Structural Characterization of Intact Glycoconjugates by Tandem Mass

[email protected], Dr. X. F. Chen, Shandong Analysis and Test Centre,. Shandong Academy of Sciences, Qilu University of Technology, Jinan, Shandong,...
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Structural Characterization of Intact Glycoconjugates by Tandem Mass Spectrometry Using Electron-Induced Dissociation Y. L. Elaine Wong, Xiangfeng Chen, Ri Wu, Y. L. Winnie Hung, and T.-W. Dominic Chan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03128 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on August 26, 2017

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Structural Characterization of Intact Glycoconjugates by Tandem Mass Spectrometry Using Electron-Induced Dissociation Y. L. Elaine Wong,† Xiangfeng Chen,†‡* Ri Wu,† Y. L. Winnie Hung,† and T.-W. Dominic Chan†*



Department of Chemistry, The Chinese University of Hong Kong, Hong Kong SAR,P. R. China ‡ Shandong Analysis and Test Centre, Shandong Academy of Sciences, Qilu University of Technology, Jinan, Shandong, P. R. China

*Address reprint requests to Professor T.-W. D. Chan, Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR. E-mail: [email protected], Dr. X. F. Chen, Shandong Analysis and Test Centre, Shandong Academy of Sciences, Qilu University of Technology, Jinan, Shandong, P. R. China, Jinan, China. E-mail: [email protected].

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ABSTRACT Characterizing the structures of glycoconjungates is important because of glycan heterogeneity and structural complexity of aglycon. The presence of relatively weak glycosidic linkages leads to preferential cleavages that limit the acquisition of structural information under typical mass spectrometry dissociation conditions, such as collision-induced dissociation (CID) and infrared multiphoton dissociation. In this paper, we explored the dissociation behaviors of different members of glycoconjugates, including glycopeptides, glycoalkaloids, and glycolipids, under electron-induced dissociation (EID) conditions. Using CID spectra as references, we found that EID is not only a complimentary method to CID but also a method that can generate extensive fragment ions for the structural characterization of all intact glycoconjugates studied. Furthermore, isomeric ganglioside species can be differentiated, and the double bond location in the ceramide moiety of the gangliosides can be identified through the MS3 approach involving sequential CID and EID processes. Keywords: glycoconjugates, glycopeptides, glycosides, gangliosides, electron induced dissociation, isomer, diagnostic ion, collision induced dissociation

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INTRODUCTION

Glycoconjugates are vital biological compounds in which carbohydrates are covalently linked with other chemical species, including peptides/proteins, lipids, and alkaloids.1 Glycoconjugates are classified into different groups according to the distinctive chemical nature of their aglycons, including glycoproteins, glycolipids, and glycosides. Glycosylation plays an essential role in various cellular activities, such as structural building, cell signaling, and recognition.2 Furthermore, glycosylation is one of the most prominent post-translational modifications in proteins. This process is responsible for protein folding and stability and facilitates the precise localization of proteins, which bind to specific receptors through sugar moieties.3–6 The interactions between glycans and protein moieties account for immune responses and pathogen interactions.7–9 Other glycan-containing compounds, including glycosides, are extremely common secondary metabolites in plants.10 Flavonoid glycosides have therapeutic potential and are used as antibiotics, antioxidant, and anti-inflammatory drugs.10,11 However, steroidal glycosides, including glycoalkaloid in potato sprouts, are human toxins.12 Glycolipids, such as gangliosides, are cell membrane components that are densely packed with cholesterol and form lipid micro-domains to modulate signal transduction events.13–15 The oligosaccharide portions of glycolipids interact with exogenous compounds, including neighboring cells, extracellular proteins, and 3 ACS Paragon Plus Environment

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pathogens. Meanwhile, the lipid parts inserted into cell walls serve as mediators for intercellular and extracellular signaling.16–19 Gangliosides are found in high concentration in mammalian central nervous system and are believed to play a vital role in brain development, maturation, and aging.20,21 Gangliosides are also used as important diagnostic markers for various neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease.22,23

Establishing an analytical platform for characterizing the primary structures of glycoconjugates is important for understanding and assessing the specific functions of various glycoconjugates because of the complexity and heterogeneity of their structures. Characterizing the structures of these biomolecules remains a challenging task.24 Tandem mass spectrometry (MS/MS) using collision-induced dissociation (CID)25 as an ion activation method has conventionally played an important role in the structural analysis of biomolecules. However, CID involves primarily vibrational activation and tends to have preferential cleavage at the weakest bond of the molecular ions, resulting in limited structural information. Meanwhile, electron-based dissociation methods, including electron capture dissociation (ECD),26 electron detachment dissociation (EDD),27 and electron-induced dissociation (EID),28 have provided alternative ion activation approaches based on electron-ion interactions. Charge-directed and radical-directed dissociation pathways are both found to operate 4 ACS Paragon Plus Environment

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under these electron-based dissociation methods. Electron-based dissociation has been a promising method for bio-analysis such as top-down protein analysis,29-31 glycopeptide analysis,32,33 and glycomics . 34-36 Notably, a combination of CID and EC/TD has been a useful approach to produce complimentary information for a nearly complete characterization of glycoproteins.37 Zubarev et al, Costello and Lin et al. have extensively studied on the application of EC/TD in oligosaccharide structural characterization.38-44 However, this approach has limited use in the analysis of small glycoconjugates, such as glycopeptides, glycoalkaloids, and glycolipids. The formation of primary singly positively charged (or negatively charged) molecular ions precluded the use of EC/TD. EID has shown to yield fruitful structural information for analysis of small natural products that are usually present as singly charged ion.45-47 In this work, we attempted to explore the EID method for the analysis of different types of glycoconjugates with various degrees of glycosylation. The EID of glycopeptides generated backbone-cleaved fragments regardless of the preservation of the glycan group in the peptide chain. The MS/MS spectra allowed a direct localization of the site of O-glycosylation. EID yielded structurally informative fragment ions, which were used to deduce the sequences and compositions of the oligosaccharide parts. EID also produced multiple cleavages across the steroidal and ceramide lipid moieties across the aglycon portions of glycoalkaloids and gangliosides. The aglycon potions 5 ACS Paragon Plus Environment

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are specific fragments that can be used for the structural characterization of different types of glycoconjugates. EXPERIMENTAL SECTION

Sample Preparation. Esculeoside A and α-tomatine were extracted from green tomatoes

according

to

the

extraction

procedure

described

in

the

literature.48 Gangliosides, GD1a and GD1b, were purchased from Avanti Polar Lipids, Inc. O-Glycopeptide standards and MUC5AC 3 was purchased from Protea Biosciences, Inc. The N-glycopeptide CSF114 was purchased from Sussex Research (Canada). These compounds were prepared at concentrations of ~0.1–0.5 µM in 1:1 methanol (LC–MS grade)/water (LC–MS grade) (v/v). Acetic acid (0.1%)was added for positive mode ESI. Permethylation is needed in most of the systems to identify the exact cross-ring cleavages in oligo- and polysaccharides. However, in the glycoconjungates systems studied, many of the cross-ring cleavages gave only one specific fragment masses. No permethylation was done in this work.

Mass Spectrometry. All experiments were performed on a Bruker SolariX FTMS equipped with a 9.4T actively shielded refrigerated magnet. Samples were directly infused and ionized in positive ESI mode for protonated or sodiated ion and negative mode for deprotonated ion through a home-built microspray source with a flow rate of 30 µL/h. Ionization was performed in a positive mode with a capillary voltage of -3 6 ACS Paragon Plus Environment

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kV or negative mode of +3 kV and a transfer capillary temperature of 180 °C. The voltage of ion funnel is -150 V and the skimmer voltage is -15 V.The trap plates voltage in analyzer cell is -1.5 V; the side kick offset voltage is 1.5 V. All mass spectra were acquired in broadband mode over a mass range of m/z 80–1500. CID experiments were performed in the collision cell, whereas EID and EDD (for doubly deprotonated gangliosides GD1a and GD1b) were performed in the analyzer cell. For the EID/EDD experiments, the precursor ions were isolated in the quadrupole and were accumulated in the collision cell for 0.2-1.5 s before being transferred into the ParaCellTM. The trapping ions were then irradiated with electrons from a heated hollow cathode dispenser operated at a heating current of 1.4 A. The bias voltage was set between -23 and -26 V and the extraction electrode was set to 0-5V and tuned for optimal fragment intensities.. The electron irradiation time was 30–60 ms for EID and 0.2 s for EDD. Background spectra were obtained by setting the bias voltage to 0 V to ensure that no emission of electrons occurred and all the other parameters are kept the same. For CID, the precursor ions were isolated in the first quadrupole and were fragmented in the collision cell with collision energies of 10V for MUC5AC 3, 35 V for CSF114, 30 V for Esculeoside A, 48–50 V for α-tomatine, and 28–35 V for gangliosides. Product ions were assigned through exact mass measurements and the Smart Formula method (Bruker) was used. Mass spectra were externally calibrated 7 ACS Paragon Plus Environment

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using the clustered ions of sodium formate and then internally calibrated using confidently assigned fragment peaks. The product ions were generally assigned with a mass accuracy within 200 ppb. For EID, 100 acquisitions were signal averaged per mass spectrum. The dataset size is 1M, padded with one zero fill and apodized using a sine-square window. Figure S-1 shows the pulse sequence of ECD/EID experiment in Bruker SolariX FTMS. The detection was in magnitude mode with Ramped Power excitation. Data were processed on a Data Analysis™ software (Version 4.4).

RESULTS AND DISCUSSION

Through EID, the distinctive classes of glycoconjugates with varied levels of glycosylation ranging from single glycan to multiple glycan attachments were characterized. The nomenclature for fragmentation and labeling used in this paper were based on the description of Costello et al.49 and followed the nomenclature proposed by the IUPAC-IUB Joint Commission on Biochemical Nomenclature forglycolipids.50

CID and EID of Glycopeptides. CID and EID results of N- and O-linked 2+

glycopeptides were compared. Figures 1a and b show the CID spectra of [M+2H] of the N-glycosylated CSF114 and O-glycosylated MUC5AC 3.The asparagine at the position 7 of CSF114 and threonine at the position 3 of MUC5AC 3 were

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glycosylated with a mono-glucose (Glc) and N-acetylgalactosamine (GalNAc) respectively. The CID of both N-and O-glycopeptides resulted in preferential cleavage of the Glc and GalNAc unit from the glycosylated site over the peptide bonds. This was indicated by the [M+2H-Glc-CH 3 COOH]2+ ion from CID of CSF114 and the intense peaks of [M+2H-Gal]2+ and [M+H-Gal]+ ions from MUC5AC 3. Furthermore, the CID spectra contained some members of the b-/y-product ions, most of which were produced from the subsequent fragmentations of the deglycosylated molecular ions. The CID of CSF114 produced more fragment ions and gave higher sequence coverage than that of MUC5AC 3. This is may be due the presence of more basic residue in the peptide, which facilitate the fragmentation. Meanwhile, only *y 16 , *y 19 , and *y 20 ions in CSF114 and *y 15 ion in MUC5AC 3 were formed from the glycopeptide molecule ions without prior loss of the Glc and GalNAc unit (where the star sign represents the retention of glycan group in fragment ion). The limited glycosylated fragment ions did not provide sufficient information for assigning the glycosylation sites in these peptides. 2+

Figures 1c and d show the EID spectra of [M+2H] of glycosylated peptides CSF114 and MUC5AC 3. Both EID spectra showed increase in structural specific ions. In the EID spectrum of CSF114, a series of Glc containing *a-/*b-/*c-ions and *y-ions was observed, including *c 7 -to *c 14 -ions and (*b 15 to *b 20 + H 2 O) ions. The presence of 9 ACS Paragon Plus Environment

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basic amino acid residue promotes a rearrangement that leads to the formation of (b n−1 + H 2 O) product ion by loss of the C-terminal amino acid.51 All other observed small b-, c-, y- and z-ions did not contain the sugar unit, implying that the glycosylation site was at the seventh asparagine. Mapping the sites of glycosylation of O-glycopeptides or glycoproteins was challenging. The preferential cleavage of glycan upon ion activation is more extensive than that of N-glycoprotein52 and hindered the localization of the modified sites. Locating the glycosylation site of MUC5AC 3 was increasingly difficult, as this peptide contained nine serine/threonine residues. In the EID spectrum of MUC5AC 3, a series of GalNAc containing b- and c-ions was observed. From the positions of these GalNAc-containing fragments, the sugar unit was attached either to threonine-2 (T-2) or to threonine-3 (T-3). The occurrence of solely b 2 that contained no sugar implied that the sugar moiety was attached to threonine-3. The fragmentation pathway of EID includes the electronic excitation of the precursor ion without altering its charge states; electron ejection by further ionization to give an electron-ionized species, and loss of hydrogen radical to give radical cation.28,53,54 The [M+2H]3+ ion in Figure 1d indicated that there was an electron-ionization process. The presence of the charged-reduced ion [M+2H]+ in EID of CSF114 and MUC5AC 3 shown in Figure 1c and d indicates that part of the high energy electron may be thermalized and captured by the precursor ion.53,54 This 10 ACS Paragon Plus Environment

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charge-reduced

ion

may

then

undergo

subsequent

dissociation

to

give

hot-ECD fragment ions.53-55 ECD (with 2 eV electron) and hot-ECD (with 10 eV electron) of [M+2H]2+ were performed for comparison (Figure S-2 a and b in Supporting Information).As shown Figure S-4, both ECD and hot-ECD results showed an intense peak of charge-reduced ions (~15% and ~35%). However, they produced little fragment ions, with only *c 16 -*c 18 , which provide limited information for peptide sequence and glycan group localization. Therefore, it is suggested that the fragments contributed by hot-ECD is not significant here. While EID produced a hybrid types of a-/b-/c-/y-/z-ions of the glycopeptides.56-58 In summary, the EID of glycopeptides were able to generate extensive backbone cleavage fragments with and without losing glycan moiety. This property allowed a fast and reliable localization of the glycosylation site. However, the possibility of acquiring sugar sequence in the glycan moiety can only be inferred from the EID spectra of other glycoconjugates (see below sections) because the model glycopeptides used in this study contained only a single Glc or GalNAc moiety.

CID and EID of Glycoalkaloids. Compared with the two glycopeptides, tomato glycoalkaloids contained more complex sugar residues. Figure 2 shows the CID and EID spectra of Esculeoside A and α-tomatine. The major dissociation channels in the CID of the [M+H]+ of α-tomatine and Esculeoside A were losses of small neutral 11 ACS Paragon Plus Environment

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molecules, including water in α-tomatine to provide ions at 1016 m/z and acetic acid in Esculeoside A to give ions at 1210 m/z. Both CID spectra showed a preferential cleavage on glycosidic bonds, producing Y and Z ions. Only one cross ring cleaved ion 2,4X 1 -CH 2 O was observed in the CID of Esculeoside A. Generally, EID produced more useful fragment ions than those of CID. EID included all cleavages induced in CID. EID generated extra multiple cleavages, including extensive cross-ring cleavages of every sugar unit to give a series of X ions. This finding provided important information on glycoalkaloids. Some characteristic ions of spirosolane aglycon moiety in α-tomatine, including C 11 H 18 NO+, C 8 H 16 N+, and C 6 H 12 N+, were produced. These ions were not observed in the EID of the [M+H]+ of Esculeoside A. This condition might be caused by the increased number of liable glycosidic bonds and an additional glucose unit attached to the other side of spirosolane aglycon moiety. However,

cleavages

[M-C 13 H 22 NO 8 ]+,

0,2

occurred

across

the

aglycon

ring

and

produced

X 0 -C 8 H 14 O 6 , and C 27 H 44 NO 3 + fragment ions. The presence of

these ions in the lower mass region were attributed to the cleavages in the aglycon entities. These ions provided useful information on the structures of the different types of glycosides.

CID and EID of Gangliosides. Ganglioside is a glycosphingolipid composed of ceramide and oligosaccharide, which contains one or more N-acetylneuraminic acids 12 ACS Paragon Plus Environment

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(sialic acid, Neu5Ac) linked to their sugar chains. GD1a and GD1b are structural isomers, which can be distinguished from each other according to the position of the two sialic acid residues. Due to the presence of acidic Neu5Ac units, gangliosides tend to form negative ions in the solution and are normally analyzed in a negative ion mode of MS analysis. The CID and EDD of the doubly deprotonated ion both produced diagnostic C 2α and B 2α ions in GD1a and B 2β ion in GD1b, which could be used to differentiate the isomers. Compared to the CID results, EDD provided more structural informative ions for deducing the glycan sequence and linkage information (Figures S-3 and S-4 in Supporting Information). Despite its low proton affinity, addition of metal adducts will allow analysis in positive mode. There has been demonstrations on the effects of different metal ions on the fragmentation of oligosaccharides.59-62 Here, the sodium salt was applied to convert the acidic ganglioside to singly sodiated precursor ions, which can be used for studying subsequent MS/MS analyses in positive ion mode. Figures 3a and b show that the CID spectra of sodium-ion-adducted GD1a and GD1b. Only two product ions were generated by the successive losses of two sialic acid moieties. The CID spectra were not able to facilitate the differentiation of the two isomers because the Y 4α /Y 2β and [Y 2β -Z 4 ]/[Y 4 -Z 2β] ions in the CID of GD1a, and Y 3β and Y 2β ions in the CID of GD1b had the same masses at 1568.8659 m/z and 1277.7705 m/z (sodium adducted). On the 13 ACS Paragon Plus Environment

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contrary, the EID of the same molecular ions produced spectra containing more structurally informative fragments, as shown in Figures 3c and d. Both EID and EDD could produce extensive fragment ions for ganglioside. However, the efficiency of EDD is lower than EID and EDD requires longer irradiation time than EID. Moreover, EDD only applied to multiply charged anion, EID could also be used to singly charged anion. The interpretation of these complex MS/MS spectra was facilitated by the use of van Krevelen diagram, which was an approach for elemental mapping and was commonly used to identify the major components of highly complex high-resolution electron impact ionization mass spectra,63,64 The van Krevelen was extended to identify the different classes of organic compounds in the high resolution ESI spectra.65–68 This approach was also used to assist the interpretation of the EID spectra of gangliosides. Figure 4 shows the van Krevelen diagram with information extracted from Figures 3c. The regional plots of the elemental compositions of the EID fragments according to their H/C ratios and O/C ratios clearly grouped the fragment ions into three regions. Regions A and C were identified as sugar-related and lipid-related fragment regions, respectively. Their positions in the van Krevelen diagram

were

further

confirmed

from

the

literature

about

lipids

and

carbohydrates.65–68 Fragment ions containing both sugar and lipid portions were plotted between the regions. The grouping of the structurally related MS2 fragment 14 ACS Paragon Plus Environment

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ions through the van Krevelen diagram facilitated the assignments of the ganglioside fragment ions at region B.

The EID spectra of both gangliosides produced a complete set of glycosidic Y-/Z-fragments, which were used to map the carbohydrate sequence and determine the monosaccharide compositions. Complementary B-/C-ions further affirmed the glycan linkage and branching information. The abundant cross-ring fragments (A-/X-ions) observed in EID were able to facilitate the localization of modifications and

establish

the

branching

pattern

within

individual

residues.

For

example, 1,4X 2α , 1,5X 3α , and 0,4X 3β observed in the EID of GD1b (Figure 3d) indicated that the linkage positions of the two sialic acid residues on the glycan chain differed from that of GD1a. Furthermore, EID spectra yielded specific diagnostic fragment ions, tri-saccharides B 3α and C 3α in GD1a (Figure 3c) and disaccharides B 2β and C 2β in GD1b (Figure 3d), thereby enabling clear differentiation between the two isomers.

Only extremely limited structural information could be extracted from the spectra for the hydrocarbon tails of the ceramide residue. A weak signal corresponding to G 18:0 ion indicated cleavage at the linkage between C 2 −C 3 of the sphingosine tail. Brodbelt et al.69 has applied UVPD for structural characterization of glycolipids and ganglioside. Although G ion and cleavages in the ceramide C-C and C-N bonds also observed, there were no extensive cleavages across the hydrocarbon tail. Kanea et 15 ACS Paragon Plus Environment

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al.70 studied the structures of glycerophospholipids by using EID and obtained a series of fragment ions corresponding to the successive cleavages along the hydrocarbon tails. We postulated that the absence of corresponding hydrocarbon cleavage signals may be attributed to the presence of weak glycosidic linkages which might lead to preferential dissociation and suppression of the cleavages at the somewhat stables phingosine tail. MS3 experiments were performed to test the hypothesis and to obtain relevant structural information. As shown in Figures 3a and b, the CIDs of GD1a and GD1b can generate strong fragment ion signal at 1277 m/z, and this signal corresponds to the loss of the two labile sialic acid residues. We attempted to perform EID experiments on these mass-selected CID fragment ions. Produced from the CID of GD1a, the EID MS3 spectrum of the aisalo-ganglioisde ion at 1277 m/z is shown in Figure 5. EID of the aisalo-ganglioisde ion generated glycosidic and cross-ring fragments. These findings were also obtained in previous spectra. O 18:1 was produced from the cleavage of the amide-linked fatty acid tail. Meanwhile, G 18:0 and O 18:0 provided specific structural information on the lengths of the two separate constituent hydrocarbon chains. A close inspection of the EID spectrum revealed a set of fragment ions from d1 to d14 (except d8 and d11). These fragment ions originated from successive cleavages along the ceramide lipid chain. An expanded region of the part of these product ions is shown in Figure 5. A mass difference of 26 Da between 16 ACS Paragon Plus Environment

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the d3 and d1 ions enabled the direct localization of the double bond in the ceramide chain. By combining the EID spectra of ganglioisde and aisalo-ganglioside, we were able to obtain a complete characterization of ganglioside, including both structural information on carbohydrate head and hydrocarbon tail.

CONCLUSIONS

Compared with CID, EID provided much more elucidative information for the characterization of different glycoconjugate structures. More structural informative fragments on glycan and aglycon portions were observed in all glycoconjugates studied. The fragments were then used to localize the glycosylation sites of glycopeptides. Localization was achieved by producing a series of fragment ions produced from an extensive cleavage of the peptide amide linkages regardless of the loss of the glycan groups. In the glycoalkaloid analysis, the presence of multiple cleavages in the aglycon moieties in the EID spectra enabled a detailed structural analysis of the different classes of glycosides. Additionally, nearly complete structural information were generated by the EID of gangliosides. This information included the fragments characterizing glycan linkages and composition and those used for characterizing lipid types and chain lengths and locating the C–C double bonds in the ceramide chain. The supplementary information provided by EID greatly facilitated the structural characterization of complex glycoconjugates. 17 ACS Paragon Plus Environment

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Supporting Information Pulse sequence of ECD/EID experiment, CID, ECD, EDD mass spectra, Summary of all the product ions, Tables

ACKNOWLEDGEMENTS Financial supports from the Research Grant Council of the Hong Kong Special Administrative Region (Research Grant Direct Allocation, Ref. 2060351), Natural Science Foundation of Shandong Province (ZR2017MB011) and National Natural Science Foundation of China (21205071) are gratefully acknowledged. REFERENCES (1) Glycoconjugates at the US National Library of Medicine Medical Subject Headings (MeSH). (2) Dennis, J.W.; Granovsky, M.; Warren, C.E. Bioessays. 1999, 21, 412-421. (3) Dwek, R.A. Chem. Rev. 1996, 96, 683-720. (4) Helenius, A. Mol. Biol. Cell. 1994, 5, 253-265. (5) Imperiali, B.; O’Connor, S.E. Curr. Opin. Chem. Biol. 1999, 3, 643-649. (6) Benting, J.H.; Rietveld, A.G.; Simons, K. J. Cell Biol. 1999, 146, 313-320. (7) Rudd, P.M.; Woods, R.J.; Wormald, M.R.; Opdenakker, G.; Downing, A.K.; Campbell, I.D.; Dwek, R.A. Biochem. Biophys. Acta. 1995, 1248, 1-10. (8) Rudd, P.M.; Endo, T.; Colominas, C.; Groth, D.; Wheeler, S.F.; Harvey, D.J.; Wormald, M.R.; Serban, H.; Prusiner, S.B.; Kobata, A.; Dwek, R.A. J. Mol. Biol. 1999, 293, 351-366. 18 ACS Paragon Plus Environment

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Figure Captions 2+

Figure 1. CID spectrum of [M+2H]

of glycosylated peptide (A) CSF114and (B) 2+

MUC5AC 3; EID spectrum of [M+2H]

of (C) CSF114 and (D) MUC5AC 3; -○

indicating the loss of H 2 O; triangle indicating the loss of NH 3 ; N-Acetylgalactosamine (GalNAc);

as

as Glucose (Glc); *product ions (blue) with

glycan retention.

Figure 2. CID spectrum of [M+H]+ of (A) α-tomatine and (B) Esculeoside A; EID spectrum of [M+H]+ of (C) α-tomatine and (D) Esculeoside A; -○ indicating the loss of H 2 O;CID and EID cleavages (blue); EID cleavages (red).

Figure 3. CID spectrum of [M+Na]+ of (A) GD1a and (B) GD1b; EID spectrum of [M+Na]+ of (C) GD1a and (D) GD1b; -○ indicating the loss of H 2 O; # indicating the loss of CO 2 ; [A–B] indicating internal fragments cleaving from A to B; fragments (blue) as diagnostic ions; Neu5Ac as N-Acetylneuraminic acid (sialic acid).

Figure 4. Van Krevelen plot for elemental data of EID fragments from ultrahigh-resolution mass spectrum of ganglioside

Figure 5. MS3 EID spectrum of 1277 m/z from CID of [M+Na]+ of GD1a; *marking the precursor ion.

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CID

CID

EID

EID

Figure 1

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(A) CID

(B) CID

(C)

(D)

EID

EID

Figure 2

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CID

CID

EID

EID

Figure 3

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Figure 4

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Figure 5

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TOC

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