off-line capillary electrophoresis/fully automated nanoelectrospray chip quadrupole time-of-flight...

JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 2004; 39: 1190–1201Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.707

Off-line capillary electrophoresis/fully automatednanoelectrospray chip quadrupole time-of-flight massspectrometry and tandem mass spectrometryfor glycoconjugate analysis†

Laura Bindila,1 Reinaldo Almeida,2 Alistair Sterling,2 Mark Allen,2 Jasna Peter-Katalinic1

and Alina Zamfir1∗

1 Institute for Medical Physics and Biophysics, University of Munster, Robert Koch Strasse 31, D-48149 Munster, Germany2 Advion BioSciences, Norwich, UK

Received 16 June 2004; Accepted 6 August 2004

Implementation and optimization of an off-line capillary electrophoresis (CE)/(−)nanoESIchip-quadrupoletime-of-flight (QTOF) mass spectrometric (MS) and tandem MS system for compositional mapping andstructural investigation of components in complex carbohydrate mixtures is described. The approach wasdeveloped for glycoscreening and applied to O-glycosylated peptides from urine of a patient suffering froma-N-acetylhexosaminidase deficiency, known as Schindler’s disease. The fundamental issue of sensitivity,previously representing a serious drawback of the off-line CE/MS analysis, could be positively addressedby the off-line conjunction of CE with automated chip-based ESI-QTOF-MS to provide flexibility forCE/chip MS coupling and enhance structural elucidation of single components in heterogeneous mixtures.Copyright 2004 John Wiley & Sons, Ltd.

KEYWORDS: capillary electrophoresis; chip nanoelectrospray ionization; quadrupole time-of-flight mass spectrometry;automation; glycoconjugates

INTRODUCTION

The current goal in the glycoanalytical field is defined asa search for highly sensitive methods to be applied to theidentification and structural elucidation of glycoconjugatesoriginating from biological sources. Nanoelectrospray ion-ization mass spectrometry (nanoESI-MS) has emerged inthe past decade as a powerful tool for investigations ofcarbohydrate structure.1 – 5

Carbohydrate mixtures from biological matrices show ahigh degree of heterogeneity regarding the composition andstructural diversity. Therefore, the combination of coupledseparation techniques such as capillary electrophoresis (CE)or liquid chromatography (LC) with MS is currently con-sidered a powerful tool for further developments.6 – 10 CEhas been established as an independent and versatile ana-lytical technique for biomolecule separation.11 – 13 Combinedwith ESI-MS, it provides an appealing platform for the fast,automated, miniaturized and highly efficient separation andidentification of peptides, proteins and nucleic acids.14 – 19

Carbohydrates, a major class of biopolymers, require in gen-eral some particular efforts in order to be analyzed by CE

ŁCorrespondence to: Alina Zamfir, Institute for Medical Physicsand Biophysics, University of Munster, Robert Koch Strasse 31,D-48149 Munster, Germany. E-mail: [email protected]†Presented at the 52nd ASMS Conference in Nashville, TN, USA.

because of restrictive conditions required for the ion forma-tion on the one hand and for their separation according tothe high level of structural complexity on the other. A highpotential of CE techniques in carbohydrate species charac-terization, however, was recently demonstrated by us andothers.20 – 25 It was reported that, under specific optimizedanalyte preparation and instrument conditions, off-line andon-line CE/MS is a viable alternative for the analysis ofcomponents from complex carbohydrate mixtures, makinguse of the high resolving power of CE on the one handand the capability of structural elucidation by MS with highsensitivity and accuracy on the other.

Off-line CE/MS might be generally considered as aconvenient approach owing to its flexibility towards systemoptimization, since the respective experimental conditionsfor the CE and MS instruments can be set separately.Additionally, it offers the possibility of using differenttypes and configurations of MS ion sources. The off-linestrategy includes fraction collection, which can be performedusing different techniques such as calculating the timewindow when a compound has migrated to the end ofthe capillary or using a pre-run to estimate the migrationvelocities. However, by using the CE instrument as a fractioncollector, the eluted sample is diluted by an electrolytein sample reservoirs which provide analyte volumes offew nanoliters collected into 5–10 µl. Lack of sensitivityis a specific and well-known drawback of this approach.

Copyright 2004 John Wiley & Sons, Ltd.

Off-line CE/MS for glycoconjugate analysis 1191

In order to increase the throughput and sensitivity, asignificant potential is offered by the approach of chip-basedMS. Fully automated nanoelectrospray chip-based MS hasbeen so far successfully implemented for peptides, proteins,and drug development.26 – 30 In the field of carbohydrateanalysis, the optimization of automated chip-based ESI-MSfor the determination of the dissociation constant of the non-covalent interaction between cellulase and oligosaccharideswas described by Zhang et al.31 and our group reported forthe first time the implementation of fully automated chip-based nanoESI-QTOF-MS and -MS/MS for glycoscreeningand identification surveys.32

As a part of our focus on the implementation ofmicrofluidic-based methods for the investigation of glyco-conjugates from biological sources, the aim of the presentstudy was the development of a reliable CE/(�)nanoESIchip-quadrupole time-of-flight (QTOF) MS platform for composi-tional mapping and structural investigation by MS/MS. Theapproach was developed for glycoscreening and applied toa complex mixture of O-glycosylated peptides and aminoacids from the urine of patients suffering from a hereditary˛-N-acetylhexosaminidase deficiency known as Schindler’sdisease. Schindler’s disease is a rare inherited metabolic dis-order characterized by a deficiency of the lysosomal enzyme˛-N-acetylgalactosaminidase, which leads to an abnormalaccumulation of sialylated and asialo-glycopeptides andoligosaccharides with ˛-N-acetylgalactosaminyl residues.The deficient N-acetylhexosaminidase causes a 100 timeshigher concentration of O-glycans in urine than in healthycontrols. For this reason, screening, structural characteriza-tion and complete identification of O-glycosylated aminoacids and peptides extracted from patients’ urine is of majordiagnostic importance.33

Negative ion detection has been shown to be advanta-geous over positive ion detection for mapping and sequenc-ing of glycoconjugates.34,35 In particular, the assignmentof already known or previously unknown components inbiological mixtures is easier owing to the lower degree of het-erogeneity caused by cation attachment to molecular and/orfragment ions.36,37

The potential of the off-line conjunction of CE withautomated chip-based negative ion ESI-QTOF-MS to addressthe issue of off-line CE/MS sensitivity, to provide flexibilityfor CE/MS coupling and to offer the structural elucidationof single molecules in complex mixture by chip ESI-MS/MSanalysis is presented.

EXPERIMENTAL

Reagents and materialsMethanol, 98% formic acid and 32% ammonia were pur-chased from Merck (Darmstadt, Germany). Distilled, deion-ized water from a Milli-Q water system (Millipore, Bedford,MA, USA) was used for the preparation of the CE bufferand sample solutions. The buffer was prepared at a concen-tration of 0.1 M formic acid as a methanol/water (6 : 4, v/v)solution and adjusted with ammonia to pH 2.8. Prior to use,all solvents were filtered through 0.2 µm disposable filterunits from Schleicher and Schuell (Dassel, Germany). Aque-ous sample solutions were dried in a SpeedVac SPD 111V

evaporator (Savant, Dusseldorf, Germany). The pH of the CEbuffer was adjusted with a Model 766 Calimatic pH-meter(Knick, Germany). Externally polyimide-coated fused-silicaCE capillaries were obtained from BGB Analytic Vertrieb(Essen, Germany). The sample and buffer solutions werecentrifuged for 1 h in an Eppendorf (Hamburg, Germany)Model 5415 C centrifuge.

Biological sampleA mixture of O-glycosylated sialylated amino acids andpeptides, denoted BQ5, was obtained from urine of patientB.P. suffering from Schindler’s disease type I as describedpreviously.38,39 Briefly, for isolation of components, thepatient’s urine was first filtered and submitted to gel filtrationchromatography on Biogel P2. The glycans were separated bygel filtration chromatography performed on Fractogel TSKHW 50 in 0.01 M pyridinium acetate (pH 5.4) as eluting bufferand pre-fractionated by anion-exchange chromatographyusing a monoQ column.40

A stock sample/water solution of 1 µg µl�1 of BQ5was prepared and stored at �70 °C. Working solutions of5 pmol µl�1 (calculated for a 2500 Da average molecularmass) for direct (�)nanoESIchip-QTOF-MS screening and5 µg µl�1 for fraction collection were prepared by drying thestock solution and further dissolving it in the CE buffersystem.

Capillary electrophoresisCE/UV experiments were carried out on a PACE 5000instrument (Beckman, Fullerton, CA, USA) equipped witha UV detector. The separated components were detectedat 214 nm. Fused-silica capillaries (375 µm o.d., 75 µmi.d.) internally uncoated were used throughout the entirestudy. CE separation was performed in reverse polarityby applying a �30 kV separation voltage over a 100 cmcapillary length. The CE components were collected inmicrovials containing 15 µl of CE buffer solution accordingto the procedure described previously.20 The CE capillarywas flushed between runs for 30 min with methanol,dried by an air flow for 15 min, followed by conditioningwith the running buffer for 20 min. The sample washydrodynamically injected into the CE capillary by applying0.5 psi pressure for 6 s.

Mass spectrometryMass spectrometry was performed on an orthogonal hybridquadrupole time-of-flight mass spectrometer (QTOF, Micro-mass, Manchester, UK) in Z-spray geometry. The QTOF massspectrometer was interfaced with a PC running MassLynxNT software to control the instrument, acquire and processthe MS data. All mass spectra were acquired in the negativeion mode, which has been shown to be advantageous tocarbohydrate ion species formation.34,35 The sampling conepotential was varied within the range 20–40 V to generateefficient ionization of the components. The ion source tem-perature was set at 80 °C. For all experiments the desolvationgas was used at a 50 l h�1 flow-rate. Collision-induced disso-ciation (CID) experiments were performed at low energies.The LM and HM values on the QTOF instrument for ion

Copyright 2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 1190–1201

1192 L. Bindila et al.

isolation were set for all the MS/MS experiments at 10 and10, respectively. The MS/MS signals were acquired within20–45 eV collision energy values using Ar as a collision gasat a pressure of 12 psi. All spectra were calibrated usingsodium iodide as a calibrant. The nomenclature of the frag-ment ions followed generally the rules established by Domonand Costello.41 For a better description of the charge state andmore precise assignment of the fragment ions, we applied forthe internal fragments, i.e. ion at m/z 469.33, the description,Y2˛/B1ˇ.2,3,22,32

Fully automated nanoESIchipThe NanoMate 100 (Advion BioSciences, Ithaca, NY, USA)is a fully automated chip-based nanoelectrospray ionizationsystem that aspirates samples from a 96-well glass-coatedplate using disposable conductive tips, to the back of anESI chip. The chip consists of a 10 ð 10 microarray ofindependent nanoelectrospray nozzles etched into the planarsurface of a silicon wafer system. Each nozzle has an internaldiameter of 10 µm. The robot is controlled and manipulatedby ChipSoft software operating under the Windows system.The position of the electrospray chip was adjusted withrespect to the sampling cone to give rise to efficient ionizationand optimal transfer of the ionic species into the massspectrometer. A volume of 5 µl of sample solution followedby 2 µl of air were aspirated into the pipette tip and thendelivered to the inlet of the microchip. The preparation ofthe microchip is described in detail elsewhere.27 A channelextends from the nozzle through the microchip to an inleton the opposite surface and, during analysis, the conductivepipette tip containing the sample is engaged against thisinlet, forming a pressure seal. Electrospray was initiatedby applying voltages in the range 1.45–1.67 kV and a headpressure between 0.3 and 0.5 psi. Following sample infusionand MS analysis, the pipette tip was ejected and a fresh tipand nozzle were used for each sample, thus preventing anycross-contamination or carry-over.

RESULTS AND DISSCUSION

Optimization of off-lineCE/(−)nanoESIchip-QTOF-MSThe off-line coupling of CE to (�)nanoESI-QTOF-MS hasbeen intensively developed and implemented by our groupfor glycoconjugate analysis. Its potential for the separation,screening and structural elucidation of different types of gly-coconjugates such as glycosaminoglycans,23,24 gangliosides22

and O-glycosylated peptides20,21 was demonstrated in detail.However, it was reported that in the case of off-line cou-pling of CE to MS, a major drawback of the method isthe lack of sensitivity, which is a critical requirement espe-cially when dealing with biological mixtures. Therefore, inthe present study, we focused on the development andimplementation of the off-line coupling of CE with a moresensitive detection mode, in particular NanoMate chip-basednanoESI-QTOF-MS. To our knowledge, a combination of CEwith NanoMate/MS has not been reported previously. Theexperiments were conducted on a mixture of glycopeptidesand glycosylated amino acids, BQ5, containing as dominant

structures already characterized O-glycosylated peptides toassess the method for biological samples.

To test the compatibility of off-line CE coupling to the(�)nanoESIchip NanoMate-QTOF-MS for the ionization anddetection of the analyte in a given electrolyte buffer system,the BQ5 mixture of O-glycosylated amino acids and peptidesfrom the urine of a patient suffering from Schindler’s diseasewas dissolved in 0.1 M formic acid/ammonia (pH 2.8) to aconcentration of 5 pmol µl�1. A constant and stable sprayover a 10 min analysis time and high total ion currentindicated that the components present in 5 µl of the buffersystem are efficiently ionized.

A solution of 5 µg µl�1 buffer of the BQ5 mixture wasinjected for 6 s into the CE capillary and collected in 15 µl ofbuffer solution as a single fraction at 95 min after injection torecover all components present as a control. The CE/UV pro-file of 1 µg µl�1 BQ5/buffer carried out in the reverse polarityat �30 kV separation voltage indicated that all the compo-nents in the analyte mixture migrate within 80 min. TheCE-collected BQ5/buffer solution was subjected, without anypost-collection treatment, to the (�)nanoESIchip-QTOF-MSexperiments. By careful alignment of the nanoelectrospraychip towards the skimmer and varying the sampling conepotential within the range 20–40 V, a fair ionization yieldand signal-to-noise ratio after only 2 min of signal acquisitionwere achieved.

The major components detected in the collected mixtureand their corresponding assignments to the structures arelisted in Table 1. The spectrum (Fig. 1) is dominated by Ser-and Thr-linked Neu5Ac2HexHexNAc observed as a doublycharged ion at m/z 525.34 and 532.34, singly charged ionsat m/z 760.33 and 774.34 corresponding to Ser- and Thr-linked Neu5AcHexHexNAc, followed by doubly chargedions at m/z 707.83 and 714.82 assigned to Ser- and Thr-linkedhexasaccharide, respectively.

Table 1. Major ions (m/z) and their corresponding assignmentto the structures detected in the BQ5 mixture by(�)nanoESIchip-QTOF-MS

[M � H]� [M � 2H]2� Structure assignment

290.36 Neu5Ac308.36 Neu5Ac469.37 HexHexNAc-Ser483.39 HexHexNAc-Thr1051.25 525.34 Neu5Ac2HexHexNAc-Ser1065.25 532.34 Neu5Ac2HexHexNAc-Thr1144.24 571.85 Neu5Ac2HexHexNAc-Thr-Pro/H2O1162.26 580.85 Neu5Ac2HexHexNAc-Thr-Pro673.34 Neu5AcHexHexNAc

707.83 Neu5Ac2Hex2HexNAc2-Ser714.82 Neu5Ac2Hex2HexNAc2-Thr

760.33 Neu5AcHexHexNAc-Ser774.34 Neu5AcHexHexNAc-Thr853.32 Neu5AcHexHexNAc-Thr-Pro/H2O871.32 Neu5AcHexHexNAc-Thr-Pro

890.27 Neu5Ac2Hex3HexNAc3-Ser964.28 Neu5Ac2HexHexNAc



300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 11500

100

%

×1.3'×2 532.34

525.34

308.36

290.36

309.37 377.33

1065.25

532.86

1051.25

774.34

543.33

707.83

580.85

543.85

544.35581.36

581.85632.34 673.34

760.33

708.33

714.82

715.33

718.82

775.32

964.28853.32796.32 890.27

1066.29

1087.241144.24

1088.25

1162.261163.221184.17

2-

2-

2-

2-

2-2-

2-

1-

1-

1- 1- 1-

1-

1-

1-

1-

1-

1-

1-

1-1-

1-

m/z

469.37 483.39

1125.23

Figure 1. (�)NanoESIchip-QTOF mass spectrum of CE-collected BQ5 at 95 min after injection. Solvent, 0.1 M formic acid bufferedwith ammonia to pH 2.8; injection time into the CE capillary, 6 s; BQ5/buffer concentration injected, 5 µg µl�1; acquisition time,2 min.

Thr-Pro-linked sialylated saccharides were visible in thespectrum at m/z 580.85 as a doubly charged ion correspond-ing to Neu5Ac2HexHexNAc-Thr-Pro and at m/z 871.36corresponding to the singly charged Neu5AcHexHexNAc-Thr-Pro. As reported previously,32 the application of chip-based nanoESI is advantageous for glycoconjugate analysisowing to two relevant factors: (a) the minimization of the in-source decay of labile groups such as sialic acids and fucosesand (b) the preferential generation of multiply charged ions.These aspects are clearly illustrated by the spectrum in Fig. 1,showing for (a) only low abundant in-source generated sialicacid C-ion at m/z 308 and a number of doubly chargedmolecular ions as predominant over the singly charged onesfor (b).

The CE/UV analysis of the BQ5 carried out in reversepolarity and on-line reverse polarity CE/(�)nanoESI-QTOF-MS42 (data not shown) together with our previouslyreported data obtained by CE/(�)nanoESI-QTOF-MS ofO-glycosylated peptides and amino acids from the urineof patients suffering from Schindler’s disease20,21 indicatedthat the order of migration is determined by the degreeof sialylation. Thus, in the first fraction the Thr-Pro-linked (disialylated) tetra- and hexasaccharide are found,

whereas the monosialo glycopeptides migrate at a slowerrate. The most abundant species found were the Ser- andThr-linked tetrasaccharides followed by the Ser- and Thr-hexasaccharides. The first fraction was collected within60 min after injection and the second fraction between 80and 115 min after injection.

The spectrum of the fraction collected at 80 min afterinjection, according to the CE/UV profile carried out inreverse polarity at �30 kV separation voltage42 (data notshown), is depicted in Fig. 2. Two major doubly charged ionsat m/z 525.28 and 532.29 are accompanied by their respectivesingly charged counterparts at m/z 1051.23 and 1065.23corresponding to Ser- and Thr-linked Neu5Ac2HexHexNAc.Ser- and Thr-linked Neu5AcHexHexNAc were observedas singly charged ions at m/z 760.24 and 774.28. Inlower abundance, the Ser-linked Neu5Ac2Hex2HexNAc2

was detected as a doubly charged ion at m/z 707.75. Twosingly charged ions at m/z 1103.18 and 1109.20 could notbe assigned. Although off-line CE/MS has been reportedto require fairly large amounts of analyte in the collectedfraction for detection, e.g. 10–20 µg µl�1 sample/solventinjected into the CE capillary15,20 for this approach, only5 µg µl�1 sample/buffer solution were injected into the CE



500 550

0

53

52

48

53

774.28

760.2454707.75

55

619.38708.25

1065.23

1051.23775.26

796.22 853.32 993.44939.42

1103.18

1109.20

1162.191177.43

2-

2-

1-

1-1-

1-2- 1-

1-

NeuAc2HexHexNAc-Ser

NeuAcHexHexNAc-Thr

NeuAc2HexHexNAc-Ser

NeuAc2HexHexNAc-Thr

NeuAc2Hex2HexNAc2-Ser

MS/MS

600 700

10053

525.28

483.34

532.78

774.28

543.77

55

%

650

m/z

532.29

774.28

555.29

673.36

2-

1-

1-

1-2-

1-

NeuAc2HexHexNAc-Thr

NeuAcHexHexNAc-Ser

MS/MS

MS/MS

750 800 850 900 950 1000 1050 1100 1150 1200

MS/MS

Figure 2. (�)NanoESIchip-QTOF mass spectrum of the second CE-collected fraction at 80 min after injection. Solvent, 0.1 M formicacid buffered with ammonia to pH 2.8; fraction/buffer concentration, ¾3 pmol µl�1.

capillary yielding a final concentration of the collectedfraction of ¾3 pmol µl�1. The ESI-QTOF-MS system wasprogrammed to record the signal at a scan speed of onescan per 2.1 s. The flow-rate of the NanoMate chip system isin the range 50–100 nl min�1. In the case of ammoniumformate, the flow-rate exhibited by the NanoMate chipsystem, calculated by spraying through it 5 µl of the CEbuffer system, was ¾75 nl min�1. Under these conditions, afair signal-to-noise ratio could still be obtained with only 30scans of signal accumulation, even for minor species (Fig. 2).Hence the sample consumption for generating the spectrumin Fig. 2 was estimated as ¾0.4 pmol. Further, using theoptimized chip-based (�)nanoESI-QTOF-MS procedure, aconstant and stable spray over the entire MS and MS/MS(see below) analysis time could be generated from only 4 µl ofthe CE-separated glycopeptides in the original CE buffer. Thetotal amount of sample consumption of the second collectedfraction for generating one MS and three MS/MS traces offair signal-to-noise ratio and sufficiently informative, wasestimated as 12 pmol.

Off-line CE/(−)nanoESIchip-QTOF-MS/MSAs the most abundant species, the doubly charged ion at m/z532.29 was subjected to low-energy CID (�) nanoESIchip-QTOF-MS/MS. The collision energy, collision gas pressureand precursor ion isolation parameters were carefullyadjusted to provide the full set of structural informationfor the molecule. The fragmentation spectrum of the doubly

charged ion at m/z 532.29 is depicted in Fig. 3. The spectrumobtained after 2 min of signal acquisition reveals a largenumber of fragment ions detected at a fairly high signal-to-noise ratio. The most abundant fragment ion detected as asingly charged ion at m/z 483.34, assigned to HexHexNAc-Thr, corresponds to the loss of both sialic acid moietiesfrom the precursor ion. The stripping of only one sialicacid moiety is indicated by the doubly charged ion at m/z386.81 and singly charged counterpart at m/z 774.28 alongwith the ion obtained by further neutral loss of H2O at m/z756.32 and of CO2 at m/z 730.36. Of particular interest forstructure assignment are the singly charged sequence ionsat m/z 673.36, assigned to Neu5AcHexHexNAc (C3/B1ˇ),at m/z 470.30, assigned to Neu5AcHex (C2˛) and atm/z 493.26, corresponding to Neu5AcHexNAc (C3/C2˛)diagnostic for the sialylation pattern of the molecule. Inaddition, the ion generated by the peptide bond cleavage,while both of the sialic acid entities remained attached,is evidenced by the doubly charged ion at m/z 481.79,assigned to Neu5Ac2HexHexNAc. The linkage positions andthe anomericity of the Ser- and Thr-linked tetrasaccharidewere described in an earlier report.43 Additionally, in Fig. 3the diagnostic ring cleavage ions detected as 0,4A2 ionat m/z 350.28 and at m/z 306.32 corresponding to the0,4A2/CO2 indicate a presence of a Neu5Ac˛2–6 linkage.44

The fragmentation pattern of the precursor ion at m/z 532.29is presented in Scheme 1. The nomenclature of the fragment



275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 825 850

100 x2 x2

290.28

386.

306.

308.

470.

387.

421.82

532.31

484.

510.

532.80

774.28

730.36756.32

775.33

m/z

%

0

x2 x2

386.81

306.32

308.30

470.30

387.32

532.31

484.32

510.30533.35

553.30673.36

B1α or B1β

Y2α or Y1β

Y2α /B1βor

Y1β/B1α

[M-2H]2-

[M-2H]2-/CO2

[M-2H]2-/H2O

C2α

C3/B1β

Y2α/CO2

orY1β/CO2

Z2α or Z1β

Y2α or Y1β

C1α or C1β

1-

1-

1-

1-

1-

2-1-

2-

2-

2-

1-1-

1-

1-

1-2-

523.29

493.26

C3/C2

481.792-

C3

483.34

0,4A2/CO2

0,4A21-350.28

Figure 3. (�)NanoESIchip-QTOF tandem mass spectrum of the doubly charged ion at m/z 532.29 corresponding toNeu5Ac2HexHexNAc-Thr detected in the second CE-collected fraction. Collision energy, 20–40 eV (Elab, the value set on the QTOFinstrument by the user); collision gas pressure, 12 psi (Plab, the value set on the QTOF instrument by the user). For precursor ionisolation, LM and HM parameters were set at 10 and 10, respectively. Nomenclature of the fragment ions is according to Domon andCostello.41

ions followed generally the rules established by Domon andCostello.41

A similar fragmentation pattern to that for the Thr-linked precursor ion species at m/z 532.29 (Fig. 3), regardingthe relative signal intensities and the generated sequenceions, was obtained in the same manner in the spectrumin Fig. 4 for the homologous Ser-linked tetrasaccharide.The fragmentation spectrum of the doubly charged ionat m/z 525.31 is presented in Fig. 4. The advantage ofthe introduction of off-line CE/chip-based ESI-QTOF-MSis highlighted here in the context of an accurate isolation ofthe precursor ion. No trace ions related to the Thr-linkedstructures were obtained, whereas in the MS/MS analysis,directly from the unseparated mixture, difficulties relatedto the fair isolation of precursor ions caused by the highcomplexity of the mixture are frequently encountered. Inanalogy with the spectrum of Thr-linked tetrasaccharide, theion at m/z 470.32 was assigned to a C2˛, although theoreticallyit could represent the isotopic peak of the ion at m/z 469.33.This assignment is supported by the presence of the same ionin the spectrum of the Thr-linked tetrasaccharide. Diagnosticring cleavage ions at m/z 350.28 and 306.32, respectively,reflect the same structural elements as in Fig. 3. The spectrum

acquired for 2 min at variable collision energies (CID-VE)24

generated a high coverage of sequence ions for reliableassignment of the molecular structure. The sequence patternof the ion detected in the second collected fraction at m/z525.31 is depicted in Scheme 2.

Owing to the spray stability and optimized conditionsfor ionization/detection of glycopeptide species in the CEfraction, the MS/MS investigation of a low-abundance iondetected as a doubly charged species at m/z 707.75 (Fig. 2),attributed to Neu5Ac2Hex2HexNAc2-Ser, was enhanced. Thelow signal-to-noise ratio of the ion was compensated by thelonger acquisition time of the MS/MS signal for about 15 min.The (�)nanoESIchip-QTOF-MS/MS of the doubly chargedion at m/z 707.75, recorded within a 20–40 eV collisionenergy range by CID-VE, is presented in Fig. 5. Even underrestrictive conditions for MS1 signal intensity, a sufficientnumber of ions as fingerprints for structural elucidation ofthe molecule could be generated and detected. Thus, thefragment corresponding to the cleavage of one sialic acidmoiety is visible as the most prominent peak at m/z 562.32(Y3˛

2� or Y2ˇ2�) detected also as a singly charged ion at m/z

1125.32 and accompanied by its counterpart at m/z 290.32.The successive stripping of the sialic acid is represented by



275 300 325 350 375 425 475 525 575 625 675 700 725

100

750400

%

550 600 650

0

Yα or Y1β

x6x4x6

290.30

379.79

306.32

308.30

350.28

414.80

380.32

415.32

525.31

470.32

503.31525.82

760.31

716.35

526.31

539.31673.27

761.31

762.28

2-

2-

2-

2-

1-

1-

1-

1-

1-

1-

2-

1-

1-

1-

1-

493.31

516.30

2-

1

Z2α or Z1β

Y2α/CO2

or

Y1β/CO2

C3/B1β

C2α

[M-2H]2-

[M-2H]2-/CO2

[M-2H]2-/H2O

Y2α or Y1β

B1α or B1β

C1α or C1β

C3

469.

742.32481.81

469.33

C3/C2α

Y2α/B1β

or

Y1β/B1α

0,4A2/CO2

0,4A2

450 500 775 900825 850 875800

m/z

Figure 4. (�)NanoESIchip-QTOF tandem mass spectrum of the doubly charged ion at m/z 525.28 corresponding toNeu5Ac2HexHexNAc-Ser detected in the second CE-collected fraction. Collision energy, 20–40 eV (Elab, the value set on the QTOFinstrument by the user); collision gas pressure, 12 psi (Plab, the value set on the QTOF instrument by the user). For precursor ionisolation, LM and HM parameters were set at 10 and 10, respectively. Nomenclature of the fragment ions is according to Domon andCostello.41

the singly charged fragment ion at m/z 834.33 (Y3˛/B1ˇ). Anumber of ions derived from neutral loss of H2O and CO2

from the precursor ion and the Ser-linked hexasaccharidesingly charged ion at m/z 1125.32 were detected (Scheme 3).The peptide bond cleavage gave rise to the singly charged ionat m/z 858.31 assigned to Neu5AcHexHexNAc2. Althoughat lower abundance, the Ser-linked trisaccharide fragmention at m/z 760.37 (Y1˛) and the singly charged ion at m/z672.32 assigned to HexHexNAc2-Ser could also be detected.The fragmentation pattern of the doubly charged ion at m/z707.75 is depicted in Scheme 3. The specific linkages andthe anomericity of the Ser-linked hexasaccharide indicatedin Scheme 3 are in accordance with evidence previouslydescribed.33,43

All data discussed above were obtained from a singleMS1 and three MS/MS data acquisitions for which only 4 µlof the analyte in the original CE buffer were consumed.

In order to test the sensitivity limit exhibited by the off-line CE/(�)nanoESIchip-QTOF-MS/MS of glycopeptides,1 µl of the second CE fraction was diluted to a ratio of1 : 5 in 0.1 M formic acid buffered with ammonia to pH 2.8and transferred into the glass plate of the robot for furtherMS/MS analysis. The singly charged ion at m/z 774.28

corresponding to Neu5AcHexHexNAc-Thr was subjectedto CID, rending the fragmentation spectrum combined over90 scans (Fig. 6). The complete set of sequence ions couldstill be obtained and detected at a concentration below1 pmol µl�1 CE fraction. The most abundant peak is thedesialylated fragment ion detected as a singly charged ion atm/z 483.34. Its counterpart was detected at m/z 290.30 as B1

and 308.32 as C1, respectively. Relevant ions for the structureelucidation were detected at a good signal-to-noise ratio atm/z 673.23 assigned to Neu5AcHexHexNAc, singly chargedat m/z 347.36 corresponding to the dehydrated HexHexNAc.Additionally, monosaccharide building block fragment ionsat m/z 202.30 and 179.29 are characteristic for HexNAc andHex, respectively. The neutral loss of H2O and CO2 from theprecursor ion is observed at m/z 756.32 and 730.36.

CONCLUSIONS

The implementation and optimization of off-line CE/fullyautomated chip-based (�)nanoESI-QTOF-MS for screeningand structural elucidation of glycopeptides originating fromurine of patients suffering from Schindler’s disease have



-CO2-H2O[M-2H]2- = 532 510(2-)523(2-)

-221

421(2-)-NeuAc

553(1-)

C3481(2-)

Y1β

774(1-)

Neu5Acα2

Neu5Acα2

HexNAcα1

Hexß1

6

3

3

Thr

B1β

290 (1-)

-44Y1β/CO2

730(1-)

OC1β

308(-1)

Z1β

756(1-)

B1α

290(1-)

O

O

C1α308(1-)

Y2α774(1- )

Z2α756(1-)

C2α470(1-)

C3/B1β673(1-)

C3/C2α493(1-)

-291

Y2α/B1β483(1- )

Y1β/B1α

483(1-)

Y1β/CO2

730(1-)-44

O

0,2X2α0,2X2α /B1β

-291

Scheme 1. Fragmentation scheme of the doubly charged ion at m/z 532.29 corresponding to Neu5Ac2HexHexNAc-Thr detected inthe second CE-collected fraction. Nomenclature of the fragment ions is according to Domon and Costello.41

6

3

3

Ser

O

O

O

O

Y1β/CO2

716(1-)-44

Neu5Acα2

Y2α760(1-)

-291

Y2α/B1β469(1-)

Z2α742(1-)

C3/C2α

493(1-)

Hexβ1

290(1-)

B1α

C1α

308(1-)

C1β308(1-)

Neu5Acα2B1β

290 (1-)

C2α470(1-)

HexNAcα1

Y1β/B1α

469(1-)-291 Y1β

760(1-)-44

Z1β742(1-)

Y1β/CO2

716(1-)

C3

481(2-)

C3/B1β

673(1-)

516(2-)-H2O

0,2X2α/B1β 539(-1)-NeuAc

414(2-)0,2X2α

-220

[M-2H]2- = 525-CO2

503(2-)

Scheme 2. Fragmentation scheme of the doubly charged ion at m/z 525.28 corresponding to Neu5Ac2HexHexNAc-Ser detected inthe second CE-collected fraction. Nomenclature of the fragment ions is according to Domon and Costello.41



350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 12500

100

%

×2 4×2×2 ×6290.32

562.32

424.33

291.30

335.35

353.37 426.32 525.31

834.33562.82

708.31

707.81

563.33

699.30

563.80672.32

575.31

708.81

709.31

760.37

835.32

1125.32

836.341107.32

1081.34858.31

1127.33

300

1-

1-

1-

2-1-

1-

2-

or

1-

or

or

1-

1- 1-

B1α or B1β

0,2A3

Y3α/B2β

Y2α/B1β

[M-2H]2-

[M-2H]2-/H2O

[M-2H]2-/CO2

Y3α or Y2β

Y2β/B1α

Y3α/B1β

Y1α

C4/C2β

Y2β/CO2

Y3α/CO2

Z3α or Z2β

Y3α or Y2β

m/z

Figure 5. (�)NanoESIchip-QTOF tandem mass spectrum of the doubly charged ion at m/z 707.75 assigned toNeu5Ac2Hex2HexNAc2-Ser detected in the second CE-collected fraction. Collision energy, 20–40 eV (Elab, the value set on theinstrument by the user); collision gas pressure, 12 psi (Plab, the value set on the QTOF instrument by the user). For precursor ionisolation, LM and HM parameters were set at 10 and 10, respectively. Nomenclature of the fragment ions is according to Domon andCostello.41

-CO2-H2O[M-2H]2- = 707 685(2-)699(2-)

Z2β1107 (1-)

Y2β1125(1-)

and 562(2-)

Neu5Acα2

3Hexβ1

6HexNAcα1

3Hexβ1

Neu5Acα2

O

O

O

O

SerO

B1β290 (1-)

C1β290 (1-)

B1α290 (1-)

C1α308 (1-)

Y3α1125(1-)

and 562(2-)

Z3α1107(1-)

C4 /C2β

858(1-)

Y1α760(1-)

-44

Y2β/CO2

1081(1-)

-44

Y3α/CO2

1081(1-)

3

O 4HexNAcα1

Scheme 3. Fragmentation scheme of the doubly charged ion at m/z 707.75 assigned to Neu5Ac2Hex2HexNAc2-Ser detected in thesecond CE-collected fraction. Nomenclature of the fragment ions is according to Domon and Costello.41



1-

120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 8000

100

%

179.29

Ο Ο Ο3Hexβ1 Thr

C3

673(1-)B3

655(1-)B1

290(1-)

Y2

483(1-)

Y1/Z0

-44

[M-H]- /CO2

730(1-)

[M-H]- = 774

-18

[M-H]- /H2O

756(1-)

C1

308(1-)

×24x4x10

774.34

483.34

142.29

126.29

347.36

290.30202.30

220.31

253.45 308.32

349.36

730.36

613.43484.35

577.45559.43

673.33655.27684.30

756.32

731.34

775.33

1-1-

1-

1-

1-

1-

1-

1-1-

1-

Y2

[M-H]-

[M-H]- /CO2

[M-H]- /H2O

B3

C3

B1

C1Y1/Z0

Y1/Y0

Y2/Z1

Y2/Z1

1-

m/z

Neu5Acα2 3HexNAcα1

220(1-)

179(1-)

Figure 6. (�)NanoESIchip-QTOF tandem mass spectrum of the singly charged ion at m/z 774.28 assigned toNeu5AcHexHexNAc-Thr detected in the second CE-collected fraction. Collision energy, 20–40 eV (Elab, the value set on the QTOFinstrument by the user); collision gas pressure, 12 psi (Plab, the value set on the QTOF instrument by the user). For precursor ionisolation, LM and HM parameters were set at 10 and 10, respectively. Nomenclature of the fragment ions is according to Domon andCostello.41

been described. The particular advantage of chip-based MSto provide efficient ionization of the glycopeptides arisingfrom CE and high sensitivity of detection was explored.By careful optimization of the ionization/detection condi-tions, a detailed description of the highly heterogeneousglycopeptide mixture entirely collected in the CE fractionwas obtained. (�)nanoESIchip-QTOF-MS turned out to becompatible with particular requirements for efficient ioniza-tion of glycopeptides desorbed directly from the CE buffersystem, promoting the ionization yield and the detectionof a large number of complex carbohydrate species presentin the mixture. Consequently, the high complexity of theglycosylated amino acids and peptide mixture with respectto the carbohydrate chain length and degree of sialylationcould be assessed. Moreover, the MS/MS analysis of speciespresent in the CE collected fraction was possible owing to thelong-lasting and stable spray generated through the Nano-Mate system. Under the optimized tandem MS parametersand acquisition by CID-VE, a complete set of sequence ionsfor the structural elucidation of molecular architecture at alimit of detection well below 1 pmol µl�1 collected fraction

can be obtained. Interestingly, the issue of sensitivity for theoff-line CE/MS method can also be fully addressed usingNanoMate chip-based (�)nanoESI-QTOF-MS. Additionally,off-line CE/automated chip-based (�)nanoESI-QTOF-MSand -MS/MS offers new perspectives for the automationand high-throughput screening of glycoconjugate fractionsarising from CE.

The off-line CE/fully automated chip-based (�)nanoESI-QTOF-MS and -MS/MS described here represents anadditional valuable method for compositional mappingand structural elucidation of the glycopeptides originatingfrom biological materials. As a potential tool of wideapplicability in the glycoanalytical area, it represents a basicreliable prerequisite for further developments toward on-lineCE/MS.

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