Higher Energy Collisional Dissociation Mass Spectrometry of Sulfated

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Higher-energy collisional dissociation (HCD) Mass Spectrometry (MS) of sulfated O-linked oligosaccharides. Samah M. A. Issa, Varvara Vitiazeva, Catherine A. Hayes, and Niclas G. Karlsson J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00376 • Publication Date (Web): 02 Aug 2018 Downloaded from http://pubs.acs.org on August 3, 2018

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Journal of Proteome Research

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Higher-energy collisional dissociation (HCD) Mass Spectrometry (MS) of sulfated O-linked oligosaccharides. Samah M. A. Issa1*, Varvara Vitiazeva1*, Catherine A. Hayes1 and Niclas G. Karlsson1** 1

Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska

Academy, University of Gothenburg, University of Gothenburg, Box 440, Medicinaregatan 9A, 405 30 Gothenburg, Sweden *Joint first author **Corresponding author: [email protected] Tel: 46-31-7866528; Fax: 46-31-41608

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ABSTRACT Sulfation is the final decoration of mucin type O-linked oligosaccharides before mucins are released into the lumen of the gastrointestinal, respiratory and genital tracts. Since only a fraction of oligosaccharides undergo this type of modifications in the Golgi apparatus, sometimes also only by dedicated cells, the glycobiology of these low abundant sulfated oligosaccharides is often overlooked. At the same time, the technology to consistently identifying and characterize them has been lagging behind. In this report we adopted Higher-energy Collisional Dissociation (HCD) to characterize sulfated oligosaccharides from porcine gastric and human salivary MUC5B mucins. With this approach we could generate conclusive spectra up to nona-saccharides. Both singly and doubly sulfated oligosaccharides were characterized. By comparing the fragmentation of low mass fragments of m/z 100-320 with standards for 6-linked and 3-linked sulfate, it could be shown that characteristic fragmentation exists, verifying that porcine gastric mucin contains mostly 6-linked sulfate to GlcNAc while human MUC5B contains mostly 3-linked Gal. When performing ion trap MS2 fragmentation, these low molecular mass fragments are usually not detected. Hence, it can be concluded that in order to be able to address biological questions of sulfation, low mass fragments are important for assignment of sulfate position.

Key words: Glycosylation, Sulfation, Orbitrap, Mucin, Fragmentation, Sulfoglycomics, Glycomics, Olinked oligosaccharides, Graphitized carbon, LC-MS

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INTRODUCTION Development and evaluation of methods for consistent analyses of sulfation of oligosaccharides is the front line of the research in glycomics and glycoproteomics. The ability to identify sulfate location is a key issue in sulfoglycomics. With reports of the ubiquitous sulfation appearing, not only on mucous glycoproteins, but also in other areas of the human body including serum(1), white blood cells(2) and synovial fluid(3, 4), the interest to investigate its biological roles has increased. As sulfation is appearing on the non-reducing end of both N-linked and O-linked oligosaccharides it is appreciated that this is an important modification for modulating oligosaccharide interactions regardless whether this is on a mucosal surface, on the glycocalyx of white blood cells, if it is expressed by carcinoma tissue or is modulating the lubrication on articular cartilage. The potential biological importance of sulfation is in stark contrast to the few examples demonstrating its biological function. The earliest example includes modulating the half life of luteinizing hormones(5). Sulfation also play a role in the selectin mediated interaction, especially the fine tuning of L-selectin interaction to its preferred ligand GlcNAc-6-sulfated sialyl Lewis x mediating homing of lymphocytes and neutrophils to site of inflammation(6). Sulfation on 6-position of galactose residues together with sialylation have been proposed to be involved in interaction with Siglec-8(7, 8) . However, since the mouse paralog, SiglecF, does not require this sulfation(9), indicates that the absolute requirement for sulfation of the human Siglec-8 ligand is unclear. The HNK-1 ligand (CD57) HSO3-3GlcAβ1-3Galβ1-4GlcNAc-(10) contains a 3-linked sulfate and is expressed on NK cells and neural cell adhesion molecules on both glycolipids and glycoproteins. Apart from this example and despite that 3-linked sulfate is frequently detected (mostly on galactose), its biological functions remain unexplored. On mucosal surfaces, the sulfation (mostly 6 linked GlcNAc and 3 linked Gal) appears to be involved in the interplay with the microbiota, participating in interaction(11) and preventing degradation of the protective mucosal surface(12). The level of colon mucin sulfation has been suggested to be associated with ulcerative colitis(13).

The various approaches for analyses of sulfated oligosaccharides by MS has been thoroughly reviewed in 2011(14). One approach relies on fragmentation analysis of underivatised oligosaccharides in combination with pre-fractionating and/or hyphenated HPLC(15-17). Advances in sample preparation 3


after permethylation are now also producing consistent results for sulfated oligosaccharides. Fast scanning high resolution MS and MSn using orbitrap technology generates fragmentation that reliably allows location of the sulfation position within a permethylated monosaccharide residue(2, 18, 19). HCD fragmentation on orbitrap instruments has shown to generate low molecular fragments for sulfate localization(20). With sensitivity comparable with linear/Paul ion trap CID fragmentation, HCD appears to be beneficial for fragmentation of sulfated oligosaccharides, without sacrificing the informative low mass region (as in linear/Paul ion trap CID), required for confident assignment of sulfate position. In this paper we set out to investigate the use of HCD fragmentation of sulfated Olinked oligosaccharides (porcine gastric and human salivary mucins) in combination with graphitized carbon chromatography, in order to allow separation of individual sulfated oligosaccharides for HCD fragmentation analysis.

EXPERIMENTAL Materials and reagents: Porcine gastric mucins were purchased from Sigma-Aldrich (St. Louis, MO). MUC5B was isolated from human saliva by SDS-PAGE(21). Oligosaccharides were released from mucins blotted onto PVDF membranes using reductive β-elimination(22). Sulfated standards HSO36Galβ1-4GlcNAcβ1-azide (6’-O-sulfo LacNAc) and Galβ1-4(HSO3-6)GlcNAcβ1-ethyl amine (6-O-sulfo LacNAc) were generous gift from Professors James Paulson, Scripps Institute and 3- HSO3-3Galβ14GlcNAc (3’-sulfo-LacNAc) were obtained from Dextra (Reading, UK).

Porous graphitized carbon LC-MS LC-MS was performed using a home made 10 cm × 250 µm ID column packed with 10 µm PGC material (Thermo Fisher, San Jose, CA) and an Agilent 1100 Series HPLC (Santa Clara, CA) using a static split from 250 µL/min down to 5-10 µl/min and a linear gradient from 0 to 40 % acetonitrile for 45 min in 20 mM ammonium bicarbonate as been described(22). Oligosaccharides were detected and fragmented in negative ion mode using either a Thermo LTQ (ion trap CID)(22) or a Thermo Orbitrap LTQ XL (HCD)(20). Specific HCD settings required a minimal signal from survey scan of 20000, isolation width of m/z 4.0 of the 3 most intense ions and a normalized collision energy of 95.0%.

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Intense background signals lacking isotopes were identified in HCD spectra and subtracted as background electronic noise.

Interpretation of fragment data Fragmentation data from CID and HCD was interpreted manually. Annotation was aided by generation of theoretical fragments using Glycoworkbench(23). For comparison of low molecular fragments for sulfate position annotation, HCD spectra were exported from the automatically generated labeled peak list obtained by the Qual browser in the Xcalibur 2.2 software (Thermo Fisher). The mass range m/z 100-320 from the porcine gastric and MUC5B fragmented sulfated oligosaccharides was compared with the same range from the HCD spectra of the standards 6-O-sulfo LacNAc, 6’-O-sulfo LacNAc and 3’-sulfo-LacNAc using the dot-product function available in the R package OrgMassSpecR (http://orgmassspecr.r-forge.r-project.org).

RESULTS AND DISCUSSION Comparison of HCD and linear ion-trap CID MS2-spectra of sulfated O-linked oligosaccharides Due to the triple quadrupole like fragmentation in the HCD collision cell, we decided to investigate the use of HCD for the analysis of reduced O-linked glycans as an additional diagnostic tool to complement CID fragmentation of sulfated structures. Although CID in linear/Paul ion traps has progressed in resolving power and mass range, the low mass cut-oﬀ (which usually limits the MS/MS spectra range up to 30% of its precursor m/z) would result in the loss of useful diagnostic ions essential for sulfate localization. Porcine Gastric Mucin (PGM) O-linked oligosaccharides were utilized to assess the use of HCD for characterization of sulfated oligosaccharides. These glycans have been found to be sulfated primarily with a 6-linkage to HexNAc (GlcNAc)(16). The HCD and CID fragmentation spectra of three 6-linked sulfated structures to the HexNAc from PGM are shown in Figure 1. The presence of the sulfate on each compound was confirmed from the high accuracy parent and fragment ions produced. The fragments in Figure 1A (HCD and CID) at m/z 505 and m/z 282 are due to a loss of the terminal Hex unit and the Hex-HexNAcol, respectively, which is indicative of sulfation on the HexNAc. In the HCD spectra, it was interesting to observe that independently of the presence of the sulfate in a terminal position or as an internal monosaccharide unit, the

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fragmentation process progressed all the way down to the single sulfated HexNAc unit of m/z 282. (Figure 1B and C). However, by ion trap CID, the low mass range cut-off prevented this to be detected. Consequently, it was obvious that analyzing sulfated O-glycans by CID in the ion trap (resonant excitation mediated collision) for high molecular mass structure resulted in not only the loss of the low mass m/z 282 fragment, but also other low molecular mass sulfate containing fragments (Figure 1). Even though this deficit by ion trap CID may to some extent be resolved by the multiple activation step capacity in the ion trap technology(24), the slow scan rate of MSn experiments in ion traps(25) makes multistage fragmentation difficult to perform for LC-MS. What was also obvious when comparing HCD with ion trap CID (Figure 1), was the HCD capability of producing consistent and efficient fragmentation throughout the whole mass range, despite an increasing molecular mass of the sulfated oligosaccharide. This HCD fragmentation feature carried right down through to the lower m/z fragments (lost in ion trap CID) and provided valuable m/z fragments that could be used for the sulfate localization. The characteristic fragment ion of m/z 139 (0,4AGlcNAc) and m/z 199 (C/0,2XGlcNAc) for a 6-linked sulfate to the HexNAc have been reported previously(26). In addition, m/z 181 (B/0,3XGlcNAc) and other low intensity fragments were found to be present. These low mass fragments were predominantly cross-ring and internal fragments of the pyranose ring. Sulfate position directed fragmentation in HCD The data from PGM showed that extended mass range in MS/MS scan mode coupled with higher collision energy of the HCD cell resulted in a number of smaller (m/z < 380) observable A-cross ring fragments as described above. These fragments have the potential to provide a mean to determining/confirming the sulfate position of O-linked oligosaccharides. Hence, we investigated if HCD could be utilized to distinguish between various types of sulfation, especially since it appeared that low molecular mass fragment arose from fragmentation of the sulfated monosaccharide unit. In order to investigate this, we also used human salivary Muc5B oligosaccharides that have been shown to have high amount of a second common sulfation of O-linked oligosaccharides, i.e. 3-linked sulfation on Gal. The HCD fragmentation patterns of two isomers with the [M – H]--ion of m/z 975 corresponding to the composition Hex2HexNAc1Sulfate1Fuc1HexNAcol from PGM (Fig 2A) and MUC5B (Fig 2B) are shown. As with 6-linked sulfation of GlcNAc, the charge remote fragmentation of 3-linked sulfation on Gal in HCD appeared to funnel down to the sulfate residue with low molecular fragments

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prominent in the m/z < 380 area. The most distinguishing feature in this area in Figure 2B is the sulfated Hex (Gal) residue (m/z ion of 241), which was the result of a single cleavage event; whereas the fragment in Figure 2A at m/z of 282 from a double cleavage event was instead indicative of sulfation on the HexNAc. In order to asses if the data in the low mass region (< m/z 380) was indeed carrying information about sulfation we performed a thorough investigation of the low mass fragment, by matching theoretically calculated cross ring fragments with observed fragments. In Figure 3 it can be seen that the majority of assigned peaks did indeed carry the negatively charged sulfate group. There were 10 out of 15 assigned fragments for the 6-linked isomer from PGM and 7 out of 11 fragments for MUC5B that were found to be carrying the sulfate group. What was also important was that 6-linked sulfates on the HexNAc and 3-linked sulfates on the Hex (Figure 2B) displayed different diagnostic ions relevant to the specific sulfate position. For instance, the fragment ion at m/z of 199 (from a combined glycosidic/cross ring fragmentation), or lack thereof, was found to be useful as a diagnostic ion for locating the primary position of the sulfate group. Its presence indicated that the sulfate was linked to C-6 (Figure 3A). Vice versa, its absence/suppression suggested a C-3 sulfation (Figure 3B). Comparison of low molecular weight fragmentation pattern to identify type of sulfation In order to investigate if the HCD fragmentation of sulfated oligosaccharides provided a consistent fragmentation pattern in the low m/z region to allow assignment of the sulfation, we generated HCD spectra from sulfated N-acetylactosamine standards and compared with the fragments from oligosaccharides from PGM and MUC5B. Rather then focus on individual diagnostic ions, the fragments from the low mass region and their intensities were selected and compared to the same region of sulfated structures from PGM (mostly 6-linked GlcNAc sulfation) and MUC5B (mostly 3linked Gal sulfation) (Fig 4). Using state-of-the-art MS spectral comparison algorithm (normalized dot product)(27), it could be shown that the scoring ranked the type of sulfation on the oligosaccharides from each tissue according the reported sulfation from these tissue (Table 1). The only exceptions to this were the presence of low abundant pentasaccharide ([M – H]--ion of 975) a hexasaccharide ([M – H]--ion of 1121) and a heptasaccharide ([M – H]--ion of 1324) from Muc5B that were assigned as a 6linked sulfated GlcNAc structures rather than 3-linked Gal. This data shows that that MUC5B contains

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a mixture of 3-linked Gal and 6-linked GlcNAc sulfated oligosaccharides. In most cases the scoring was in the range of 0.65-0.85 but in some exceptional cases it was as low as 0.43-0.47 for some of the doubly charged large molecular mass structures. Despite these lower scorings, the higher rank and the presence of distinct diagnostic ions (Fig 5A), indicated that 6-GlcNAc sulfation was present rather than 3-linked Gal in these structures. We also found that when mixtures of structures were present in the HCD spectra (e.g. as with the m/z 975 from MUC5B), the data was inconclusive whether 3-linked Gal, 6-linked Gal or 6-linked GlcNAc was present (Table 1). This shows the importance of efficient isomeric separation before HCD. The similarity between 6-linked Gal and 6-linked GlcNAc cross ring fragments was reflected in the fragmentation of the PGM data, where the highest ranked 6-linked GlcNAc is mostly followed by the second ranked 6-linked Gal. However, the high intense B-type ion of m/z 282 (sulfated GlcNAc) in the former versus the B-type ion m/z 241 (sulfated Gal) in the latter, allows the dot product to make a clear distinction between the two types of sulfation (Figure 4A versus 4C).

HCD for characterization of complex sulfated structures We further explored the capacity of HCD to allow us to characterize sulfated oligosaccharides that are currently challenging using CID/ion trap, in particular high molecular mass structures and structure with multiply charged residues. In Figure 5A the HCD spectra of a branched core 2 nonasaccharide containing 6-sulfation and blood group H from PGM is shown. Previous attempts using ion trap CID to sequence a structure this size would have provided limited information regarding the structure and the location of the sulfate. The evenly distributed intensities of the fragments across the spectrum were found to be dominated primarily by Y series remote from sulfate. The fragments in the lower m/z range indicated sulfation on the 6-position of a single HexNAc. The sulfated monosaccharide residue on this structure was found to be a terminating residue on the core 2 6-C branch. There were no B fragments detected that suggested there was an extension of this branch. With a high quality HCD spectra as generated in Figure 5A, we could be confident that other potential non detected Bions such as m/z 444 (sulfated LacNAc) and m/z 590 (sulfated fucosylated LacNAc), would be of medium or high intensity, if co-eluting isomers were present. The even distribution of glycosidic

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fragment intensities as is demonstrated by HCD, increases confidence in assignment of sequences. However, co-eluting low abundant isomer makes interpretation of spectra difficult or impossible, where potentially only major isomer can be assigned, as illustrated in the assignment of structures in Table 1 of the [M – H]- - ions of 1121 (RT 20.45 min ) and 975 (RT 20.53 min) from Muc5B.

Figure 5B shows the fragmentation of an oligosaccharide from MUC5B containing both a sulfated and sialylated residue. Typically, such structures are very difficult to analyze by MS due to the highly labile sialic acid. Generally, the most dominant fragment ion for these types of structures is the neutral loss of the sialic acid(15). Also by HCD this is the dominant fragmentation. The only detected sialic acid containing HCD fragment was the monosaccharide residue, while most of the other fragments contained only the sulfate group. The HCD fragments detected were mainly Y fragments, and could confidently validate the assigned sequence of the oligosaccharide after the loss of the sialic acid residue. The MS/MS spectrum in the lower range also shows fragment ions highlighting the position of the sulfate on the C-3 of a Hex residue.

Conclusions HCD fragmentation of sulfated mucin oligosaccharides in combination with graphitized carbon LC-MS provides an efficient and informative way to assess the type of sulfation that is available in a sample. Specific fragmentation in the low mass region provides information that allowed distinction of the most prominent type of mucin oligosaccharide sulfation i.e. 6 -linked sulfation of Gal and GlcNAc and 3-linked sulfation of Gal. Fragment ions (m/z 139, 181, 199) with their intensities gave confident assignment of 6-linked sulfation, while 3-linked sulfation was characterized by only a single m/z 139 fragment ion. The assignment of the type of sulfation in complex mixtures relies on the selectivity of the LC for oligosaccharides. Graphitized carbon has shown to be able to separate different sulfated isomers(15, 28), but the possibility of co-elution can never be excluded The fact that the absence of large intense fragment ions m/z 181 and 199 are indicative for 3 sulfated Gal and the same ions with high intensity indicate 6-sulfation (Gal, distinguished by B-ion of 241 or GlcNAc, B-ion of 282), using the normalized dot product to identify sulfate position in spectra from isomeric mixtures represents a challenge., The fact that also 4-sulfated GalNAc also give rise to intense m/z 181 and 199

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(supplementary Figure 1), illustrate that implementation of normalized dot product for automatic interpretation needs to be validated also for this location. For instance, 4 sulfated LacdiNAc has been shown to be present on N-linked glycans(5). The fragmentation pattern of sulfated oligosaccharides in the low mass region did not appear to be dependent on whether the sulfate was terminal or internal GlcNAc as illustrated in Table 1. We could not identify any structures in the samples we analyzed that contained an internal 6 sulfated and fucosylated (3 or 4 position) GlcNAc residue (i. e. a 6-Sulfo Lewis a/x epitope). However, the fragmentation of 3-sulfated terminal Gal appeared to be independent of the fucosylation status of the adjacent GlcNAc (i.e. a 3´-Sulfo Lewis a/x epitope) residue as illustrated by examples among the MUC5B oligosaccharides.

In summary, using the normalized dot product, assignment of 3 sulfation of Gal and 6 sulfation of GlcNAc could be performed on mucin oligosaccharides from PGM and MUC5B. SUPPORTING INFORMATION: The following supporting information is available free of charge at ACS website http://pubs.acs.org Supplementary Figure 1. HCD fragmentation of chondroitin sulfate dervived 4 sulfated disaccharide. Acknowledgements This work was supported by the Swedish Research Council (621-2013-5895), Kung Gustav V:s 80-års foundation, Petrus and Augusta Hedlund’s foundation and the AFA insurance research fund (dnr 150150). The LTQ-Orbitrap mass spectrometer was obtained by a grant from the Knut and Alice Wallenberg foundation (KAW2007.0118) and the LTQ ion trap from the Swedish Research Council (342-2004-4434). Professor James Paulson is gratefully acknowledged for providing sulfated oligosaccharide standards. FIGURE LEGENDS

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Figure 1 Comparison of HCD and CID fragmentation for characterization of sulfated O-linked oligosaccharides. (A) Core 2 trisaccharide sulfated on the C-6 position of the HexNAc. (B) Core 2 tetrasaccharide sulfated on the C-6 position of the HexNAc and fucosylated only on the Hex residue attached to the C-3 branch of the HexNAcol. (C) Core 2 hexasaccharide sulfated on the C-6 position of the HexNAc and fucosylated on both the C-3 and C-6 branch of the HexNAcol. The MS spectra in blue and red represent the characteristic fragmentation of the HCD and the CID cell, respectively. Oligosaccharides were released from PGM. Samples were analysed by LC-MS using graphitized carbon and RT recorded in Table 1.

Figure 2 HCD tandem mass spectra of two O-linked oligosaccharide isomers with [M - H]- -ion of 975 identified by LC-MS2 exemplifying 3-linked and 6-linked sulfation. (A) A core 1 blood group H pentasaccharide from PGM sulfated on the 6 position of the HexNAc and (B) a core 2 blood group H pentasaccharide of MUC5B sulfated on the 3 position of the Hex. For key of monosaccharide residue symbols, see Figure 1.

Figure 3 Identification of sulfate containing fragments of 3-linked Gal and 6-linked GlcNAc sulfated O-linked oligosaccharides. Zoomed in region between m/z 100-400 of Figure 2. The data shows that the HCD fragmentation of 6-linked (A) and 3-linked (B-inverted). The cross ring fragments are most predominant fragments containing the sulfated residues. For key of monosaccharide residue symbols, see Figure 1.

Figure 4 Scoring of HCD spectra of PGM and MUC5B oligosaccharides using standards for 3-linked and 6-linked sulfation. Examples of spectral matching and scoring of the m/z 100-320 region of 3 Gal sulfated oligosaccharide from MUCB ([M – H]--ion of 1324, RT 14.17 min ) with 3’-SulfoLacNAc standard (A) and 6 GlcNAc sulfated oligosaccharide from PGM (M – H]--ion of 1178, RT 21.72 min) with 6-SulfoLacNAc(B). In C the lower matching and scoring of the 3 linked Gal structure in A with the 6´-SulfoLacNAc standard is shown. For key of monosaccharide residue symbols, see Figure 1.

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Figure 5 Characterization of challenging sulfated oligosaccharides. (A) shows a HCD spectrum of a blood group H containing oligosaccharide with an [M – H]-- ion of m/z 884. (B) shows the [M – H]-- ion of m/z 1120 of a sialylated core 2 structure. In both cases distinct fragmentation in the lower mass region allowed confident assignment of the sulfate to a 3 position of a Hex residue (Table 1).

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transferase-deficient mice highlights differences compared with human MUC2. Glycobiology 2012, 22, (8), 1128-39. Thomsson, K. A.; Karlsson, N. G.; Hansson, G. C., Liquid chromatography-electrospray mass spectrometry as a tool for the analysis of sulfated oligosaccharides from mucin glycoproteins. J Chromatogr A 1999, 854, (1-2), 131-9. Wheeler, S. F.; Harvey, D. J., Extension of the in-gel release method for structural analysis of neutral and sialylated N-linked glycans to the analysis of sulfated glycans: application to the glycans from bovine thyroid-stimulating hormone. Anal Biochem 2001, 296, (1), 92-100. Cheng, C. W.; Chou, C. C.; Hsieh, H. W.; Tu, Z.; Lin, C. H.; Nycholat, C.; Fukuda, M.; Khoo, K. H., Efficient Mapping of Sulfated Glycotopes by Negative Ion Mode nanoLC-MS/MS-Based Sulfoglycomic Analysis of Permethylated Glycans. Anal Chem 2015, 87, (12), 6380-8. Hsiao, C. T.; Wang, P. W.; Chang, H. C.; Chen, Y. Y.; Wang, S. H.; Chern, Y.; Khoo, K. H., Advancing a High Throughput Glycotope-centric Glycomics Workflow Based on nanoLC-MS(2)-product Dependent-MS(3) Analysis of Permethylated Glycans. Mol Cell Proteomics 2017, 16, (12), 22682280. Kenny, D. T.; Issa, S. M. A.; Karlsson, N. G., Sulfate migration in oligosaccharides induced by negative ion mode ion trap collision-induced dissociation. Rapid Commu Mass Spectrom 2011, 25, (18), 2611-2618. Karlsson, N. G.; Schulz, B. L.; Packer, N. H., Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn. J Am Soc Mass Spectrom 2004, 15, (5), 659-72. Hayes, C. A.; Nemes, S.; Issa, S.; Jin, C.; Karlsson, N. G., Glycomic work-flow for analysis of mucin O-linked oligosaccharides. Methods Mol Biol 2012, 842, 141-63. Damerell, D.; Ceroni, A.; Maass, K.; Ranzinger, R.; Dell, A.; Haslam, S. M., The GlycanBuilder and GlycoWorkbench glycoinformatics tools: updates and new developments. Biol Chem 2012, 393, (11), 1357-62. Karlsson, H.; Larsson, J. M.; Thomsson, K. A.; Hard, I.; Backstrom, M.; Hansson, G. C., Highthroughput and high-sensitivity nano-LC/MS and MS/MS for O-glycan profiling. Methods Mol Biol 2009, 534, 117-31. Wong, S. H.; Cooks, R., G. C, Ion Trap Mass Spectrometry. Curr. Separations 1997, 16, (85), 1-8. Karlsson, N. G.; Karlsson, H.; Hansson, G. C., Sulphated mucin oligosaccharides from porcine small intestine analysed by four-sector tandem mass spectrometry. J Mass Spectrom 1996, 31, (5), 560-72. Campbell, M. P.; Nguyen-Khuong, T.; Hayes, C. A.; Flowers, S. A.; Alagesan, K.; Kolarich, D.; Packer, N. H.; Karlsson, N. G., Validation of the curation pipeline of UniCarb-DB: building a global glycan reference MS/MS repository. Biochim Biophys Acta 2014, 1844, (1 Pt A), 108-16. Estrella, R. P.; Whitelock, J. M.; Packer, N. H.; Karlsson, N. G., Graphitized carbon LC-MS characterization of the chondroitin sulfate oligosaccharides of aggrecan. Anall Chem 2007, 79, (10), 3597-606.

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Table 1 Sulfated oligosaccharides identified from Human Salivary MUC5B and Porcine Gastric Mucin (PGM) including normalised dot product score compared to standards with 3-linked Gal and 6-linked GlcNAc in the m/z 100-320 mass range. Assignments of structures are based on fragmentation as well as known biosynthetic pathways for linkage position and configuration. Human Salivary MUC5B Mol. ion

RT min

Structure

Porcine Gastric Mucin (PGM) RT Structure min

Mol. ion a

0.46

0.79

667.20

1-

16.09

0.30

0.89

0.41

12.71

0.69

0.41

0.65

667.20

1-

17.47

0.24

0.83

0.35

13.25

0.86

0.38

0.77

813.30

1-

29.91

0.25

0.87

0.35

1-

14.46

0.89

0.36

0.75

884.28

2-

27.7

0.37

0.47

0.30

1324.43

1-

15.30

0.40

0.86

0.39

884.28

2-

30.7

0.33

0.43

0.30

782.72

2-

15.19

0.71

0.34

0.65

975.30

18.22

0.44

0.84

0.55

1178.38

1-

16.15

0.65

0.40

0.61

975.30

1-

19.23

0.38

0.88

0.43

1178.37

1-

21.06

0.86

0.40

0.77

975.30

1-

28.39

0.34

0.77

0.35

1032.32

1-

16.44

0.85

0.39

0.76

1121.36

1-

20.28

0.45

0.79

0.52

709.69

2-

19.05

0.66

0.50

0.66

1178.38

1-

21.29

0.39

0.75

0.47

464.11

1-

9.91

667.19

1-

829.24

1-

1324.43

0.83

16


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1120.33

18.92

0.72

0.40

0.72

1178.38

1-

21.72

0.31

0.89

0.39

628.67

2-

19.60

0.74

0.43

0.69

1178.38

1-

24.39

0.34

0.88

0.41

1121.35

1-

19.97

0.91

0.31

0.75

1324.44

1-

23.17

0.44

0.69

0.49

1121.35

1-

20.45

0.76

0.63

0.73

1324.44

1-

25.11

0.36

0.84

0.42

975.30

1-

20.18

0.81

0.41

0.75

1324.44

1-

25.92

0.45

0.72

0.51

0.69

0.64

0.70

b

b

975.30

1-

20.53

a

Bold indicate consistence between highest normalized dot product score and manual assigned position. Manual interpretation indicate that 3-linked Gal component co-elute in the LC-MS.

b

17



Figure 1


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Figure 2



Figure 3


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Figure 4



Figure 5


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Page 23 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41


For TOC only

p

itra b r O D C H

CID Ion t

rap


Higher Energy Collisional Dissociation Mass Spectrometry of Sulfated

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