Research Article

Received: 10 June 2013 Revised: 28 August 2013 Accepted: 6 September 2013 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2013, 27, 2625–2630

Deglycosylation induces extensive dynamics changes inα-amylase revealed by hydrogen/deuterium exchangemass spectrometry

Chengjie Ji and Gary Wei*NovaBioAssays, 52 Dragon Ct, Woburn, MA 01801, USA

RATIONALE: N-Linked glycosylation plays important roles in modulating protein structure and function. The directimpact of the modification on protein conformation is not yet well understood.METHODS:Here we probed the dynamic changes following Endo H trimming of high mannose glycans in α-amylase bymeans of amide hydrogen/deuterium exchange mass spectrometry.RESULTS: The results revealed that deglycosylation elicited extensive alterations in backbone dynamics, affectingregions both adjacent to and distal from the glycosylation site.CONCLUSIONS: The overall exchange rate is reduced in the glycosylated state, which indicates rigidity enhancementdue to the attached carbohydrates. Copyright © 2013 John Wiley & Sons, Ltd.

(wileyonlinelibrary.com) DOI: 10.1002/rcm.6732

The covalent attachment of oligosaccharides to proteinasparagines, a process termed N-linked glycosylation, is aubiquitous post-translational modification that occurs in botheukaryotic and prokaryotic cells.[1,2] N-Linked glycans areimplicated in a multitude of cellular processes includingprotein folding, intracellular targeting, intercellular recognitionand immune response.[3–6] While substantial knowledge hasbeen accumulated in glycobiology, precise understanding ofthe structural consequences of N-glycosylation is still lacking.The bulky carbohydrate moiety can profoundly influenceprotein conformation.[7,8] Biophysical studies using circulardichroism (CD) anddifferential scanning calorimetry (DSC) haveconcluded that glycosylation enhances protein stability.[9,10] Togain site-specific information, X-ray crystallography and nuclearmagnetic resonance (NMR) were applied. The crystal structuresof bovine pancreatic ribonuclease (RNAase) in theglycosylated and nonglycosylated form have been solvedand found to be virtually identical,[11] suggesting thatglycosylation does not significantly alter the proteinconformation in the static state. Importantly, a hydrogen/deuterium exchange (HDX) study by NMR detectedincreased exchange rate in the nonglycosylated RNAase,which indicated that glycans decreased the structuralflexibility.[12–14] An NMR relaxation experiment on thedeglycosylated uster of differentiation protein 2 (CD2)revealed that the glycan stabilized the region surroundingthe glycosylation site.[15,16] Extensive nuclear Overhauserenhancement (NOE) interactions between the carbohydrateand the protein were observed in human chorionicgonadotropin hormone (hCG), which have important effects

* Correspondence to: G. Wei, NovaBioAssays, 52 Dragon Ct,Woburn, MA 01801, USA.E-mail: gary.wei@novabioassays.com

Rapid Commun. Mass Spectrom. 2013, 27, 2625–2630

onmaintaining the protein conformation.[17–19] A stable-isotope-assisted NMR study of the Fragment crystallizable (Fc) portionfrom immunoglobulin (IgG) showed stabilization of theFcγRIII-binding region by the glycans.[20]

While NMR techniques enable the detection ofconformational changes with spatial resolution, it issuitable for only small glycoproteins and glycopeptides.Recently, hydrogen/deuterium exchange (HDX) of peptidylN-glycosidase F (PNGase F)-treated IgG was successfullymonitored by mass spectrometry (MS) and revealeddisturbances around the glycosylation sites.[21] HDX MSis becoming increasingly popular for probing proteindynamic changes, owing to the technical improvement inseparation and MS.[22,23] In a typical HDX MS experiment,the protein is incubated in deuterium dioxide (D2O) toallow exchange of the amide hydrogens with deuterons,the rate of which is determined by solvent accessibilityand dynamics. Since the protein is digested prior to MSanalysis, the technique circumvents the size limitation ofNMR. In the present study, α-amylase, a 55 kDaglycoprotein, is selected as a model system to comparethe exchange profile for the glycosylated anddeglycosylated forms. Some insights regarding thestructural role of the glycans were obtained frominspecting the affected areas in the context of thecrystal structure.

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EXPERIMENTAL

Preparation of deglycosylated amylase

α-Amylase (100 μg) from Aspergillus oryzae (Sigma, St.Louis, MO, USA) in phosphate-buffered saline pH 7.4(PBS) was incubated with 10 μL of Endo H (New EnglandBiolabs, Ipswich, MA, USA) at 4 °C for 48 h. The

Copyright © 2013 John Wiley & Sons, Ltd.

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deglycosylated amylase was purified by TSKgel size-exclusion chromatography (Tosoh Bioscience, King ofPrussia, PA, USA) and concentrated with a spin filter.

Hydrogen/deuterium exchange

Glycosylated or deglycosylated amylase (3 μg) at aconcentration of 1 mg/mL in PBS was incubated with37 μL PBS prepared in D2O (Sigma) at 4 °C for 1 min,5 min, 1 h and 4 h. The exchange was quenched byadding an equal volume of 100 mM tris(2-carboxyethyl)phosphine (TECP), 2 M guanidine hydrochloride and100 mM sodium phosphate buffer with a final pH at 2.6.The quenched samples were digested with 5 μg porcinepepsin (Sigma) at 4 °C for 2 min. The deuterium recoverywas determined by a mixture of peptide standards thatwere 50% deuteriated. The average recovery rate wasabout 75%.

10 20 30 40 50

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mAU

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m/z

b7y6 y7b6y5y4

HexHexNAc

y3b3

y4 y5

y6

y7b6

y9

b7

Figure 1. Preparation of deglycosylatedoryzae was treated with Endo H and purithe glycopeptides containing high-mannbottom: MS/MS spectra of the Man6deglycosylated peptide. (B) Extractedglycopeptide before and after Endo H tchromatograms of native and deglycosyla

wileyonlinelibrary.com/journal/rcm Copyright © 2013 John Wil

Mass spectrometry

The protein digestswere analyzed on a liquid chromatography/mass spectrometry (LC/MS) system comprised of an AcquityUPLC system (Milford, MA, USA) coupled to a MicroTOF-Q IImass spectrometer (Bruker, Billerica, MA, USA). The LC systemwas enclosed in a chromatography refrigerator maintained at0 °C. Thepeptideswere separatedon aC8BEHcolumn (1.0mm×50 mm; Waters). A 15 min linear gradient of 5–40% acetonitrile/0.1% formic acid was employed. The capillary voltage was4500 V. The ISCID voltage was kept at 0 and the drying gastemperature was kept at 120 °C to minimize back-exchange.

Data analysis

Sequence coverage of the non-exchanged protein was obtainedby tandem mass spectrometric (MS/MS) search with Mascot(Matrix Science, Boston, MA, USA) against the protein sequencewith manual validation. Themass accuracy for precursor ions is

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Endo H treated

min9 9.5 10 10.5

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Man 5

1200 1400 1600

Man 6

Man 7Man 8

M+HexNAc

M++

y13+HexNAcy9+HexNac

y9+HexNAc

y10y13

y14y15 y16

α-amylase. α-amylase from Aspergillusfied by SEC. (A) Top: MS spectrum ofose glycosylated Asn197; middle andglycopeptide and the correspondingion chromatograms of the Man6

reatment. (C) Overlaid size-exclusionted α-amylase.

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Figure 2. Sequence coverage for pepsin cleavage of α-amylase. (A) Total ionchromatograms of peptic digests of the native and deglycosylated α-amylase. (B)Sequence coverage map from peptic digestion and MS/MS analysis.

Glycosylation effect probed by HDX

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10 ppm and 20 ppm for the fragment ions. The Mascot cut-offscore used for positive identification is 20. The data for theexchanged samples were collected in the MS mode. The rawdata was processed to generate extracted ion chromatograms,and the combined mass spectra across the peak were smoothedand used to calculate the centroid mass. The deuterium contentwas calculated from the shift in centroid mass.

Copyright © 2013 JRapid Commun. Mass Spectrom. 2013, 27, 2625–2630

RESULTS AND DISCUSSION

We selected α-amylase from Aspergillus oryzae as a modelsystem for its simple glycosylation profile. The proteincontains a single N-glycosylation site at Asn197.[24] Theglycans comprise high mannose type that is amenable todeglycosylation with endoglycosidase H (Endo H), leaving

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one N-acetylglucosamine (HexNac) residue remaining on theasparagine.[25] We believe that Endo H is superior to PNGase Ffor glycan removal, as the latter converts an asparagine residueinto an aspartate, introducing a strong negative charge thatmay confound the results. First, the high mannose glycanstructures at Asn197 were confirmed by tryptic digestionfollowed by LC/MS analysis. The major glycan structuresrange fromMan 5 to Man 8, with Man 6 as the most abundantisoform (Fig. 1(A), top panel). These ions are predominantlytriply charged and the MS/MS spectrum of the Man 6glycopeptide is shown in the middle panel of Fig. 1(A).Endo H cleavage converted the glycan into a single HexNacand its structure was confirmed by MS/MS (Fig. 1(A),lower panel). In the MS/MS spectra for both the Man6 andHexNac glycopeptides, the carbohydrates were cleaved fromthe majority of the b and y ions upon collisional activationexcept for the y9 ion. The completeness of the glycan removalwas confirmed by the extracted ion chromatogram (±0.05 Damass window) in Fig. 1(B), where the Man6 glycopeptidedisappears upon Endo H treatment. The extracted ionchromatograms for other glycopeptide forms (Man 5 and 7)are shown in Supplementary Fig. S1(A), see SupportingInformation, which also disappear following deglycosylation.The deglycosylated amylase was purified by size-exclusionchromatography (SEC) and the chromatograms of the native

1-12

13-1

920

-33

34-5

051

-68

67-8

2

91-1

0710

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414

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515

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417

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teri

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take

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0Deu

teri

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take Segment 134-144 144-1

Exchange

A

B

Figure 3. Deuterium uptake for peptide segamylase. (A) Deuterium incorporation follodeuterium incorporation vs. exchange time for

wileyonlinelibrary.com/journal/rcm Copyright © 2013 John Wil

and deglycosylated proteins are shown in Fig. 1(C). Aslight shift in retention time was observed as a result ofglycan removal.

The deuterium exchange rate was compared for the nativeand deglycosylated amylase following incubation in D2O for2 min, 10 min, 1 h and 4 h. The exchange reactions werequenched with low pH, and digested with pepsin followedby LC/MS analysis. The digestion was complete after the2 min incubation indicated by the absence of an intact proteinpeak in the chromatogram. Prior to deuterium exchange, thepeptic digest of the non-exchanged protein was analyzed todetermine the sequence coverage. The total ionchromatograms for the native and deglycosylated proteinsare very similar (Fig. 2(A)). The sequence coverage map fromMS/MS analysis is depicted in Fig. 2(B): 95% sequencecoverage was achieved, with many overlapping peptidesegments typically found for the non-specific pepsin. Peptidesegments were selected to span the entire sequence and thedeuterium incorporation was calculated from the increase incentroid mass. Charted in Fig. 3(A) is the deuterium contentafter 1 h exchange from triplicate measurement. Because theglycans may affect the back-exchange rates, directcomparison of the exchange behaviors of the glycopeptidescontaining Asn197 with the deglycosylated peptide can bemisleading. Therefore, the exchange plot for the Man6

273-

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60 240 2 10 60 240

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55 152-164

time (min)

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ments from native and deglycosylated α-wing a 1-h exchange time. (B) Plots ofselected peptide segments.

ey & Sons, Ltd. Rapid Commun. Mass Spectrom. 2013, 27, 2625–2630

Glycosylation effect probed by HDX

glycopeptide and the deglycosylated peptide is shown inSupplementary Fig. S1(B) (Supporting Information) forinformation purposes only. Notably, five peptide segments,including residues 34–50, 91–107, 152–164, 174–194 and329–349, showed increased exchange rates upondeglycosylation, whereas two peptides, including residues134–144 and 248–259, exhibited decreased exchange rates.Also shown in Fig. 3(B) is the deuterium buildup plot vs.exchange time for several selected segments, where the sametrend was observed across the entire incubation window. Theresults indicate that deglycosylation induced profoundreshuffling in the molecule structure. Glycosylationapparently enhanced the structural rigidity as there are morepeptides experiencing faster exchange upon deglycosylation.Additional insights were learned by inspection of the

crystal structure of α-amylase[26] (PDB ID: 7TAA, Fig. 4).Among the fragments with increased flexibility, fragment34–50 constitutes an α-helix adjacent to Asn197 whereasfragment 174–194 resides on the same helix as Asn197.Segment 91–107 is a helix close to residues 34–50. Residues152–164 and 329–349 are loops remote from the glycosylationsite. As for the two segments with decreased flexibility,segment 134–144 is an adjacent loop whereas segment248–259 is remote from Asn197. These results suggested thatglycosylation can affect protein dynamics both locally andremotely. The two segments adjacent to Asn197, residues34–50 and 174–194, are expected to have direct interaction

Figure 4. Structures of α-amylase highlighting reg(A) Linear sequence of α-amylase. The segments wideglycosylated form are annotated with solid and dAsn197 is highlighted in grey. (B) Crystal structure osite; yellow: regions with increased exchange ratedecreased exchange rate; the rest of the molecule is

Copyright © 2013 JRapid Commun. Mass Spectrom. 2013, 27, 2625–2630

with the glycans, and thus deglycosylation removed the sterichindrance and increased structural flexibility. It is difficult toexplain the mechanism for the long-range effect withoutextensive computational study. Nonetheless it has beenproposed that the stabilizing role of carbohydrate duringprotein folding may be entropy driven: the increased entropyof the highly flexible carbohydrate moiety can compensate forthe reduced entropy of the newly folded polypeptide andthus lower the total energy.[27] The similar process mayunderlie the stabilizing effect observed here. Previous studieshave suggested that a single acetylglucosamine residue issufficient to stabilize protein conformation[16] whereas thedata here indicated that the outer antennae also have asignificant structural role. The scope of glycan-inducedchanges in α-amylase appeared to be greater than in Fc andIgG, which could be reconciled by the difference in theenvironment surrounding the glycosylation sites: theFc/IgG glycan is partially buried at the CH2 dimer interfacewhereas Asn197 in amylase is extending away from themolecular surface.

Collectively, the study demonstrated the utility of massspectrometry based hydrogen/deuterium exchange inanalyzing the structural dynamics of glycoproteins. Systemswith more complex glycan populations can be investigatedwith the same approach for a more complete understandingof protein-carbohydrate interactions. It is notable that thenative protein contains a mixture of several glycoforms

ions of altered dynamics upon deglycosylation.th increased and decreased exchange rate in theashed boxes, respectively. The glycosylation sitef α-amylase (PDB ID: 7TAA). Red: glycosylationin the deglycosylated form; blue: regions within green.

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(Man5, 6, 7, etc.), so, after pepsin digestion, each peptide is amixture of all of these isoforms. Unlike the top-down HDXapproach where the structure of a specific PTM isoform canbe selectively analyzed,[28] the peptide-based bottom-upHDX data obtained here actually represent an average of allof the glycoforms.

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SUPPORTING INFORMATION

Additional supporting information may be found in theonline version of this article at the publisher’s web site.

ey & Sons, Ltd. Rapid Commun. Mass Spectrom. 2013, 27, 2625–2630