We report collision cross sections (CCS) of high-mannose N-glycans as [M + Na]+, [M + K]+, [M + H]+, [M + Cl]-, [M + H2PO4]- and [M − H]- ions, measured by drift tube (DT) ion mobility-mass spectrometry (IM-MS) in helium and nitrogen gases. Further analysis using traveling wave (TW) IM-MS reveal the existence of distinct conformers exclusive to [M − H]- ions

Benesch, J.

Harvey, D.

Pagel, K.

Struwe, W.

English

MPG.PuRe

AnalystCOMMUNICATIONCite this: Analyst, 2015, 140, 6799Received 1st June 2015,Accepted 6th July 2015DOI: 10.1039/c5an01092fwww.rsc.org/analystCollision cross sections of high-mannoseN-glycans in commonly observed adduct states –identification of gas-phase conformers unique to[M − H]− ions†W. B. Struwe,*a J. L. Benesch,a D. J. Harveyb and K. Pagel*c,dWe report collision cross sections (CCS) of high-mannose N-glycans as [M + Na]+, [M + K]+, [M + H]+, [M + Cl]−, [M + H2PO4]−and [M − H]− ions, measured by drift tube (DT) ion mobility-massspectrometry (IM-MS) in helium and nitrogen gases. Further analy-sis using traveling wave (TW) IM-MS reveal the existence of distinctconformers exclusive to [M − H]− ions.N-glycans represent the most common carbohydrate proteinposttranslational modification and are attached to the proteinbackbone via asparagine residues. The extent of protein N-gly-cosylation in eukaryotes has been estimated up to 50% 2 andthe involved carbohydrates are important for protein structure,signalling, protein–protein recognition and protection fromproteolysis. N-glycan structures contain a common core con-sisting of two N-acetylglucosamine (GlcNAc) residues at thereducing end (termed the chitobiose core) and three additionalmannose (Man) residues. Individual structures are extendedfrom the mannose residues and form elaborate branchedstructures; characterized as high-mannose, hybrid or complextype.Due to the complexity inherent in their structure, thecharacterization of glycans remains challenging. Typically,their analysis relies on liquid chromatography techniques(with or without exoglycosidase digestions) for separation ofcomplex mixtures and/or tandem mass spectrometry (MS) toproduce fragment ions that facilitate structural assignments.3,4A more recent approach is the application of ion mobilitycoupled to mass spectrometry (IM-MS) in which glycan mix-tures are separated in the gas phase. The use of IM-MS in gly-comics studies is still comparatively limited, yet several reportshave demonstrated its potential using both travelling-wave(TW) and drift-tube (DT) IM-MS instruments.5–12 The empha-sis of these studies has been on the separation of isomersfrom synthetically derived oligosaccharides or N-glycan mix-tures purified from glycoprotein standards with alkali adductions being interrogated in the majority of cases.5,6,10–12 Overrecent years, we have explored IM-MS of negative glycan ionsfor both deprotonated or phosphate adducts. Using glycansreleased from biologically derived material, we demonstratedthe capability of negative polarity IM-MS to differentiate glycanisomers in complex mixtures6,8 and to extract glycan ions fromnon-carbohydrate material in the sample.7IM separates ions based on the time required to traverse aregion of inert neutral gas under the influence of a weak elec-tric field. The separation is driven by differences in mass,charge, size and shape of each analyte and thereby providesinformation about the underlying three-dimensional structure.IM-MS data typically consists of mass-to-charge values (m/z)and arrival times which can be further converted into orienta-tionally averaged collision cross sections (CCS).8,9,13 TheseCCSs are intrinsic to a particular glycan and are influenced byboth the ionic state (i.e. positive/negative mode and adduction)as well as the particular drift gas (commonly helium or nitro-gen). In a successful IM-MS glycomics experiment it can there-fore be crucial to simultaneously consider multiple adductstates, instrument polarities and IM gases.CCS values calculated from theoretical structures obtainedby molecular dynamic simulations (MD) have been comparedto experimental CCS data of native oligosaccharides11 and per-methylated N-glycans10 as [M + Na]+ and [M + 2Na]2+ adducts,respectively. In both cases theoretical and experimental valueswere comparable providing insight into the gas-phase struc-ture of the glycan adduct ions. These studies are valuable, butraise the questions as to whether, and how other adduct typesalter the CCSs. In this context protonated and deprotonatedions are of particular interest, since the location of charge mayvary, making MD simulations rather challenging. The simplest†Electronic supplementary information (ESI) available: Detailed methodsincluding instrument acquisition settings and complete list of CCS valuesdescribed in the main text. See DOI: 10.1039/c5an01092faDepartment of Chemistry, University of Oxford, Oxford, UK, OX1 3TA.E-mail: weston.struwe@chem.ox.ac.ukbDepartment of Biochemistry, Oxford Glycobiology Institute, University of Oxford UK,OX1 3QU, UKcFritz Haber Institute of the Max Planck Society, Faradayweg 4-6, 14195 Berlin,GermanydInstitut für Chemie und Biochemie der Freien Universität Berlin, Takustr. 3, 14195Berlin, Germany. E-mail: kevin.pagel@fu-berlin.deThis journal is © The Royal Society of Chemistry 2015 Analyst, 2015, 140, 6799–6803 | 6799Open Access Article. Published on 07 July 2015. Downloaded on 09/02/2016 13:53:40. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal | View Issueoligo-mannose structure Man3GlcNAc2 (or “Man3”, denotingthe number of mannoses) for example carries 15 hydroxylgroups, all of which are potential sites for deprotonation.Although quantum chemical approaches may help to predictthe location of the charge, and the three-dimensional struc-ture, the computational resources required are very demandingand therefore often not practical for investigating a largevariety of glycan structures. The separation power of IM restssolely in differences in glycan-adduct gas-phase structures andso a comprehensive study examining the CCSs of pure, knownstandards in all commonly observed ion states will aid theutility of the method and facilitate further development.Here we report absolute DTCCS values of pure, synthetically-derived high-mannose N-glycans in six commonly observedion adduct states ([M + H]+, [M + Na]+, [M + K]+, [M − H]−, [M +Cl]− and [M + H2PO4]−) using helium and nitrogen drift gases(Fig. 1). The investigated N-glycan standards were purchasedfrom Dextra Laboratories (Reading, UK) and absolute DTCCSmeasurements were performed using a modified SynaptHDMS fitted with a linear drift tube as described previously.13Glycan adducts were generated by the addition of respectivesalt solutions to promote adduct formation.The absolute DTCCS values of the investigated high-mannose structures ranged from 190 Å2 to 355 Å2 in heliumand 269 Å2 to 455 Å2 in nitrogen drift gases. In general, theDTCCSs of proton, sodium and potassium adducts are rathersimilar especially so for the larger glycans Man8 (DTCCSN2 (H)413 Å2, (Na) 415 Å2, (K) 415 Å2), Man9 (DTCCSN2 (H) 436 Å2,(Na) 435 Å2, (K) 437 Å2) and Man9Glc (DTCCSN2 (H) 453 Å2,(Na) 453 Å2, (K) 454 Å2). This is consistent with the specificcation having only minimal influence on the overall gas-phaseconformation. Molecular dynamics simulations of [M + Na]+ions have shown that glycans tend to “wrap” around the metalcation encouraging more compact gas-phase ions10,11 opposedto possible conformations where the different branching armsof the glycan are extended away from one another. Interest-ingly [M + H]+ ions had similar DTCCSs to sodium and potass-ium adduct ions yet the effect of protonation is unknown. It islikely that protons are located on nitrogens of N-acetylglucosa-mine residues. As these moieties are exclusively present in thechitobiose core, this would restrict charge effects in gas-phasestructure formation. Overall, the CCS differences between pro-tonated and cationated glycans are modest which suggests thatthey adopt rather similar gas-phase structures.When examining positive ions we find that the similarityin CCS values of the different adducts/protomers contrastsconsiderably to negative ion DTCCS measurements where[M − H]− values were generally significantly smaller than thosefor chloride and phosphate anion adducts most notably forthe smallest and largest N-glycans investigated. For example,the deprotonated Man3 DTCCSN2 is 269 Å2, the chloride adduct279 Å2 (3.8% larger) and the phosphate adduct 290 Å2 (7.6%larger). Similarly, the Man9Glc DTCCSN2 values for [M − H]−(439 Å2) were significantly smaller compared to [M + H2PO4]−(455 Å2), [M + H]+ (453 Å2) and [M + K]+ (454 Å2) adducts.Unlike positive ions where the addition of a proton shows aminimal effect to the CCS (<1% change for higher massglycans) compared to cationated glycans, where the removal ofa proton significantly affects CCSs for high-mannose glycanstructures.To understand why CCS values of deprotonated ions differsignificantly to other negative as well as positive ions we inves-tigated synthetic standards as [M + H]+, [M − H]−, [M + Cl]−and [M + H2PO4]− ions by TW IM-MS using a Synapt G2-Siinstrument which has higher IM resolution than the DTIM-MS instrument used for absolute DTCCS measurements.The extracted arrival time distributions (ATDs) of [M − H]−ions of the synthetically-derived glycans show at least two dis-tinct features for Man5, Man6, Man9 and Man9Glc structuresthat were absent in the respective [M + H]+ ions (Fig. 2).Fig. 1 Absolute collision cross sections (DTCCS) of N-glycan standardsmeasured in nitrogen and helium as [M + H]+, [M + Na]+, [M + K]+, [M −H]−, [M + Cl]− and [M + H2PO4]− adducts using drift tube ion mobility-mass spectrometry. For ATDs that exhibit multiple features the mostabundant feature was used for CCS determination. Mannose residuesare shown in green circles, N-acetylglucosamine residues are bluesquares, and glucose residues are blue circles. Glycosidic linkages arebased on the Oxford Glycan Nomenclature.1Communication Analyst6800 | Analyst, 2015, 140, 6799–6803 This journal is © The Royal Society of Chemistry 2015Open Access Article. Published on 07 July 2015. Downloaded on 09/02/2016 13:53:40. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineThe shape and relative intensity of these distributions are notaffected by activation until the threshold of dissociation isreached. In addition, the DTCCS and TWCCS values of [M − H]−ions are systematically lower than those of [M + H]+ ions (Fig. 1and 2 and ESI Table 1†) for which the nearly Gaussian shapeof the ATDs suggest the existence of only one species. Chlorideand phosphate adducts ATDs also had single species (ESIFig. 1†). Interestingly, the Man7 and Man8 [M − H]− ATDpeaks are also nearly Gaussian but the estimated TWCCSs werelower (376 Å2 and 406 Å2) than those of protonated ions(392 Å2 and 421 Å2).Multiple features in the ATDs could in principle arise fromstructural isomers or distinct gas-phase conformers that areonly detectable as [M − H]− ions. It is well documented thatCID of negative ions generates informative fragment ionsspecifically cross-ring (A-type) and D-type cleavages that can beused to differentiate high-mannose isomers.14 MS/MS of [M −H]− ions with collision-induced dissociation (CID) applied inthe transfer region (i.e. MS/MS after IM separation) showedthat the doublet peaks of Man5, Man6 and Man9 yield similarfragmentation patterns (Fig. 3). The CID spectra of the Man5ATD peaks 1 and 2 match and the D′ (m/z 323), D (m/z 657) aswell as cross ring A fragments (m/z 545 and 575) verify theexistence of a single structure. Similarly, for Man6 we observedthe same D-type fragment series, which indicates that theadditional mannose residue is on the 3-arm. This is further-more supported by the occurrence of characteristic 2,4A4/Y4βfragments m/z 869. The CID spectra of both Man9 ATDs alsoindicate a single isomer that is commonly observed on eukary-otic glycoproteins.15 Ions at m/z 971 (D), 827 (D-18), 809 (B3α)and 485 (D′) confirm this structure with five mannose residueson the 6-arm and three mannose residues substituted on the3-arm. However, some ions (e.g. the parent ion and the m/z 971D ion) are less abundant in ATD peak 2. It is unclear why theparent ions intensities differ here as equivalent CID voltageswere applied. A possible explanation is the difference in sizeand CCS of both conformers, which could lead to considerablydifferent internal energy deposition during CID.In order to investigate whether these conformations couldbe impurities deriving from the synthesis of the syntheticglycans, we examined N-glycans released from the glycoproteinstandards porcine thyroglobulin, bovine ribonuclease B(RNase B) and chicken ovalbumin, which have been studiedpreviously by IM-MS as [M + Na]+ and [M + H2PO4]− ions.8,9The presence of multiple Man5, Man6 and Man9 [M − H]−Fig. 3 Negative ion MS/MS spectra of synthetically derived Man5, Man6and Man9 [M − H]− ions. Fragmentation is identical between ATD peaks1 and 2 from each sample confirming the presence of a single structure.Fig. 2 Arrival time distributions (ATDs) of synthetic high-mannoseN-glycans as [M − H]− and [M + H]+ ions. Multiple features are observedin the ATDs of Man5, Man6, Man9 and Man9Glc [M − H]− ions, whichpoints to the coexistence of multiple conformers. Measurements wereperformed using a TW IM-MS instrument.Analyst CommunicationThis journal is © The Royal Society of Chemistry 2015 Analyst, 2015, 140, 6799–6803 | 6801Open Access Article. Published on 07 July 2015. Downloaded on 09/02/2016 13:53:40. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlineconformers was observed in all cases (Fig. 4). However [M +Cl]− and [M + H2PO4]− ion ATDs indicated single conformers(ESI Fig. 2–4†) although the presence of structural isomers,particularly Man5 from RNase B16 may affect ATD peak widths.Interestingly the relative intensity of Man5, Man6 and Man9[M − H]− conformers remained consistent between samples(i.e. ATD peak shapes). For example, the most abundant Man5conformer had a lower DTCCS (thyroglobulin 321 Å2, RNase B319 Å2, ovalbumin 322 Å2, synthetic 318 Å2) than the secondspecies. This consistent ratio was also true for Man9 butthe opposite was seen for Man6 where the most abundant con-former had a larger CCS (thyroglobulin 358 Å2, RNase B359 Å2, ovalbumin 359 Å2, synthetic 357 Å2). MS/MS of thyro-globulin Man5 and Man6 confirmed the presence of singlestructures (ESI Fig. 5†). While it remains unclear what causesthese N-glycans to adopt multiple distinct gas-phase confor-mations, it is clear that IM-MS provides an exquisitely sensitivemeans to separate, identify, and interrogate N-glycanstructures.ConclusionsWe have reported the absolute DTCCSs of pure high-mannoseN-glycans as commonly observed adducts by DT IM-MS in Heand N2. These and other previously published values are de-posited in the recently launched glyoconjugate CCS databaseGlycoMob (http://www.glycomob.org) housed within Uni-CarbKB, a glycomics mass spectrometry resource.17 CCS valuesdeposited in GlycoMob were measured both by DT IM-MS andTW IM-MS and are well within instrument error of approxi-mately 1%. We have found that DTCCS of [M − H]− were notice-ably smaller than both [M + H]+ and anion/cationated glycans.Analysis by TW IM-MS furthermore revealed the presence ofdistinctive gas-phase conformers, specifically for Man5, Man6and Man9 N-glycans among four sample sets. The presence ofsuch conformers may lead to misinterpretation of IM-MS dataunless careful attention is given to MS/MS data to discriminatebetween structural isomers and conformers. As IM-MS appli-cations for glycomics studies are in their early stages furthersystematic studies are needed to understand how these bio-molecules fold into their specific, adduct-dependent gas-phasestructure.AcknowledgementsFinancial support for this work was provided by the Biotech-nology and Biological Sciences Research Council (BBSRC)[BB/L017733/1 to W.B.S., D.J.H. J.L.P.], the Free UniversityBerlin and the Fritz Haber Institute of the Max Planck Society.Johanna Hofmann is gratefully acknowledged for reading themanuscript and fruitful comments.References1 D. J. Harvey, A. H. Merry, L. Royle, M. P. Campbell,R. A. Dwek and P. M. Rudd, Proteomics, 2009, 9, 3796–3801.2 R. Apweiler, H. Hermjakob and N. Sharon, Biochim.Biophys. Acta, 1999, 1473, 4–8.3 L. Royle, C. M. Radcliffe, R. A. Dwek and P. M. Rudd,Methods Mol. Biol., 2006, 347, 125–143.4 J. Zaia, OMICS, 2010, 14, 401–418.5 L. S. Fenn and J. A. McLean, Phys. Chem. Chem. Phys., 2011,13, 2196–2205.6 D. J. Harvey, C. A. Scarff, M. Edgeworth, M. Crispin,C. N. Scanlan, F. Sobott, S. Allman, K. Baruah, L. Pritchardand J. H. Scrivens, Electrophoresis, 2013, 34, 2368–2378.7 D. J. Harvey, F. Sobott, M. Crispin, A. Wrobel, C. Bonomelli,S. Vasiljevic, C. N. Scanlan, C. A. Scarff, K. Thalassinos andJ. H. Scrivens, J. Am. Soc. Mass Spectrom., 2011, 22, 568–581.8 J. Hofmann, W. B. Struwe, C. A. Scarff, J. H. Scrivens,D. J. Harvey and K. Pagel, Anal. Chem., 2014, 86, 10789–10795.9 K. Pagel and D. J. Harvey, Anal. Chem., 2013, 85, 5138–5145.10 M. D. Plasencia, D. Isailovic, S. I. Merenbloom, Y. Mechref,M. V. Novotny and D. E. Clemmer, J. Am. Soc. Mass Spec-trom., 2008, 19, 1706–1715.11 J. P. Williams, M. Grabenauer, R. J. Holland,C. J. Carpenter, M. R. Wormald, K. Giles, D. J. Harvey,R. H. Bateman, J. H. Scrivens and M. T. Bowers, Int. J. MassSpectrom., 2010, 298, 119–127.Fig. 4 Arrival time distributions of [M − H]− high-mannose N-glycansfrom ovalbumin, RNase B and thyroglobulin. The presence of multipleconformers is evident for Man5, Man6 and Man9. Measurements wereperformed using a TW IM-MS instrument.Communication Analyst6802 | Analyst, 2015, 140, 6799–6803 This journal is © The Royal Society of Chemistry 2015Open Access Article. Published on 07 July 2015. Downloaded on 09/02/2016 13:53:40. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online12 F. Zhu, S. Lee, S. J. Valentine, J. P. Reilly andD. E. Clemmer, J. Am. Soc. Mass Spectrom., 2012, 23, 2158–2166.13 M. F. Bush, Z. Hall, K. Giles, J. Hoyes, C. V. Robinson andB. T. Ruotolo, Anal. Chem., 2010, 82, 9557–9565.14 D. J. Harvey, J. Am. Soc. Mass Spectrom., 2005, 16, 631–646.15 H. Schachter, Glycoconjugate J., 2000, 17, 465–483.16 J. M. Prien, D. J. Ashline, A. J. Lapadula, H. Zhang andV. N. Reinhold, J. Am. Soc. Mass Spectrom., 2009, 20, 539–556.17 M. P. Campbell, R. Peterson, J. Mariethoz, E. Gasteiger,Y. Akune, K. F. Aoki-Kinoshita, F. Lisacek and N. H. Packer,Nucleic Acids Res., 2014, 42, D215–D221.Analyst CommunicationThis journal is © The Royal Society of Chemistry 2015 Analyst, 2015, 140, 6799–6803 | 6803Open Access Article. Published on 07 July 2015. Downloaded on 09/02/2016 13:53:40. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

Collision cross sections of high-mannose N-glycans in commonly observed adduct states – identification of gas-phase conformers unique to [M − H]- ions

We report collision cross sections (CCS) of high-mannose N-glycans as [M + Na]+, [M + K]+, [M + H]+, [M + Cl]-, [M + H2PO4]- and [M − H]- ions, measured by drift tube (DT) ion mobility-mass spectrometry (IM-MS) in helium and nitrogen gases. Further analysis using traveling wave (TW) IM-MS reveal the existence of distinct conformers exclusive to [M − H]- ions

https://pure.mpg.de/pubman/item/item_2174935_10/component/file_2248152/c5an01092f-1.pdf

Collision cross sections of high-mannose N-glycans in commonly observed adduct states – identification of gas-phase conformers unique to [M − H]<sup>-</sup> ions

Abstract

Similar works

Full text

Available Versions

MPG.PuRe

MPG.PuRe