano-LC and mass spectrometry for the elucidation of post-translational modifications of Trichoderma reesei cellobiohydrolase I

N-Glycosylation of cellobiohydrolase I from the fungus Trichoderma reesei (strain Rut-C30) is studied using a combination of electrophoretic, chromatographic and mass spectrometric techniques. As four potential N-glycosylation sites and several uncharged and phosphorylated high-mannose glycans are present, a large number of glycoforms and phospho-isoforms can be expected. Isoelectric focusing both in gel and in capillary format was successfully applied for the separation of the phospho-isoforms. They were extracted in their intact form from the gel and subsequently analysed by nanospray-Q-TOF-MS, thereby making use of a powerful two-dimensional technique. Nano-LC/MS/MS on a Q-Trap MS further allowed the determination of the glycosylation sites. As a novel approach, an oxonium ion was used in precursor ion scanning for selective detection of glycopeptides containing phosphorylated high-mannose glycans.     

Structure elucidation of glycoproteins by direct nanoESI MS and MS/MS analysis of proteolytic glycopeptides

Bovine ribonuclease B (RNAse B) and asialofetuin (FETUA) were subjected to in-capillary tryptic digest (Pohlentz et al. Proteomics. 2005, 5, 1758-1763) and the obtained glycopeptides were analyzed, respectively, by nanoelectrospray ionization mass spectrometry and collision-induced dissociation (CID) during the ongoing digest. For RNAse, B glycans of the high-mannose type (Man(4) to Man(9)) attached to either a tetra- or a hexapeptide containing the sole N-glycosylation site of the protein were detected. Glycopeptides derived from all three N-glycosylation sites of FETUA were observed, and the corresponding CID spectra proved the respective glycans to be oligosaccharides of the triantennary complex type. Moreover, an O-glycopeptide carrying Gal-GalNAc at T(280) could be unambiguously identified. An in-solution tryptic/chymotryptic digest of human transferrin (TRFE) was analyzed directly for glycopeptides subsequent to the addition of methanol and formic acid. Disialylated diantennary glycans were observed in glycopeptides of both N-glycosylation sites of TRFE. These results demonstrate the feasibility of direct structure determination of glycopeptides in proteolytic mixtures without any further refurbishment.     

Mass spectrometric characterization of glycosylation of hepatitis C virus E2 envelope glycoprotein reveals extended microheterogeneity of N-glycans

Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The polyprotein encoded in the HCV genome is co- and post-translationally processed by host and viral peptidases, generating the structural proteins Core, E1, E2, and p7, and five nonstructural proteins. The two envelope proteins E1 and E2 are heavily glycosylated. Studying the glycan moieties attached to the envelope E2 glycoprotein is important because the N-linked glycans on E2 envelope protein are involved in the interaction with some human neutralizing antibodies, and may also have a direct or indirect effect on protein folding. In the present study, we report the mass spectrometric characterization of the glycan moieties attached to the E2 glycoprotein. The mass spectrometric analysis clearly identified the nature, composition, and microheterogeneity of the sugars attached to the E2 glycopeptides. All 11 sites of glycosylation on E2 protein were characterized, and the majority of these sites proved to be occupied by high mannose glycans. However, complex type oligosaccharides, which have not been previously identified, were exclusively observed at two N-linked sites, and their identity and heterogeneity were determined.     

Product ion spectral simplification using time-delayed fragment ion capture with tandem linear ion traps

A new technique to generate product ion spectra as the internal energy of a collisionally activated precursor ion evolves is described. The precursor ion is activated by acceleration into a mass-selective linear ion trap under conditions whereby some of the fragment ions formed are unstable within the trap. After a time delay the stability parameters of the ion trap are changed to allow capture of fragments that that were previously unstable. The result is a product ion spectrum that originates from precursor ions with a modified internal energy distribution. It is possible to follow the evolution of the precursor internal energy distribution for many milliseconds after admittance of the precursor ions into the linear ion trap. Time-delayed fragmentation product ion spectra typically display reduced sequential fragmentation products leading to spectra that are more easily interpreted. Several important experimental parameters important to time-delayed fragmentation have been identified and are discussed. The technique has applications for both small precursor ions and multiply charged peptides.     

Mass spectrometry of proton adducts of fucosylated N-glycans: fucose transfer between antennae gives rise to misleading fragments

Fragmentation behavior of fucosylated N-glycans in both protonated and sodiated form was studied by low-energy collision-induced dissociation with an ion trap mass spectrometer as well as by laser-induced dissociation with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Diantennary, core-(alpha1-6)-fucosylated N-glycans with Lewis X (Gal(beta1-4)[Fuc(alpha1-3)]GlcNAcbeta1-) and/or fucosylated LacdiNAc antennae (GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAcbeta1-) were obtained from the human parasite Schistosoma mansoni and used as model substances, after labeling with 2-aminobenzamide, or as native reducing glycans. While fragment spectra of sodiated as well as protonated species obtained in both mass spectrometers resulted in B- and Y-type ions, fragmentation of proton adducts additionally gave rise to various fragment ions which had acquired fucose residues from other parts of the molecule. In particular, fucose was transferred efficiently to the Lewis X antennae suggesting the occurrence of difucosylated antennae, which could erroneously be interpreted as Lewis Y epitopes. By studying two additional model substances, this fucose gain was shown to occur by transfer of fucose between the antennae, but not by transfer of a core-(alpha1-6)-fucose. Despite the drastically different lifetimes of the ions, protonated species analyzed on the ion trap (millisecond range) and by MALDI-TOF/TOF-MS (microsecond range) showed similar rearrangement patterns, suggesting that the fucose mobility goes hand in hand with decomposition. Notably, permethylation of the model N-glycans seemed to completely preclude fucose migration. This study indicates that caution should be applied with the interpretation of tandem mass spectrometric (MS/MS) data of protonated glycoconjugates, including glycopeptides, because of the potential occurrence of fucose rearrangements.     

CHARMM additive all-atom force field for carbohydrate derivatives and its utility in polysaccharide and carbohydrate-protein modeling

Monosaccharide derivatives such as xylose, fucose, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GlaNAc), glucuronic acid, iduronic acid, and N-acetylneuraminic acid (Neu5Ac) are important components of eukaryotic glycans. The present work details development of force-field parameters for these monosaccharides and their covalent connections to proteins via O-linkages to serine or threonine sidechains and via N-linkages to asparagine sidechains. The force field development protocol was designed to explicitly yield parameters that are compatible with the existing CHARMM additive force field for proteins, nucleic acids, lipids, carbohydrates, and small molecules. Therefore, when combined with previously developed parameters for pyranose and furanose monosaccharides, for glycosidic linkages between monosaccharides, and for proteins, the present set of parameters enables the molecular simulation of a wide variety of biologically-important molecules such as complex carbohydrates and glycoproteins. Parametrization included fitting to quantum mechanical (QM) geometries and conformational energies of model compounds, as well as to QM pair interaction energies and distances of model compounds with water. Parameters were validated in the context of crystals of relevant monosaccharides, as well NMR and/or x-ray crystallographic data on larger systems including oligomeric hyaluronan, sialyl Lewis X, O- and N-linked glycopeptides, and a lectin:sucrose complex. As the validated parameters are an extension of the CHARMM all-atom additive biomolecular force field, they further broaden the types of heterogeneous systems accessible with a consistently-developed force-field model.     

Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro‐heterogeneity) and evaluate the molar site occupancy (macro‐heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N‐glycans was chemically synthesised by solid‐phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N‐acetylglucosamine‐linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well‐defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI‐IT, ESI‐Q‐TOF, MALDI‐TOF, ESI/MALDI‐FT‐ICR‐MS). Depending on the ion source/mass analyser, glycopeptides carrying complex‐type N‐glycans exhibited clearly lower signal strengths (10–50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano‐ESI and medium‐pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro‐heterogeneity and macro‐heterogeneity by label‐free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics. Copyright © 2013 John Wiley & Sons, Ltd.