Structural glycomics using hydrophilic interaction chromatography (HILIC) with mass spectrometry

STRUCTURAL GLYCOMICS USING HYDROPHILICINTERACTION CHROMATOGRAPHY (HILIC) WITHMASS SPECTROMETRY

Manfred Wuhrer,* Arjen R. de Boer, and Andre M. DeelderLeiden University Medical Center, Biomolecular Mass Spectrometry Unit,Department of Parasitology, P.O. Box 9600, 2300 RC Leiden,The Netherlands

Received 15 June 2007; received (revised) 18 March 2008; accepted 1 May 2008

Published online 31 October 2008 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20195

Hydrophilic interaction chromatography (HILIC) with massspectrometry is a versatile technique for structural glycomics.Glycans are retained by hydrogen bonding, ionic interactions,and dipole–dipole interactions. Glycopeptides as well asglycans with various modifications and reducing-end labelscan be efficiently separated, which often results in theresolution of isobaric species. Chromatography is usuallyperformed with solvent mixtures of organic modifier (oftenacetonitrile) and volatile (acidic) buffer which are suitable foronline-electrospray ionization-mass spectrometry. When per-formed at the nano-scale, this results in a detection limit foroligosaccharides of approximately 1 femtomol. Alternatively,glycans may be analyzed by offline-MALDI-MS(/MS) in bothnegative-ion mode and positive-ion mode, which allows theregistration of informative fragment ion spectra from deproto-nated species and sodium adducts, respectively. # 2008 WileyPeriodicals, Inc., Mass Spec Rev 28:192–206, 2009Keywords: glycopeptide; hydrophilic interaction chromato-graphy; LC-MALDI; normal phase; oligosaccharide

I. INTRODUCTION

Hydrophilic interaction chromatography (HILIC) has beenintroduced in the 1970s and is defined by Alpert (1990) as avariant of normal phase chromatography in which analytesinteract with a hydrophilic stationary phase. Analytes areretained on the hydrophilic stationary phase by hydrogenbonding, ionic interactions and dipole–dipole interactions.Elution is performed by a binary eluent of organic modifier/water, in which water is the stronger eluting solvent (Alpert,1990). HILIC has recently been comprehensively reviewed byHemstrom and Irgum (2006), and HILIC of carbohydrates hasbeen reviewed by Churms (1996).

HILIC of derivatized oligosaccharides with fluorescencedetection is a widely applied analytical technique for the profilingand structural analysis of glycans (reviewed by Anumula, 2000,2006; Lamari, Kuhn, & Karamanos, 2003). The technique isalternatively referred to as normal-phase HPLC (Guile et al.,1996; Rudd et al., 1997, 2001), but differs from organic normalphase by using polar aqueous mobile phases. Glycans comprising

a reducing end are commonly labeled with a fluorescent tag byreductive amination, followed by a sample clean-up step andHPLC analysis using water or an aqueous buffer as eluent. Thelarger the glycans the later they tend to elute, which is why HILICof glycans is sometimes referred to as ‘‘size fractionation.’’ Forthe structural assignment of glycans, the elution positions inHILIC are expressed in glucose units which are defined using aglucose polymer reference ladder (e.g., dextran hydrolysate).Next to the size of the glycan, charges influence retention.Moreover, HILIC is influenced by steric properties of theanalytes, resulting in the separation of isomers.

Often a single HILIC run may not be sufficient for structuralassignment. Therefore, glycans may be analyzed by two- orthree-dimensional mapping with additional anion exchange-LCand reversed phase-HPLC separation steps (Ohara et al., 1991;Hase, 1994; Takahashi, 1996). Alternatively, HILIC may berepeated after various enzymatic cleavage steps which result incharacteristic shifts in elution positions (Rudd et al., 1997, 2001).Moreover, (tandem) mass spectrometry may be applied to obtaindecisive structural information. This is often performed by peakfractionation followed by offline-MALDI-TOF-MS (Rudd et al.,2001).

In recent years, HILIC with online-electrospray ionization(ESI)-MS has evolved as a powerful analytical tool in structuralglycomics at the level of released glycans as well as glyopeptides.Moreover, HILIC with automated MALDI-TOF-MS analysis hasbeen established (Maslen et al., 2006, 2007). Moreover, HILIC-based enrichment techniques for glycans and glycopeptides haveevolved as valuable tools in glycoproteomics. These approachesallow the analysis of glycans with or without reducing end-tagas well as glycopeptides by tandem mass spectrometry withsensitivities down to the low femtomol range, as reviewed here.

II. PRINCIPLES OF HILIC OF GLYCOCONJUGATES

A. HILIC Stationary Phases

A variety of HILIC stationary phases comprising ionic and non-ionic phases are used for the separation of glycoconjugates.Almost all HILIC stationary phases are silica-based, as shown inthe detailed overview by Hemstrom and Irgum (2006). Mostlyused non-ionic columns contain amide and diol stationaryphases, and the ionic columns silica, using underivatized silica

Mass Spectrometry Reviews, 2009, 28, 192– 206# 2008 by Wiley Periodicals, Inc.

————*Correspondence to: Manfred Wuhrer, E-mail: [email protected]

(silicon dioxide) particles or monoliths, amine, aminopropyl andthe zwitterionic ZIC-HILIC of SeQuant AB (Umea, Sweden;Takegawa et al., 2006a,b).

B. Hydrogen Bonding

HILIC of glycoconjugates with online-electrospray ionization(ESI) and MS detection may be performed on glycans in theirnative form, after reduction to oligosaccharide alditols, and afterreductive amination, with the latter resulting in the introductionof a fluorescent or UV-absorbing label at the reducing end(Wuhrer, Deelder, & Hokke, 2005) (Fig. 1). Moreover, HILIC-ESI-MS may be applied to glycopeptides. In the case of neutralglycans and non-ionic stationary phases, retention is believed tobe caused by partitioning (hydrogen bonding) of the glycanbetween a water-enriched layer that is supposed to cover the polarhydrophilic stationary phase, and a less hydrophilic bulk eluent(Alpert, 1990; Hemstrom & Irgum, 2006).

C. Ionic Interactions and Charge Status ofthe Analytes

1. Ionic Interactions

Ionic stationary phases may, next to hydrogen bonding,contribute to the retention of charged glycans in HILIC by ionicand dipole–dipole interactions (Alpert, 1990; Hemstrom &Irgum, 2006). The stationary phase of silica(-based) andaminopropyl columns may contain negative or positive charges,respectively, which is generally pH-dependent. Silica-basedcolumn materials can exhibit charges at basic pH due todeprotonated silanol groups, which means that the HILICretention is increased when pH is below pKa of basic compounds.Meanwhile, negatively charged compounds like sialic acids areelectrostatically repelled by the negatively charged surface. An

acidic pH and/or ion pairing reagent is required to preventunwanted interactions between deprotonated silanol groups andbasic compounds. The opposite effect occurs for amine andaminopropyl phases, which act as weak anion-exchange columnsbecause of the positively charged amino groups at pH below �9.Furthermore, HILIC at higher ionic strength does result in thereduction of ionic interactions between analyte and stationaryphase (elimination of the ion exchange effect), leaving hydrogenbonding as the major retentive effect. Ions in the mobile phasewill act as counterions for both charged analytes as well ascharged groups of the stationary phase and will, therefore,weaken ionic interactions (Fig. 1). However, higher ionicstrengths and ion pairing agents often also decrease the ionizationefficiency of ESI. Moreover, a pH shift may likewise alter ionicinteractions by reducing or introducing charged groups onanalyte and/or stationary phase.

A disadvantage of bare silica and aminopropyl columnsis the limited stability in aqueous solvents (dissolution andhydrolysis) and the irreversible adsorption of compounds to thesestationary phases. Irreversible adsorption was observed on silicacolumns due to the highly polar character of this stationary phase(Li & Huang, 2004) and on aminopropyl columns due to a morereactive stationary phase compared to alkyl phases. For example,the separation of reducing carbohydrates by aminopropylcolumns may result in Schiff-bases (imines) by on-columnreaction of the primary amines with aldehydes (Brons &Olieman, 1983). Alternatively, diol columns can be used as theyare more stable and do not form Schiff’s bases with reducingsugars (Herbreteau et al., 1992).

Noteworthy, zwitterionic stationary phases (ZIC-HILICof SeQuant AB, Umea, Sweden; Takegawa et al., 2006a,b)exhibit permanent positive and negative charges. These chargedstationary phases act, in addition to the water-retaining property,as weak-cation exchange HPLC columns (Hemstrom & Irgum,2006). Takegawa et al. (2006b) demonstrated the potentialof ZIC-HILIC by the separation of 2-aminopyridine-labeled

hydrogen bonding

size separation

ionic interactions

charge separation

cationic functional groups

of stationary phase

high ionic strength

and acidity of mobile phase

separation of isobaric structures

HILIC

MALDI-MS

-TOF/TOF-MS

-Q-TOF-MS

-FT-ICR-MS

nano-RP-ESI-MS

ESI-MS

-Q-TOF-MS

-IT-MS

-FT-ICR-MS

native glycans labeled glycans

- fluorescent tag

- biotin

glycopeptides

MS analysisonline offline

FIGURE 1. Scheme of HILIC-MS approaches in structural glycomics.

STUCTURAL GLYCOMICS BY HILIC-MS &

Mass Spectrometry Reviews DOI 10.1002/mas 193

N-glycans of human serum IgG (Fig. 2). Fluorescence chromato-grams of 10 sequential runs (Fig. 2A) show a good repeatabilityof the ZIC-HILIC separation method. Moreover, it should benoted that both pairs of isomeric N-glycans (b/c and f/g) could beseparated by the ZIC-HILIC column (Fig. 2B).

2. Charge Status of the Analytes

Glycans may have a negative charge due to the presence ofcarboxyl groups (e.g., sialic acid, muraminic acid, glucuronic

acid), sulfate groups, or phosphate groups (see Essential ofGlycobiology by Varki et al. (1999); online accessible via http://www.ncbi.nlm.nih.gov/books). A major source of chargedN-glycans are mammalian glycoproteins, which often exhibitcomplex-type N-glycans with multiple sialic acid residues.Positive charges may occur due to choline substituents(permanent positive charge; found in glycoconjugates ofnematodes) (Nyame, Kawar, & Cummings, 2004) or aminesugars (such as glucosamine which occurs in the oligosaccharidechains of bacterial lipopolysaccharides (LPS); Holst, 2007).

FIGURE 2. ZIC-HILIC separation of 2-aminopyridine-labeled N-glycans of human serum IgG. A: Fluo-

rescence (FL) chromatograms (Em, 400 nm; Ex, 320 nm) in sequential 10 runs. Retention time RSD (%)

values of peaks a–h are 0.37–0.71. B: Mass chromatograms (MCs) of molecular ions of major eight peaks

(a–h). Mass chromatograms of d–g are a sum of MCs for their single- and double-protonated ions which are

relatively abundant (adapted from Takegawa et al. (2006b), with permission from Elsevier, copyright 2006).

[Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

& WUHRER, de BOER, AND DEELDER

194 Mass Spectrometry Reviews DOI 10.1002/mas

Moreover, derivatization by reductive amination results in theintroduction of a secondary aromatic amine, which may beprotonated at acidic pH. Labeling of glycans with 2,6-diaminopyridine (DAP; Xia et al., 2005) results in additionalpositive charge(s) at acidic pH, whilst labels such as 2-aminobenzoic acid (anthranilic acid) and 8-Aminopyrene-1,3,6-trisulfonic acid (APTS) confer also negative charges(Anumula, 2006; Shilova & Bovin, 2003).

When (tryptic) glycopeptides are analyzed by HILIC, thepeptide moieties often exhibit several charges, which may bepositive charges at amino acid residues arginine, lysine andhistidine and at the N-terminus. Moreover, negative charges maybe observed due to the carboxylic acid side chains of aspartateand glutamate as well as the C-terminus.

The occurrence of charges in the glycan moiety, thereducing-end tag, or the peptide moeity can often be modulatedby changes in pH. These changes often likewise effect the chargestate of the stationary phase. A shift in pH will therefore representa method for modulating the influence of ionic interactionsin glycan separations (Li & Huang, 2004). Another mean forchanging the influence of ionic interactions in HILIC separationsis the adjustment of the buffer concentration. This has impres-sively been demonstrated by Takegawa et al. (2006a), whoobserved a profound dependence of the elution properties of2-aminopyridine-labeled, sialylated N-glycans on the concen-tration of the ammonium acetate buffer.

For glycopeptides, the influence of the peptide moietyhas not been studied in detail. Peptide moieties may, however,undergo various interactions in HILIC, which may be ionicinteractions or hydrophilic interactions with the stationary phase.The influence of HILIC interactions of the peptide moiety hasbeen demonstrated by Takegawa et al. (2006b) for IgG1 and IgG2glycopeptides, as discussed in Section V.

D. Gradient Elution

HILIC normally works with a binary gradient, consisting of anaqueous part (containing some salt and/or acid) and a less polar,organic part (Hemstrom & Irgum, 2006). Mostly acetonitrile isused as organic and weakly eluting solvent, but also other watermiscible aprotic solvents are an option. Methanol can also beused, but has a higher solvent strength and is therefore neededat higher concentrations in order to achieve retention timescomparable to those for aprotic solvents (Li & Huang, 2004). Thegradient starts at high organic solvent concentrations with anaqueous part of approximately 20%. Under these conditions,polar interactions of the analytes within the mobile phase arerestricted, and glycoconjugates tend to be retained by hydrophilicinteractions with the stationary phase (see above). By applyinga gradient to an aqueous percentage of 50 or higher, thepossibilities for hydrophilic interactions within the mobilephase strongly increase. These interactions now successfullycompete with the polar interactions of analyte and stationaryphase, resulting in the elution of polar glycans and glycanderivatives. The initial high amount of organic modifiermay result in solubility problems (precipitation) of the polaroligosaccharides, which is a specific problem of HILIC, incontrast to for example, reversed phase and graphitized carbon

HPLC. However, HILIC is still in advantage over normal phase,because normal phase uses non-polar eluents (often hexane-based) which are even worse for dissolution of oligosaccharides.In addition, interfacing of normal phase with ESI-MS is also aproblem because ionization is not easily achieved in totallyorganic, non-polar eluents (Hemstrom & Irgum, 2006).

Using ionic stationary phases, ionic additives to the mobilephase are usually applied to control the pH and ionic strength, andso to prevent or stimulate ionic interactions between glycans andthe stationary phase. Separations on non-ionic stationary phasesare in many cases performed at pH around 4.4 (obtained by50 mmol/L ammonium formate). However, for all silica(-based)columns a pH between 2 and 7 has to be used to preventhydrolysis and dissolution of the silica, respectively.

III. HILIC SPE-METHODS FOR SAMPLEPREPARATION

It is a well-known phenomenon that glycans adsorb to variouspolar chromatographic stationary phases when mobile phaseswith high organic solvent content are used, as reviewed byChurms (1996): several silica-based and polymeric stationaryphases used in size exclusion chromatography show conventionalgel filtration behavior for oligosaccharides at acetonitrileconcentrations of up to 40%. Acetonitrile concentrations of50% or higher, however, result in an inversion of the elutionsequence, and retention increased with decreasing polarity of themobile phase. Obviously, at these elevated acetonitrile concen-trations, the hydrophilic interaction of oligosaccharides with thestationary phase is favored, and these HILIC effects dominateover the gel filtration effects. This phenomenon does notonly occur for oligosaccharides, but is likewise observed forN-glycopeptides: the separation of a tryptic digest of horseradishperoxidase (HRP) on a Superdex 75 HiLoad column (600 mm�16 mm, Amersham Biosciences, Uppsala, Sweden) in 25 mMammonium bicarbonate, 50% acetonitrile, resulted in theseparation of glycoforms. In the case of glycopeptides whichshare the same peptide moiety but differ in glycan moiety,the glycoforms exhibiting disaccharides attached to the N-glycosylation site eluted earlier than the glycoforms with aheptasaccharide N-glycan structure, following HILIC rather thansize exclusion chromatography principles (M. Wuhrer, unpub-lished results).

Wada and colleagues have recently used HILIC for the solidphase extraction (SPE) of oligosaccharides and glycopeptidesfrom complex mixtures containing salts, non-glycosylatedpeptides and proteins using polysaccharide-based solid phases(Sepharose and cellulose; Wada, Tajiri, & Yoshida, 2004;Tajiri, Yoshida, & Wada, 2005; see Table 1). They have obtainedimpressive separations when applying Sepharose and cellulose inbatch mode. A possible future modification of the protocolapplying Sepharose or cellulose cartridges or a packed Sepharosecolumn is expected to result in an even better performance ofthese polysaccharide-based solid phases in glycan and glyco-peptide purification.

Alternatively, silica-based ZIC-HILIC material (Hagglundet al., 2004) and aminopropyl silica (Yu et al., 2005, 2007)have been successfully applied for the selective extraction of



glycopeptides from a tryptic digest and for the work-up ofglycans after PNGase F release for MALDI-MS analysis(Table 1). Both these approaches are micro-methods suitablefor the analysis of low amounts of biological materials: Hagglundet al. used gel-loader tips which they packed with ZIC-HILICmaterial (particle size 10 mm). Their method is nicely compatiblewith MALDI-TOF-MS analysis of the glycopeptides. Yu et al.used a 96-well HILIC microelution plate with 5 mg aminopropylsilica sorbent packed into wells to form micro-SPE cartridges,and the eluates containing glycans or glycopeptides can beanalyzed by MALDI-Q-TOF-MS.

The HILIC-SPE approaches with polysaccharide-basedsolid phases as well as silica-based chromatography materialshare the following advantageous features: (1) glycans areretained, whilst salt, detergent, peptides, and proteins are mostlyfound in the flow-through; (2) elution is accomplished with wateror solvent mixtures with high water content. MALDI-MSanalysis can be performed either directly or after a drying step.

Other HILIC methods for glycan sample work-up includethe polyamide DPA-6S resin which has been reported forsample preparation for HILIC HPLC analysis with fluorescencedetection, but not in combination with mass spectrometry(Neville et al., 2004; Prater, Anumula, & Hutchins, 2007;Anumula, 2008).

IV. HILIC-MS AT THE GLYCAN LEVEL

The analysis of complex mixtures of oligosaccharides can beachieved by direct mass spectrometric analysis of the mixture,either in native or permethylated form, applying MALDIor electrospray ionization (Harvey, 1999; Zaia, 2004). Theinterpretation of obtained tandem mass spectrometric data mayhowever be especially challenging when mixtures of structuralisomers are analyzed, which may be present in unknown ratios(Ashline et al., 2007; Prien et al., 2008). Recently, we havereviewed the advantages of various LC-MS methods (reversephase, graphitized carbon, high-performance anion exchangechromatography, and HILIC) for the analysis of complex glycanmixtures with a (partial) separation and differentiation ofstructural isomers (Wuhrer, Deelder, & Hokke, 2005). In thefollowing the use of HILIC-MS will be reviewed for thecharacterization of glycans.

A. HILIC of Glycans with Online-ESI-MS

1. Analytes

HILIC-ESI-MS has been applied to neutral plant oligosacchar-ides (xyloglucans; Alpert et al., 1994), reduced O-glycans(Thomsson, Karlsson, & Hansson, 1999, 2000) and fluorescentlylabeled N-glycans (Charlwood et al., 2000; Saba et al., 2001,2002; Wuhrer et al., 2004a,b; Geyer et al., 2005). Furthermore,stereoisomers (anomers) of the reducing end carbohydrate ring ofN-glycans may be partially separated by this method (Wuhreret al., 2004a,b). This effect may often be undesirable and can beovercome by a reduction step and subsequent analysis ofoligosaccharide alditols, or by the addition of a small amountof ammonia to accomplish the mutarotation (Alpert et al., 1994).A particular strength of HILIC-ESI-MS for the analysis ofoligosaccharides is the tolerance of the chromatographic systemto modifications: because the retention is predominantlydetermined by the multiple hydrogen bonding of the glycanmoiety, polar substituents (e.g., sulfate) as well as varioushydrophobic tags are all compatible with HILIC separation ofoligosaccharides.

2. LC-MS

Avariety of HILIC stationary phases are used for online-ESI-MSof glycans, comprising silica-based ion exchange, zwitterionic,as well as non-ionic phases, of which the non-ionic amidemodified columns are mostly used (Table 2). All these columnsallow the separation in an MS-compatible manner, that is, atlow concentrations of volatile salts and high concentrations oforganic solvents.

Oligosaccharides generally elute at acetonitrile concentra-tions which are above 50%. In the case of online coupling withESI-MS, the aqueous part usually contains low concentrations ofvolatile weak acids (formic acid, acetic acid) of bases (ammonia)and/or salts. These conditions are suitable for very efficient ESI,making online HILIC-ESI-MS a favorable choice for the massspectrometric analysis of glycans and glycan derivatives. Theamount of ion pairing agents like TFA has to be as low as possible,because these agents have been shown to significantly reduce theESI-MS signal (Naidong, 2003).

Mass spectrometric detection has hitherto predominantlybeen performed in the positive-ion mode with the registration of

TABLE 1. HILIC-SPE methods for the analysis of glycoconjugates by MS

Reference Mass spectrometry Samples Conditions Sorbent(s) Sepharose CL-4B and microcristalline cellulose

adsorption/wash with 1-butanol/ethanol/water (4;1;1, v/v) elution with ethanol/water (1:1, v/v)

glycans and tryptic glycopeptides

MALDI-TOF-MS in linear mode and MALDI-Q-ion trap-TOF-MS: DHB matrix

Wada et al., 2004 Tajiri et al., 2005

self-made microcolumns of 10 µM ZIC-HILIC material (Sequant; Umeå, Sweden)

adsorption/wash in 80% acetonitrile, 0.5% formic acid elution with 99.5% water, 0.5% formic acid

(sialylated) glycopeptides

negative linear mode MALDI-TOF-MS: DHB matrix

Hägglund et al., 2004

96-well HILIC microelution plate packed with 5 mg of aminopropyl silica (MassPREP; Waters, Milford, MA)

adsorption/wash in 90% acetonitrile elution with 10 mM ammonium citrate (p, 25% acetonitrile

glycans and pronase-generated glycopeptides

positive mode MALDI-quadrupole-TOF-MS:DHB matrix

Yu et al. 2005, 2007



proton, sodium and potassium adducts, often as multiply chargedspecies. HILIC with negative-mode MS analysis may beadvantageous, however, for multiply sialylated or sulfatedoligosaccharides. Most of the applied buffers in HILIC arecompatible with negative-mode MS analysis (Table 2), which isexpected to become more important in the near future. Thedevelopment is expected to be similar to that in the field ofgraphitized carbon-HPLC-ESI-MS of glycans, where theimportant role of negative mode MS is already obvious fromliterature (Karlsson, Schulz, & Packer, 2004; Karlsson et al.,

2004, 2005). Notably, negative-mode MS of glycans in native orlabeled form not only allows the sensitive detection but also theacquisition of informative fragment ion spectra with character-istic cross-ring cleavages (Harvey, 2005a,b,c).

In terms of sensitivity, the reduction of the diameter andconsequently flow of the HILIC system have resulted in detectionlimits of around 1 femtomol (Wuhrer et al., 2004a,b). It has to benoted, however, that analyses of very low levels of oligosacchar-ides have to overcome the MS/background noise caused by thebuffer (50 mM ammonium formate) which is frequently used in

TABLE 2. HILIC-electrospray-MS of glycans

Column LC starting conditions Samples Mass spectrometry Reference PolyGLYCOPLEX (PolyLC, Columbia, MD) 150 mm x 1 mm (polysuccinimide coated silica)

50 µl/min; isocratic run in acetonitrile: water (70:30, v/v), with 8 µl/min post-column addition of 0.1% acetic acid

xyloglucans (neutral oligosaccharides) labeled with p-nitrobenzene

positive-mode ESI-triple quadrupole-MS

Alpert et al., 1994

Hypersil APS-2 (Hypersil, Runcorn, UK) 3 µm, 250 mm x 2.1 mm (propylamine coated silica)

120 µl/min; 5 mM ammonium hydrogencarbonate pH 8.0, 80% acetonitrile

sulfated, reduced, oligosaccharides

positive-mode ESI-quadrupole-TOF-MS

Thomsson et al., 1999, 2000

GlycoSep N (Prozyme, San Leandro, CA), 150 mm x 1 mm (amide based)

40 µl/min; 50 mM ammonium formiate pH 4.4, 75 % acetonitrile

2-aminoacridone-labeled oligosaccharides


Charlwood et al., 2000

GlycoSep N (Prozyme, San Leandro, CA), 150 mm x 1 mm (amide based)

40 µl/min; 50 mM ammonium formiate pH 4.4, 80 % acetonitrile

predominantly N-glycans and O-glycans after labeling with 2-aminobenzamide


Mattu et al., 2000 Garner et al., 2001 Royle et al., 2002, 2003 Butler et al., 2003

Ultremex 5 NH2 (Phenomenex, Torrance, CA), 250 mm x 4.6 mm

1 ml/min; water adjusted to pH 3.5 with formic acid, acetonitrile varying between 50 % and 90 %

oligosaccharides labeled with 1-phenyl-3-methyl-5-pyrazolone (PMP) and 2-aminonaphthalene trisulfone (ANTS)

positive-mode and negative-mode ESI-quadrupole-TOF-MS

Saba et al., 2001, 2002

Nucleosil NH2

(Phenomenex, Torrance, CA) 5 µm, 150 mm x 1 mm

150 µl/min; 6.5 mM ammonium acetate pH 5.5, 85 % acetonitrile at 10 min

predominantly low-molecular weight carbohydrates

positive-mode ESI-ion trap-MS

Tolstikov & Fiehn, 2002

(propylamine coated silica) Polyhydroxylethyl A (PolyLC Inc, Columbia, MD) 5 µm, 150 mm x 1 mm (poly(aspartamide) coated silica)





TSK amide 80 (Tosoh Biosciences, Montgomeryville, MA) 5 µm, 80 Å pore size, 250 mm x 2 mm (silica with carbamoyl groups)





TSK amide 80 (Tosoh Biosciences, Montgomeryville, MA) 5 µm, 80 Å pore size, 100 mm x 75 µm

300 nl/min; 50 mM ammonium formiate pH 4.4, 80% acetonitrile

native, reducing N-glycans; N-glycans labeled with 2-aminobenzamide or 2-aminopyridine

positive-mode electrospray- ion trap-MS

Wuhrer et al., 2004a, b; Geyer et al., 2005

TSK amide 80 (Tosoh Biosciences, Montgomeryville, MA) 5 µm, 80 Å pore size, 120 mm x 200 µm

3 µl/min; 50 mM ammonium formiate pH 4.5, 80% acetonitrile

native, reducing N-glycans

positive-mode ESI-TOF-MS

Zhao et al., 2007

ZIC-HILIC (Sequant; Umeå, Sweden) 3.5 µm, 200 Å pore size; 150 mm x 2.1 mm

200 µl per min; between 20 and 80 mM ammonium acetate in aqueous part, approximately 75% acetonitrile

N-glycans labeled with 2-aminopyridine

positive-mode and negative-mode ESI-ion trap-MS

Takegawa et al., 2006a, b



HILIC chromatography. Current improvements of mass spectro-metric equipment as well as further downscaling of the columndimensions should allow additional, significant sensitivitygains in the near future. Moreover, the development of ultra-high-pressure HPLC materials with HILIC functional groups(range of 1–2 mm spherical silica), similar to the popularreversed phase-stationary phases (Plumb et al., 2004; Guillarmeet al., 2007), would be expected to result in a better chromato-graphic resolution and, hence, a further reduction of thedetection limit.

B. HILIC of Glycans with Offline MS Detection

HILIC of fluorescently labeled glycans with (peak) fractionationand MALDI-TOF-MS analysis of collected fractions is a well-established approach (see for example Wuhrer et al., 2002,2004c). This is conventionally performed with analytical-scaleHILIC columns resulting in a detection limit of approximately100 femtomol for glycans with 2-aminobenzamide in MS mode(Rudd et al., 2001). For very complex samples, this approach maybe varied by performing the HILIC separation of fluorescentlylabeled glycans offline with fluorescence detection, followed bythe subsequent analysis of the collected fractions by reversedphase-nano-LC-ESI-ion trap-MS(/MS), which gives a verydetailed picture of the occurring glycan structures (Wuhreret al., 2006).

Recently Stephens and co-workers have presented anattractive 1-dimensional offline approach, which is capillary-scale HILIC using an amide-80 column with automatic spottingon a target plate and automatic MALDI-TOF-MS measurement(Maslen et al., 2006, 2007). They have used this systemto separate oligosaccharides labeled with 2-aminobenzamideor 2-aminobenzoic acid, which allowed registration ofUV-absorbance next to mass spectrometric detection. Samplespots were overlayed with dihydroxybenzoic acid (DHB) matrixsolution and rapidly dried in a vacuum desiccator in order toproduce small crystals for easy automatic MALDI-TOF-MSanalysis with reproducible spectral quality. Maslen et al. (2006,2007) chose to analyze the glycans by high-energy CID MALDI-TOF/TOF-MS of sodium adducts with manual data acquisition.Resulting mass spectra are very informative as they featurecharacteristic cross-ring cleavages, which often allow thededuction of linkage positions (Lewandrowski, Resemann, &Sickmann, 2005; Mechref, Novotny, & Krishnan, 2003; Spinaet al., 2004; Wuhrer & Deelder, 2005).

Notably, next to the analysis of alkali adducts in positive-ionmode, oligosaccharides labeled with a 2-aminobenzoic acid tagadditionally allow the efficient analysis by MALDI-TOF-MS innegative-ion mode (deprotonated form) (Qian et al., 2007). Veryinformative fragment spectra may be obtained from [M-H]� ions,either by MALDI-Q-TOF-MS (Qian et al., 2007) or by MALDI-TOF/TOF-MS with laser-induced dissociation (LID), as dem-onstrated for oligosaccharides labeled with 2-aminobenzoic acid(Harvey, 2005a) and 2-aminobenzamide (Wuhrer & Deelder,2005). Negative-mode fragmentation characteristics of oligo-saccharides have recently been elaborated in detail by Harvey(2005b,c) for ions with multiple negative charges generated byESI, but many of these principles seem to be directly transferableto the fragmentation of singly deprotonated ions generated by

MALDI ionization (Morelle et al., 2005b; Qian et al., 2007;Wuhrer & Deelder, 2005).

C. HILIC of Glycans with MALDI-MS Versus ESI-MS

A comparison of the HILIC offline LC-MALDI-MS approachwith HILIC-online ESI-MS shows the following specificadvantages/disadvantages of the two methods.

1. In-Source Decay

MALDI as the harsher ionization technique results in a highdegree of fragmentation of sialylated glycans as well as otherlabile oligosaccharides. This laser-induced dissociation phenom-enon is particularly pronounced for hot matrices like a-cyano-4-hydroxycinnamic acid (HCCA) and is less pronounced for DHB(Lewandrowski, Resemann, & Sickmann, 2005). Resulting in-source decay of sialylated glycans is particularly prominent inreflectron mode with delayed extraction, and is less visible inlinear mode without an extraction delay. Moreover, the pulsedapplication of cooling gas together with a low extraction voltagein MALDI-FT-ICR-MS experiments helps to largely retain sialicacid residues on glycans (M. Wuhrer, unpublished results).In HILIC-ESI-MS, in contrast, in-source decay of multiplysialylated glycans can be efficiently avoided by choosingappropriate transfer voltages in the ion transfer region of themass spectrometer.

2. Detection Bias

Due to the very big differences in ionization efficacies in MALDIfor different glycans and glycan derivatives, some glycans maybe massively underrepresented or even missed in a HILIC-MALDI-MS experiment. Glycans carrying negative charges(e.g., sialic acid, sulfate, or glucuronic acid) are discriminated inpositive-mode MALDI ionization. Together with the above-mentioned lability and loss of sialic acid residues, this makesthe analysis of multiply sialylated glycans by MALDI-MS in thepositive ion mode a very difficult task. In negative mode MALDI-MS of native glycans or glycans with a fluorescent tag like2-aminobenzamide (no acidic groups), neutral glycan specieswill be detectable only at reduced sensitivity (Wuhrer & Deelder,2005). In HILIC-ESI-MS, on the other hand, the efficient analysisof both sialylated and neutral glycans with various (fluorescent)tags is possible in both positive ion and negative ion mode, anddifferences in ionization efficacies are much less prominent.

3. MS/MS of Sodium Adducts

The MALDI approach gives the opportunity to perform high-energy MS/MS of sodium adducts. Under these conditions,cross-ring fragmentation is regularly observed. The pattern ofcross-ring fragments observed is indicative for the presence ofsubstituents in specific positions and does therefore allow to drawstructural conclusions (Mechref, Novotny, & Krishnan, 2003;Spina et al., 2004; Zaia, 2004; Lewandrowski, Resemann, &Sickmann, 2005; Wuhrer & Deelder, 2005; Morelle, Page, &Michalski, 2005). With the electrospray ionization approach,proton adducts are generally observed in positive-ion mode



(Wuhrer et al., 2004a,b; Takegawa et al., 2006a,b). CID MS/MSof protonated glycan species results predominantly in the frag-mentation of glycosidic linkages and does not easily provideinformation on linkage positions. This drawback can beovercome by doping the running solvent with sodium salt (forexample 0.6 mmol/L sodium hydroxide added to the slightlyacidic, buffered aqueous part of the mobile phase), which oftenresults in the observation of multiply sodiated oligosaccharidespecies (M. Wuhrer, unpublished results). With the same means ashift from proton adducts to sodium adducts can be obtainedin reverse-phase nano-LC-MS of 2-aminobenzamide labeledglycans (Wuhrer et al., 2006). The sodium adducts obtained byHILIC-ESI-MS likewise allow the acquisition of MS/MS spectrafeaturing cross-ring cleavages with linkage information, inaccordance with literature data (Morelle, Page, & Michalski,2005; Zaia, 2004).

4. Offline Versus Online MS

HILIC-MALDI-MS in offline mode has the advantage that isallows performing both positive mode and negative mode MS/MS experiments from a single HILIC run. In HILIC-online-ESI-MS, the need for additional MS or MS/MS data may oftenbecome evident after a first run, which will make additionalHILIC-MS runs necessary for a complete, detailed analysis ofcomplex samples.

V. HILIC-MS AT THE GLYCOPEPTIDE LEVEL

The analysis of protein glycosylation at the level of chemically orenzymatically released glycans by MS/MS allows the verydetailed characterization of these oligosaccharide moieties. Noinformation is obtained, however, on the identity of the carrierprotein and the specific glycan attachment sites. Analysis ofprotein glycosylation at the glycopeptide level can overcomethese limitations.

HILIC has been applied for the separation of glycopeptidesfrom non-glycosylated peptides in a two-dimensional LCapproach, with reversed phase-HPLC as a first dimension, andUV detection. While the first dimension regularly results inheterogeneous fractions containing both glycopeptides and non-glycosylated peptides, the second dimension HILIC results inthe pronounced retention of only the glycopeptides (http://www.nestgrp.com/pdf/Pp1/HILIC_example.pdf; Zhang & Wang,1998).

The selective retention of glycopeptides, whilst non-glycosylated peptides elute, has made HILIC of tryptic digestsan interesting new method (Wuhrer, Deelder, & Hokke, 2005;Takegawa et al., 2006a,b). Figure 3 shows the ZIC-HILICseparation of trypic digest of human serum IgG, where glyco-peptides are separated both on glycoform and peptide sequence.The retention times of the IgG1 glycopeptides (a-1–h-1) arelonger than those of the IgG2 (a-2–h-2). The reason is probably

FIGURE 3. ZIC-HILIC separation of tryptic peptides of human serum IgG. Mass chromatograms of

molecular ions of major N-glycopeptides of IgG1 (a-1–h-1) and IgG2 (a-2–h-2). For glycan structures, see

Figure 2 (from Takegawa et al. (2006b), with permission from Elsevier, copyright 2006).



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FIGURE 5.



the more hydrophilic character of the two tyrosines (Y) present inthe IgG1 glycopeptides compared to the two phenylalanines (F)present in the IgG2 glycopeptides. In addition, it should be notedthat the N-glycopeptide isomers (b-1/c-1, b-2/c-2, and f-2/g-2)were also sufficiently separated from each other, similar to theseparation of the corresponding PA-oligosaccharides. In con-clusion, Figure 3 indicates that the ZIC-HILIC separation ofN-glycopeptides is primarily based on the polarity of both thepeptide and the neutral N-glycan.

Moreover, enzymatic cleavage of glycoproteins can beperformed with pronase, a mixture of bacterial proteases whichcleaves most proteins to single amino acids and small peptidemoieties, but often leaves glycans with a peptide tag of three ormore amino acids (Juhasz & Martin, 1997). These pronaseglycoprotein digests can efficiently be analyzed by nano-HILIC-ESI-MS, as schematically shown in Figure 4 for the modelglycoprotein ribonuclease B (Wuhrer et al., 2005). Tandem massspectrometry of the eluting glycopeptides allows the identifica-tion of the glycan moiety as well as the peptide mass and/orsequence (Fig. 4). The characteristic peptide mass usually allowsthe identification of the peptide within a purified analyteglycoprotein using the FindPept tool (http://expasy.org/tools/findpept.html) and thus the allocation of the glycan to a specificglycosylation site. Figure 4 demonstrates the particular value ofthe ion trap-mass spectrometer with its option for multiple ion

isolation/fragmentation cycles for the analysis of the glycopep-tides obtained by pronase treatment. The first fragmentationexperiment performed with a tetrapeptide containing a pentam-annosidic N-glycan moiety mainly provides information on theglycan moeity (MS2 in Fig. 4). A subsequent fragmentation stepleads to the complete deglycosylation of the peptide (MS3),followed by the peptide sequence determination after a lastfragmentation step (MS4). This approach has been successfullyapplied to register the glycosylation of horseradish peroxidase,which contains 9 N-glycosylation sites and of the Dolichosbiflorus lectin, for which no glycosylation information had beenavailable before (Wuhrer et al., 2005).

Next to N-glycosylation (Fig. 4; Wuhrer et al., 2005),O-glycosylation can be analyzed by pronase digestion of apurified glycoprotein followed by HILIC-ESI-MS, as shown inFigure 5. Analysis of the b-chain of human choriogonadotropin(bHCG, SwissProt entry P01233) showed a variety ofO-glycosylated peptides in the glycopeptide elution range(Fig. 5A,B). Most of these O-glycopeptides were fragmented inthe ion trap-MS, revealing information on the glycan composi-tion as well as peptide mass, which allowed the identification ofthe peptide moieties and the allocation of the glycans to the fourO-glycosylation sites of b-HCG known from literature (Kessleret al., 1979). While most of the registered glycopeptide ions areconsidered to reflect glycoforms present on the intact b-HCG

FIGURE 5. O-glycosylation analysis of the b-chain of human choriogonadotropin (bHCG) by nano-

HILIC-ESI-MS after pronase treatment. bHCG was treated with pronase and separated by HILIC on a nano-

amide-80 column (flow 300 nl/min; solvent A; 50 mM ammonium formiate pH 4.4; solvent B; 80%

acetonitrile, 20% solvent A; gradient: t¼ 0 min, 100%B; t¼ 2 min, 100%B; t¼ 15, start of MS; t¼ 22 min,

52%B; t¼ 25 min, 52%B; t¼ 26 min, 100% B). Glycopeptides were detected by online-electrospray with an

ion trap mass spectrometer working in the automatic MS/MS mode. A: Base peak chromatogram (BPC)

and extracted ion chromatograms (EIC) of several detected glycopeptide species. B: Sum mass spectrum

of the whole glycopeptide elution range. C: MS/MS spectrum of the glycopeptide species at m/z 774. *,

single-charged contaminant. [Color figure can be viewed in the online issue, which is available at

www.interscience.wiley.com.]



protein, some observed glycopeptide species could be attributedto in source-decay based on the chromatographic separation: thedouble-protonated glycopeptide species at m/z 720.3 (peptideL154–P161 with a trisaccharide glycopeptide), for example, elutedas a distinct, separated peak at 15.9 min and did not arise from in-source decay of the disialylated glycoform of this O-glycopep-tide (m/z 865.9 at 17.0 min; Fig. 5A). The signal at m/z 1,148.4,in contrast, which corresponds to the peptide L154–P161 with adisaccharide O-glycan moiety, showed an extracted ion chroma-togram with three peaks, with the first peak (14.9 min)representing a glycoform of the intact b-HCG protein, andthe second (15.9 min) and third peak (17.0 min) reflecting in-source decay products of the monosialylated and disialylatedglycoforms (m/z 720.3 and m/z 865.9, respectively).

Whilst these experiments were performed on an ion trap-MSwith collision-induced fragmentation (CID), other ESI-MS/MStechniques, such as quadrupole-TOF-MS and in particular iontrap-MS with electron-transfer dissociation (ETD) wouldprovide valuable peptide sequence information directly at theMS/MS stage and are, therefore, expected to present usefulcombinations with HILIC for structural glycomics at theglycopeptide level (Wuhrer et al., 2007).

Recently, two modifications of the pronase treatmentapproach with nano-HILIC-MS have been reported. First,Temporini et al. (2007) have established an automated workflowfor the analysis of protein glycosylation in a standardizedmanner. In their approach, glycoproteins are first treated withpronase using an HPLC column with covalently immobilizedpronase (pronase bioreactor). Generated glycopeptides aresubsequently collected on a graphitized carbon trap column,followed by their separation on an Amide-80 HILIC column withESI-MS. Their optimized work-flow and protocol will allow theanalysis of protein glycosylation within 1 hr. Second, Yu et al.(2007) have replaced the HILIC HPLC-online-ESI-MS byHILIC-SPE with subsequent MALDI-MS/MS of the glyco-peptides, which was successfully applied to various standardglycoproteins.

VI. CONCLUDING REMARKS

HILIC is in various respects an ideal method to combine withthe mass spectrometric analysis of glycans and glycoconju-gates. The pronounced retention of glycoconjugates in HILICis mainly due to the multiple hydrogen bonding, whilstmost contaminants, non-glycosylated peptides, salts, detergentsetc. show little or no retention. This makes the hydrophilicinteraction principle suitable for the purification/enrichment ofglycoconjugates by solid phase extraction, which is expected tobe a growing field of applications. In HILIC-MS, the pronouncedretention of glycans and glycopeptides allows the definition of alate elution window in HILIC-MS, where these species are highlyenriched, which facilitates the identification and monitoring ofglycosylation.

A specific advantage of HILIC is the tolerance to manymodifications and substitutions on oligosaccharide chains, whichmay be reducing end tags, peptide or lipid moieties, and sulfate,phosphocholine, etc. Moreover, HILIC regularly results in theseparation of structural isomers, which helps in the structural

elucidation of complex mixtures, as separate tandem massspectra of the structural isomers may be obtained. HILIC elutionconditions are ideal for online-ESI-MS, which may be performedin positive-ion or negative-ion mode. Alternatively, HILICmay be performed with offline MALDI-MS/MS allowing theacquisition of very informative fragment ion spectra fromsodiated and deprotonated glycan species.

Other powerful separation approaches for glycans andglycoconjugates comprise capillary electrophoresis, graphitizedcarbon HPLC, and high-pH anion exchange chromatography(requires on-line desalting), which are all compatible with onlineMS detection. The future impact of HILIC on glycoconjugateanalysis, as compared to alternative separation techniques, willdepend on the further improvements of the chromatography. Inorder to obtain faster separations with a better chromatographicresolution, new column material will have to be established,either silica-based with smaller particle size, or monolithic.

ACKNOWLEDGMENTS

We thank Carolien A.M. Koeleman for expert technical assis-tance. We thank Prof. Dr. J.P. Kamerling (Utrecht University) forthe kind gift of human b-HCG.

REFERENCES

Alpert AJ. 1990. Hydrophilic-interaction chromatography for the separation

of peptides, nucleic acids and other polar compounds. J Chromatogr

499:177–196.

Alpert AJ, Shukla M, Shukla AK, Zieske LR, Yuen SW, Ferguson MA,

Mehlert A, Pauly M, Orlando R. 1994. Hydrophilic-interaction

chromatography of complex carbohydrates. J Chromatogr A 676:

191–202.

Anumula KR. 2000. High-sensitivity and high-resolution methods for

glycoprotein analysis. Anal Biochem 283:17–26.

Anumula KR. 2006. Advances in fluorescence derivatization methods for

high-performance liquid chromatographic analysis of glycoprotein

carbohydrates. Anal Biochem 350:1–23.

Anumula KR. 2008. Unique anthranilic acid chemistry facilitates profiling

and characterization of Ser/Thr-linked sugar chains following hydra-

zinolysis. Anal Biochem 373:104–111.

Ashline DJ, Lapadula AJ, Liu YH, Lin M, Grace M, Pramanik B, Reinhold

VN. 2007. Carbohydrate structural isomers analyzed by sequential

mass spectrometry. Anal Chem 79:3830–3842.

Brons C, Olieman C. 1983. Study of the high-performance liquid chromato-

graphic separation of reducing sugars, applied to the determination of

lactose in milk. J Chromatogr 259:79–86.

Butler M, Quelhas D, Critchley AJ, Carchon H, Hebestreit HF, Hibbert RG,

Vilarinho L, Teles E, Matthijs G, Schollen E, Argibay P, Harvey DJ,

Dwek RA, Jaeken J, Rudd PM. 2003. Detailed glycan analysis of serum

glycoproteins of patients with congenital disorders of glycosylation

indicates the specific defective glycan processing step and provides an

insight into pathogenesis. Glycobiology 13:601–622.

Charlwood J, Birrell H, Bouvier ES, Langridge J, Camilleri P. 2000. Analysis

of oligosaccharides by microbore high-performance liquid chromatog-

raphy. Anal Chem 72:1469–1474.

Churms SC. 1996. Recent progress in carbohydrate separation by high-

performance liquid chromatography based on hydrophilic interaction.

J Chromatogr A 720:75–91.



Garner B, Merry AH, Royle L, Harvey DJ, Rudd PM, Thillet J. 2001.

Structural elucidation of the N- and O-glycans of human apolipopro-

tein(a): Role of o-glycans in conferring protease resistance. J Biol Chem

276:22200–22208.

Geyer H, Wuhrer M, Resemann A, Geyer R. 2005. Identification and

characterization of keyhole limpet hemocyanin (KLH) N-glycans

mediating cross-reactivity with Schistosoma mansoni. J Biol Chem

280:40731–40748.

Guile GR, Rudd PM, Wing DR, Prime SB, Dwek RA. 1996. A rapid

high-resolution high-performance liquid chromatographic method for

separating glycan mixtures and analysing oligosaccharide profiles. Anal

Biochem 240:210–226.

Guillarme D, Nguyen DT, Rudaz S, Veuthey JL. 2007. Recent developments

in liquid chromatography-Impact on qualitative and quantitative

performance. J Chromatogr A 1149:20–29.

Hagglund P, Bunkenborg J, Elortza F, Jensen ON, Roepstorff P. 2004. A new

strategy for identification of N-glycosylated proteins and unambiguous

assignment of their glycosylation sites using HILIC enrichment and

partial deglycosylation. J Proteome Res 3:556–566.

Harvey DJ. 1999. Matrix-assisted laser desorption/ionization mass spectro-

metry of carbohydrates. Mass Spectrom Rev 18:349–450.

Harvey DJ. 2005a. Collision-induced fragmentation of negative ions from N-

linked glycans derivatized with 2-aminobenzoic acid. J Mass Spectrom

40:642–653.

Harvey DJ. 2005b. Fragmentation of negative ions from carbohydrates: Part

2. Fragmentation of high-mannose N-linked glycans. J Am Soc Mass

Spectrom 16:631–646.

Harvey DJ. 2005c. Fragmentation of negative ions from carbohydrates: Part

3. Fragmentation of hybrid and complex N-linked glycans. J Am Soc

Mass Spectrom 16:647–659.

Hase S. 1994. High-performance liquid chromatography of pyridylaminated

saccharides. Methods Enzymol 230:225–236.

Hemstrom P, Irgum K. 2006. Hydrophilic interaction chromatography. J Sep

Sci 29:1784–1821.

Herbreteau B, Lafosse M, Morin-Allory L, Dreux M. 1992. High performance

liquid chromatography of raw sugars and polyols using bonded silica

gels. Chromatographia 33:325–330.

Holst O. 2007. The structures of core regions from enterobacterial

lipopolysaccharides—An update. FEMS Microbiol Lett 271:3–11.

Juhasz P, Martin SA. 1997. The utility of nonspecific proteases in the

characterization of glycoproteins by high-resolution time-of-flight

mass spectrometry. Int J Mass Spectrom Ion Processes 169:217–230.

Karlsson NG, Schulz BL, Packer NH. 2004. Structural determination of

neutral O-linked oligosaccharide alditols by negative ion LC-electro-

spray-MSn. J Am Soc Mass Spectrom 15:659–672.

Karlsson NG, Wilson NL, Wirth HJ, Dawes P, Joshi H, Packer NH. 2004.

Negative ion graphitised carbon nano-liquid chromatography/mass

spectrometry increases sensitivity for glycoprotein oligosaccharide

analysis. Rapid Commun Mass Spectrom 18:2282–2292.

Karlsson NG, Schulz BL, Packer NH, Whitelock JM. 2005. Use of graphitised

carbon negative ion LC-MS to analyse enzymatically digested

glycosaminoglycans. J Chromatogr B Analyt Technol Biomed Life

Sci 824:139–147.

Kessler MJ, Mise T, Ghai RD, Bahl OP. 1979. Structure and location of the

O-glycosidic carbohydrate units of human chorionic gonadotropin.

J Biol Chem 254:7909–7914.

Lamari FN, Kuhn R, Karamanos NK. 2003. Derivatization of carbohydrates

for chromatographic, electrophoretic and mass spectrometric structure

analysis. J Chromatogr B Analyt Technol Biomed Life Sci 793:15–36.

Lewandrowski U, Resemann A, Sickmann A. 2005. Laser-induced

dissociation/high-energy collision-induced dissociation fragmentation

using MALDI-TOF/TOF-MS instrumentation for the analysis of

neutral and acidic oligosaccharides. Anal Chem 77:3274–3283.

Li RP, Huang JX. 2004. Chromatographic behavior of epirubicin and its

analogues on high-purity silica in hydrophilic interaction chromato-

graphy. J Chromatogr A 1041:163–169.

Maslen S, Sadowski P, Adam A, Lilley K, Stephens E. 2006. Differentiation

of isomeric N-glycan structures by normal-phase liquid chromatog-

raphy-MALDI-TOF/TOF tandem mass spectrometry. Anal Chem 78:

8491–8498.

Maslen SL, Goubet F, Adam A, Dupree P, Stephens E. 2007. Structure

elucidation of arabinoxylan isomers by normal phase HPLC-MALDI-

TOF/TOF-MS/MS. Carbohydr Res 342:724–735.

Mattu TS, Royle L, Langridge J, Wormald MR, Van den Steen PE,

Van Damme J, Opdenakker G, Harvey DJ, Dwek RA, Rudd PM. 2000.

O-glycan analysis of natural human neutrophil gelatinase B using a

combination of normal phase-HPLC and online tandem mass

spectrometry: Implications for the domain organization of the enzyme.

Biochemistry 39:15695–15704.

Mechref Y, Novotny MV, Krishnan C. 2003. Structural characterization of

oligosaccharides using MALDI-TOF/TOF tandem mass spectrometry.

Anal Chem 75:4895–4903.

Morelle W, Page A, Michalski JC. 2005. Electrospray ionization ion trap mass

spectrometry for structural characterization of oligosaccharides

derivatized with 2-aminobenzamide. Rapid Commun Mass Spectrom

19:1145–1158.

Morelle W, Slomianny MC, Diemer H, Schaeffer C, van Dorsselaer A,

Michalski JC. 2005. Structural characterization of 2-aminobenzamide-

derivatized oligosaccharides using a matrix-assisted laser desorption/

ionization two-stage time-of-flight tandem mass spectrometer. Rapid

Commun Mass Spectrom 19:2075–2084.

Naidong W. 2003. Bioanalytical liquid chromatography tandem mass

spectrometry methods on underivatized silica columns with aqueous/

organic mobile phases. J Chromatogr B Analyt Technol Biomed Life

Sci 796:209–224.

Neville DC, Coquard V, Priestman DA, te Vruchte DJ, Sillence DJ, Dwek RA,

Platt FM, Butters TD. 2004. Analysis of fluorescently labeled

glycosphingolipid-derived oligosaccharides following ceramide glyca-

nase digestion and anthranilic acid labeling. Anal Biochem 331:275–

282.

Nyame AK, Kawar ZS, Cummings RD. 2004. Antigenic glycans in parasitic

infections: Implications for vaccines and diagnostics. Arch Biochem

Biophys 426:182–200.

Ohara K, Sano M, Kondo A, Kato I. 1991. Two-dimensional mapping by

high-performance liquid chromatography of pyridylamino oligosac-

charides from various glycosphingolipids. J Chromatogr A 586:35–41.

Plumb R, Castro-Perez J, Granger J, Beattie I, Joncour K, Wright A. 2004.

Ultra-performance liquid chromatography coupled to quadrupole-

orthogonal time-of-flight mass spectrometry. Rapid Commun Mass

Spectrom 18:2331–2337.

Prater BD, Anumula KR, Hutchins JT. 2007. Automated sample preparation

facilitated by PhyNexus MEA purification system for oligosaccharide

mapping of glycoproteins. Anal Biochem 369:202–209.

Prien JM, Huysentruyt LC, Ashline DJ, Lapadula AJ, Seyfried TN, Reinhold

VN. 2008. Differentiating N-linked glycan structural isomers in

metastatic and non-metastatic tumor cells using sequential mass

spectrometry. Glycobiology 18:353–366.

Qian J, Liu T, Yang L, Daus A, Crowley R, Zhou Q. 2007. Structural

characterization of N-linked oligosaccharides on monoclonal antibody

cetuximab by the combination of orthogonal matrix-assisted laser

desorption/ionization hybrid quadrupole-quadrupole time-of-flight

tandem mass spectrometry and sequential enzymatic digestion. Anal

Biochem 364:8–18.

Royle L, Mattu TS, Hart E, Langridge JI, Merry AH, Murphy N, Harvey DJ,

Dwek RA, Rudd PM. 2002. An analytical and structural database

provides a strategy for sequencing O-glycans from microgram

quantities of glycoproteins. Anal Biochem 304:70–90.



Royle L, Roos A, Harvey DJ, Wormald MR, Gijlswijk-Janssen D, Redwan E,

Wilson IA, Daha MR, Dwek RA, Rudd PM. 2003. Secretory IgA N- and

O-glycans provide a link between the innate and adaptive immune

systems. J Biol Chem 278:20140–20153.

Rudd PM, Guile GR, Kuster B, Harvey DJ, Opdenakker G, Dwek RA. 1997.

Oligosaccharide sequencing technology. Nature 388:205–207.

Rudd PM, Colominas C, Royle L, Murphy N, Hart E, Merry AH, Hebestreit

HF, Dwek RA. 2001. A high-performance liquid chromatography based

strategy for rapid, sensitive sequencing of N-linked oligosaccharide

modifications to proteins in sodium dodecyl sulphate polyacrylamide

electrophoresis gel bands. Proteomics 1:285–294.

Saba JA, Shen X, Jamieson JC, Perreault H. 2001. Investigation of different

combinations of derivatization, separation methods and electrospray

ionization mass spectrometry for standard oligosaccharides and glycans

from ovalbumin. J Mass Spectrom 36:563–574.

Saba JA, Kunkel JP, Jan DC, Ens WE, Standing KG, Butler M, Jamieson JC,

Perreault H. 2002. A study of immunoglobulin G glycosylation in

monoclonal and polyclonal species by electrospray and matrix-assisted

laser desorption/ionization mass spectrometry. Anal Biochem 305:16–31.

Shilova NV, Bovin NV. 2003. Fluorescent labels for the analysis of mono- and

oligosaccharides. Russ J Bioorg Chem 29:309–324.

Spina E, Sturiale L, Romeo D, Impallomeni G, Garozzo D, Waidelich D,

Glueckmann M. 2004. New fragmentation mechanisms in matrix-

assisted laser desorption/ionization time-of-flight/time-of-flight tan-

dem mass spectrometry of carbohydrates. Rapid Commun Mass

Spectrom 18:392–398.

Tajiri M, Yoshida S, Wada Y. 2005. Differential analysis of site-specific

glycans on plasma and cellular fibronectins: Application of a hydro-

philic affinity method for glycopeptide enrichment. Glycobiology 15:

1332–1340.

Takahashi N. 1996. Three-dimensional mapping of N-linked oligosacchar-

ides using anion-exchange, hydrophobic and hydrophilic interaction

modes of high-performance liquid chromatography. J Chromatogr A

720:217–225.

Takegawa Y, Deguchi K, Ito H, Keira T, Nakagawa H, Nishimura S. 2006a.

Simple separation of isomeric sialylated N-glycopeptides by a

zwitterionic type of hydrophilic interaction chromatography. J Sep

Sci 29:2533–2540.

Takegawa Y, Deguchi K, Keira T, Ito H, Nakagawa H, Nishimura SI. 2006b.

Separation of isomeric 2-aminopyridine derivatized N-glycans and N-

glycopeptides of human serum immunoglobulin G by using a

zwitterionic type of hydrophilic-interaction chromatography. J Chro-

matogr A 1113:177–181.

Temporini C, Perani E, Calleri E, Dolcini L, Lubda D, Caccialanza G,

Massolini G. 2007. Pronase-immobilized enzyme reactor: An approach

for automation in glycoprotein analysis by LC/LC-ESI/MSn. Anal

Chem 79:355–363.

Thomsson KA, Karlsson NG, Hansson GC. 1999. Liquid chromatography-

electrospray mass spectrometry as a tool for the analysis of sulfated

oligosaccharides from mucin glycoproteins. J Chromatogr A 854:131–

139.

Thomsson KA, Karlsson H, Hansson GC. 2000. Sequencing of sulfated

oligosaccharides from mucins by liquid chromatography and electro-

spray ionization tandem mass spectrometry. Anal Chem 72:4543–

4549.

Tolstikov VV, Fiehn O. 2002. Analysis of highly polar compounds of plant

origin: Combination of hydrophilic interaction chromatography and

electrospray ion trap mass spectrometry. Anal Biochem 301:298–307.

Varki A, Cummings R, Esko JD, Freeze HH, Hart GW, Marth J. 1999.

Essential of glycobiology. Cold Spring Harbour Laboratory Press, NY.

Wada Y, Tajiri M, Yoshida S. 2004. Hydrophilic affinity isolation and MALDI

multiple-stage tandem mass spectrometry of glycopeptides for

glycoproteomics. Anal Chem 76:6560–6565.

Wuhrer M, Deelder AM. 2005. Negative-mode MALDI-TOF/TOF-MS of

oligosaccharides labeled with 2-aminobenzamide. Anal Chem 77:

6954–6959.

Wuhrer M, Kantelhardt SR, Dennis RD, Doenhoff MJ, Lochnit G, Geyer R.

2002. Characterization of glycosphingolipids from Schistosoma

mansoni eggs carrying Fuc(a1-3)GalNAc-, GalNAc(b1-4)[Fuc(a1-3)]-

GlcNAc- and Gal(b1-4)[Fuc(a1-3)]GlcNAc- (Lewis X) terminal

structures. Eur J Biochem 269:481–493.

Wuhrer M, Koeleman CAM, Deelder AM, Hokke CH. 2004a. Normal-phase

nanoscale liquid chromatography-mass spectrometry of underivatized

oligosaccharides at low-femtomole sensitivity. Anal Chem 76:833–838.

Wuhrer M, Koeleman CAM, Hokke CH, Deelder AM. 2004b. Nano-scale

liquird chromatography-mass spectrometry of 2-aminobenzamide-

labeled oligosaccharides at low femtomole sensitivity. Int J Mass Spec

232:51–57.

Wuhrer M, Robijn ML, Koeleman CA, Balog CI, Geyer R, Deelder AM,

Hokke CH. 2004c. A novel Gal(beta1-4)Gal(beta1-4)Fuc(alpha1-6)-

core modification attached to the proximal N-acetylglucosamine of

keyhole limpet haemocyanin (KLH) N-glycans. Biochem J 378:625–

632.

Wuhrer M, Deelder AM, Hokke CH. 2005. Protein glycosylation analysis by

liquid chromatography-mass spectrometry. J Chromatogr B 825:124–

133.

Wuhrer M, Koeleman CAM, Hokke CH, Deelder AM. 2005. Protein

glycosylation analyzed by normal-phase nano-liquid chromatography–

mass spectrometry of glycopeptides. Anal Chem 77:886–894.

Wuhrer M, Koeleman CAM, Deelder AM, Hokke CH. 2006. Repeats of

LacdiNAc and fucosylated LacdiNAc on N-glycans of the human

parasite Schistosoma mansoni. FEBS J 273:347–361.

Wuhrer M, Catalina MI, Deelder AM, Hokke CH. 2007. Glycoproteomics

based on tandem mass spectrometry of glycopeptides. J Chromatogr B

849:115–128.

Xia B, Kawar ZS, Ju T, Alvarez RA, Sachdev GP, Cummings RD. 2005.

Versatile fluorescent derivatization of glycans for glycomic analysis.

Nat Methods 2:845–850.

Yu YQ, Gilar M, Kaska J, Gebler JC. 2005. A rapid sample preparation

method for mass spectrometric characterization of N-linked glycans.

Rapid Commun Mass Spectrom 19:2331–2336.

Yu YQ, Fournier J, Gilar M, Gebler JC. 2007. Identification of N-linked

glycosylation sites using glycoprotein digestion with pronase prior to

MALDI tandem time-of-flight mass spectrometry. Anal Chem 79:

1731–1738.

Zaia J. 2004. Mass spectrometry of oligosaccharides. Mass Spectrom Rev

23:161–227.

Zhang J, Wang DIC. 1998. Quantitative analysis and process monitoring

of site-specific glycosylation microhetereogeneity in recombinant

human interferon-gamma from Chinese hamster ovary cell culture

by hydrophilic interaction chromatography. J Chromatogr B 712:

73–82.

Zhao J, Qiu WL, Simeone DM, Lubman DM. 2007. N-linked glycosylation

profiling of pancreatic cancer serum using capillary liquid phase

separation coupled with mass spectrometric analysis. J Proteome Res

6:1126–1138.



Manfred Wuhrer graduated in Biochemistry (1995) at the University of Regensburg,

Germany. He did his Ph.D. (1996–1999) and a postdoc (2000–2002) at the University of

Giessen, Germany in the field of glycoconjugate structural analysis. He is working as a

Research Associate (2003–2004) and Assistant Professor (since 2005) at the Biomolecular

Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center.

His research covers method development for glycosylation analysis, the study of protein

glycosylation in various autoimmune diseases, infectious diseases, and cancer, as well as

the use of natural glycan microarray technology for the characterization of protein-

carbohydrate interactions.

Arjen R. de Boer received his MSc and his Ph.D. in Chemistry from VU University

Amsterdam, in 2001 and 2007, respectively. Currently, as a scientific researcher at the

Biomolecular Mass Spectrometry Unit (Department of Parasitology, Leiden University

Medical Center), he is performing research related to the development of glycan

microarrays with an emphasis on the immobilization of carbohydrates isolated from

biological materials. The glycan microarrays are applied for research in cancer as well in

bacterial and parasitic infections.

Andre M. Deelder obtained his master in Biology (Zoology, Parasitology) at Leiden

University in 1971. In 1973 he obtained his Ph.D. in Leiden on research on the

immunology of helminth infections. In 1978 he became associate professor and group

leader of the schistosomiasis group and in 1985 he was appointed as full professor and head

of the Department of Parasitology of the LUMC. His research has focused on the

immunology and epidemiology of schistosomiasis, the glycobiology of schistosomiasis

and on structural and functional studies of parasite glycoconjugates. A special interest was

the development of anti-glycan monoclonal antibodies and their application in immuno-

assays for detection of schistosome circulating antigens. The last seven years he has set up

the LUMC Biomolecular Mass Spectrometry Unit with research focused on clinical

(glyco)proteomics and metabolomics.



Documents

Structural glycomics using hydrophilic interaction chromatography (HILIC) with mass spectrometry