Enrichment of O-GlcNAc Modified Proteins by the Periodate

Oxidation-Hydrazide Resin Capture Approach

Eva Klement,† Zoltan Lipinszki,‡ Zoltan Kupihar,§ Andor Udvardy,‡ andKatalin F. Medzihradszky*,†,|

Proteomics Research Group, Institute of Biochemistry, Biological Research Center of the Hungarian Academy ofSciences, Szeged, Hungary, Institute of Biochemistry, Biological Research Center of the Hungarian Academy of

Sciences, Szeged, Hungary, Department of Medical Chemistry, University of Szeged, Szeged, Hungary, andDepartment of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94158

Received October 30, 2009

A chemical derivatization approach has been developed for the enrichment of O-GlcNAc modifiedproteins. The procedure is based on the isolation technique used for N-glycoproteins with appropriatemodifications because of the differences in the two types of glycosylation: a prolonged periodateoxidation is followed by hydrazide resin capture, on-resin proteolytic digestion, and release of themodified peptides by hydroxylamine. This enrichment strategy offers a fringe benefit in massspectrometry analysis. Upon collisional activation, the presence of the open carbohydrate ring leadsto characteristic fragmentation facilitating both glycopeptide identification and site assignment. Theenrichment protocol was applied to the Drosophila proteasome complex previously described asO-GlcNAc modified. The O-GlcNAc modification was located on proteasome interacting proteins,deubiquitinating enzyme Faf (CG1945) and a ubiquitin-like domain containing protein (CG7546). Threeother proteins were also found GlcNAc modified, a HSP70 homologue (CG2918), scribbled (CG5462)and the 205 kDa microtubule-associated protein (CG1483). Interestingly, in the HSP70 homologue theGlcNAc modification is attached to an asparagine residue of a N-glycosylation motif.

Keywords: post-translational modification • O-glycosylation • O-GlcNAc • glycopeptides • enrichment• mass spectrometry • CID • ETD

Introduction

O-linked �-N-acetylglucosamine (O-GlcNAc) is a monomericcarbohydrate modification attached to serine and threonineresidues of cytosolic and nuclear proteins.1,2 It is a dynamicmodification incorporated by O-GlcNAc transferase and re-moved by O-GlcNAcase.3 This post-translational modification(PTM) is involved in a number of cellular processes, forexample, signal transduction,3-5 transcription,1,6-9 translation3,7

or nuclear transport.10 Its importance in the brain was shownby extensive modification of pre- and postsynaptic proteinsinvolved in vesicle cycling, dendritic spine formation, cytosk-eletal organization or microtubule assembly.11,12 Perturbationsin O-GlcNAc levels have been related to diseased states.Increased levels of O-GlcNAc modification have been associ-ated with diabetes, whereas decreased level of O-GlcNAc

glycosylation of tau protein is assumed to contribute toAlzheimer’s disease.3,13

The O-GlcNAc modification has also been linked to theproteasomal degradation pathway.14,15 The ubiquitin-protea-some system (UPS) is responsible for the removal of damagedor unfolded proteins. In addition, by the degradation of keyregulatory proteins it modulates cell cycle, cell differentiationor apoptosis.16-18 UPS comprises the ubiquitination machinerytagging the proteins for degradation and the 26S proteasomeas the executive unit. In the ubiquitination pathway, theubiquitin activating enzyme E119 and a deubiquitinating en-zyme UCH-L120 has been shown as O-GlcNAc modified. In the26S proteasome, different subunits of the 20S catalytic coreparticle as well as of the 19S regulatory complex were foundto be O-GlcNAc modified.14,15 An inverse relationship betweenthe modification and proteasome function has been demon-strated, increased levels of O-GlcNAc modification decreasedproteasomal activity.15 Additionally, a number of interactingproteins copurify with the proteasome,21-23 of which HSP7024

has been shown to carry this PTM. However, the exact site ofthe modification could not be located on any of these proteins.

Being a substoichiometric modification, the analysis ofO-GlcNAc glycosylation necessitates enrichment. The isolationtechniques so far utilize chemical25 or enzymatic derivati-zation12,26,27 and affinity chromatography.11,28 In the chemical

* To whom correspondence should be addressed. Katalin F. Medzihrad-szky, phone number (415)-476-5160, fax number (415)-502-1655, e-mailaddress: folkl@cgl.ucsf.edu.

† Proteomics Research Group, Institute of Biochemistry, Biological Re-search Center of the Hungarian Academy of Sciences.

‡ Institute of Biochemistry, Biological Research Center of the HungarianAcademy of Sciences.

§ Department of Medical Chemistry, University of Szeged.| Department of Pharmaceutical Chemistry, University of California San

Francisco.

2200 Journal of Proteome Research 2010, 9, 2200–2206 10.1021/pr900984h 2010 American Chemical SocietyPublished on Web 02/11/2010

derivatization approach the sugar moiety is eliminated underalkaline conditions, the resulting double bond is then used tointroduce a thiol function or a biotin tag for the enrichmenton a thiol reactive resin or by avidin chromatography, respec-tively. In chemoenzymatic labeling, a ketone or azido moietyis added enzymatically to the O-GlcNAc modification. Then abiotin tag is attached through this ketone or azido functionalitythat enables affinity purification of the modification. Lectinweak affinity chromatography (LWAC) is based on the weakaffinity of O-GlcNAc to the lectin wheat germ agglutinin (WGA).An adequately long WGA column ensures separation of non-glycosylated peptides from O-GlcNAc modified ones.

Here, we propose an enrichment method that is analogousto the isolation of N-glycosylated proteins by the hydrazideresin capture approach.29-31 N-linked complex glycoconjugatesare readily oxidized by periodate mainly due to their sialic acidand mannose content, the resulting aldehydes are then selec-tively bound to a solid support with hydrazide functionalgroups and the modified peptides released enzymatically byPNGase F. The method has recently been extended for theenrichment of sialylated O-linked glycoproteins by replacingthe PNGase F cleavage with acid hydrolysis of the sialic acidglycosidic bond.32 Though the periodate oxidation is mostlyeffective for exocyclic vicinal diols and cyclic cis-diol com-pounds, cellulose33sa linear polymer of �-D-glucosesandcyclodextrins34,35scyclic oligomers of R-D-glucose, both con-taining trans-diol moieties, were also found to be oxidized byperiodate. This observation encouraged us to experiment withthe periodate oxidation-hydrazide resin capture approach forthe enrichment of O-GlcNAc modified proteins.

The enrichment method was developed using an O-GlcNAcmodified peptide standard and R-Crystallin. Then the strategywas applied to the 26S proteasome purified from Drosophilamelanogaster to locate the modification on proteins associatedwith the UPS.

Materials and Methods

Chemicals. Sequencing grade side-chain protected porcinetrypsin (modified by reductive methylation) was ordered fromPromega (Madison, WI). ZipTip C18 tips were from Millipore(Billerica, MA), OMIX C18 tips were from Varian (Lake Forest,CA). Click-iT O-GlcNAc peptide standard was obtained fromInvitrogen (Eugene, OR). Affi-Gel Hz was from BioRad (Her-cules, CA). Amino-functionalized controlled pore glass (LCA-CPG, pore size 513 Å) was ordered from CPG Inc. (Lincoln Park,NJ). High purity solvents (HPLC grade) were purchased fromSigma (Steinheim, Germany) and Merck (Darmstadt, Germany).All other chemicals were obtained from Sigma (Steinheim,Germany).

Controlled pore glass hydrazide (H-CPG) was synthesizedfrom LCA-CPG. First, the amino functions of LCA-CPG werederivatized to yield free carboxyls,36 then the resulting carboxylswere reacted with hydrazine.37

26S proteasome subcomplexes were purified from Droso-phila embryos as previously described.38

O-GlcNAc Enrichment by Hydrazide Resin Capture. Theproteins were solubilized with 0.5% SDS in 50 mM sodiumacetate. The pH was adjusted to pH 5-6 and sodium periodatewas added at a final concentration of 20 mM. The oxidationwas performed at 37 °C for 6 h in the dark, then it wasterminated by 5 equiv of sodium sulfite solution (freshlyprepared) for 20 min. If needed, the pH of the solution wasreadjusted to pH 5-6. The hydrazide resin (stored in isopro-

panol) was washed three times with water and added to thereaction mixture. The settled slurry was ca. 1/8 to 1/10 of thetotal volume. The coupling proceeded overnight with gentleagitation by vertical rotation. The supernatant was discardedand the resin was washed five times with 0.5 M triethylaminephosphate, pH 8.5 in 30% acetonitrile, then with 100 mMtriethylamine phosphate, pH 8. The disulfide bridges werereduced with dithiothreitol (20 mM in 100 mM triethylaminephosphate, pH 8) at 56 °C for 1 h and the free sulfhydrylsderivatized with iodoacetamide at a final concentration of 50mM at pH 8 for 1 h in the dark. The resin was washed threetimes with 50 mM triethylamine phosphate, pH 7.5. Thentrypsin was added, and the digestion proceeded at 37 °Covernight. Nonspecifically bound tryptic peptides were re-moved by washing the resin five times with each 0.5 Mtriethylamine phosphate, pH 8.5 in 30% acetonitrile and 0.1%trifluoroacetic acid in 50% acetonitrile then three times withisopropanol and finally with 50 mM sodium acetate. Themodified peptides were cleaved by the addition of 200 mMhydroxylamine hydrochloride in 50 mM sodium acetate, pH 5overnight with vertical rotation. The eluate was collected, thenthe resin was washed with 50 mM sodium acetate and addedto the eluate. The sample was desalted on a C18 ZipTip priorto the MS analysis.

BEMAD of O-GlcNAc Modified Peptides. The sample wasdried and redissolved in a solution of 250 mM sodiumhydroxide and 50 mM cysteamine hydrochloride. The reactionproceeded at 37 °C for 3 h. Then the sample was desalted ona C18 ZipTip.

MALDI-TOF MS. Data were acquired on a Bruker Reflex IIImass spectrometer in reflectron mode. 2,5-Dihydroxy-benzoicacid served as the matrix.

LC-MS/MS. CID analysis was performed on a PremierQ-TOF mass spectrometer online coupled to a nanoAQUITYUPLC system (Waters Micromass). HPLC conditions: Sampleswere injected onto a Symmetry trap column (C18, 5 µm, 180µm × 20 mm) at a flow rate of 15 µL/min for 3 min in 3% ofsolvent B and separated on an AQUITY UPLC BEH C18 column(C18, 1.7 µm, 75 µm × 200 mm) at a flow rate of 300 nL/minwith a gradient of 10-40% B in 90 min. Solvent A was 0.1%formic acid in water, solvent B 0.1% formic acid in acetonitrile.MS conditions: The spray voltage was set at 3-3.5 kV and thecone voltage at 26-28 V. Desolvation temperature was 180 °C.Data were acquired in a data-dependent fashion: 1 s MS surveyswere followed by 5 s CID experiments on computer-selectedmultiply charged ions. Collision energy was adjusted to the m/zand charge of the precursor ion. A 30 s dynamic exclusion wasused. Data were processed by Mascot Distiller (v2.2.1.0). In ETDanalysis peptide fractionation was performed similarly as above,only a 60 min gradient was used. The data acquisition wascarried out in a linear ion trap - Orbitrap hybrid massspectrometer. MS survey measurements were performed in theOrbitrap at a resolution of 60 000, CID and ETD experimentswere carried out in the linear trap. Ion populations within thetrap were controlled by integrated automatic gain control(AGC). For CID, AGC target was set to 10 000, with dissociationat 35% of normalized collision energy, activation time: 30 ms.For ETD, the AGC target values were set to 10 000 and 100 000for the isolated precursor cations and fluoranthene anions,respectively, and allowing 200 ms of ion/ion reaction time.Supplemental activation for the ETD experiments was enabled.A 60 s dynamic exclusion was used. Data were processed byan in-house peak-picking software: PAVA.

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Database Search. Q-TOF CID data were searched againstDrosophila melanogaster proteins in the NCBI 20080718 data-base (52 783 sequences) using in-house Mascot (v2.2.05) andProtein Prospector (v5.2.2, prospector.ucsf.edu) search engines.Monoisotopic masses with precursor mass tolerance of (50ppm and fragment mass tolerance of (0.1 Da were used andsemitryptic cleavages were allowed. ETD data were searchedagainst the UniProtKB 20090707 database (32 646 Drosophilamelanogaster entries) using Protein Prospector. Monoisotopicmasses with precursor mass tolerance of (15 ppm and frag-ment mass tolerance of (0.6 Da were used. Only trypticpeptides were considered, with one missed cleavage permitted.

Cysteine carbamidomethylation was considered as fixedmodification, and acetylation of protein N-termini, methionineoxidation and pyroglutamic acid formation from N-terminalglutamine residues as variable modifications. Additionally, anew modification with a mass of +231 Da on serine andthreonine residues was defined considering oxidation of theO-GlcNAc ring and conversion of the aldehyde groups tooximes, enabling neutral loss of 126 or 231 Da (see detailedstructural description in Figure 2 in Results and Discussion).This modification was included into the modifications list andused as a variable modification. Conversion of serine andthreonine residues to S-aminoethyl-cysteine and �-methyl-S-aminoethyl-cysteine, respectively, was considered as a variablemodification in BEMAD experiments. N-terminal serine/threo-nine oxidation and conversion to oxime (-16 and -30 Da,respectively) was also taken into account as variable modifica-tion. Results of the database searches were manually validated.

Results and Discussion

Adaptation of the Periodate Oxidation-Hydrazide ResinCapture Approach to the O-GlcNAc Modification. Based onthe original isolation technique developed for N-linked glyco-proteins the method consists of the following steps: (i) perio-date oxidation of the carbohydrate moiety, (ii) capture tohydrazide-functionalized solid phase, (iii) removal of nongly-cosylated proteins, (iv) enzymatic digestion, (v) removal ofnonglycopeptides, and (vi) release of the modified peptides.However, appropriate changes had to be introduced becauseof the differences between N-linked complex glycoconjugatesand O-GlcNAc modification.

The first main difference is the reduced reactivity of theO-GlcNAc moiety in the periodate oxidation due to the transconfiguration of the vicinal hydroxyls at positions C3 and C4.However, at elevated temperatures and longer incubation timesthe GlcNAc ring can also be oxidized to the dialdehydederivative. Unfortunately, N-terminal serine and threonineresiduessbeing vicinal amino alcoholssare also oxidized bythe periodate to the respective glyoxylyl derivatives39 and willbe captured by the hydrazide resin. Even if the oxidation isperformed at the protein level the N-terminal peptides ofproteins starting with serine or threonine will contribute to thenonglycosylated background.

Another important difference to the original protocol is inthe release of the modified peptides. In the enrichment ofN-glycoproteins, the release of the modified peptides is per-formed by PNGase F treatment cleaving the peptide from thehydrazide captured sugar structure.29-31 The site of the modi-fication is “labeled” by the conversion of the respectiveasparagine to aspartic acid. The oxidized and hydrazidecaptured O-GlcNAc cannot be removed by an enzyme. At thesame time the liberation of the Ser/Thr-modified peptides by

�-elimination is analogous to the PNGase F treatment of theN-linked glycopeptides. This approach provides two advan-tages: (i) the modified amino acid is marked and (ii) peptidesattached N-terminally to the hydrazide resin are unaffected.However, the relatively harsh conditions that have to be usedfor the reaction may lead to partial degradation of the sample.That is especially likely when a threonine is modified, sincethis residue is much less prone to �-elimination than aglycosylated serine. Moreover, �-elimination of unmodifiedserine, threonine, or alkylated cystein residues of nonspecifi-cally bound peptides can lead to false positive results. There-fore, nondestructive alternatives were considered: peptides arereleased by the cleavage of the hydrazone bond with the sugarmodification still attached, thus providing a direct evidence ofglycosylation. However, peptides captured N-terminally to theresin are also released by this method impairing selectivity.

The enrichment protocol was developed using a standardpeptide and then optimized on R-Crystallin, known to beO-GlcNAc modified. In the periodate oxidation performed withthe O-GlcNAc modified peptide standard several conditionswith respect to periodate concentration, time, temperature andpH were tested. The initial oxidation product was R-N-glyoxylyl-APTSgTIAPG, however, a minor peak appeared at -2 Da withina few hours at room temperature indicating partial oxidationof the carbohydrate ring. The ring-opening was complete inan overnight reaction. At elevated temperatures the reactionproceeded faster, at 37 °C the sugar ring was completelyoxidized to the dialdehyde derivative in 4-6 h (SupplementalFigure 1, Supporting Information). The reaction took place onlyunder acidic conditions (pH 3-6.5), under alkaline conditions(pH ∼9) no oxidation of the sugar structure was observed evenin a 24 h reaction. Periodate oxidation at the protein level wasperformed in the presence of denaturing agents, GuHCl or SDS.Both were found to be compatible with the enrichment;however, SDS proved to be superior over GuHCl probably dueto electrostatic interactions with the resin. Oxidative damageof the peptides was not investigated, however, partial oxidationof cysteine and methionine residues was observed. The oxida-tion reaction was terminated by sulfite,31 then oxidized O-GlcNAc was reacted with hydrazide resin overnight. Two typesof solid support were tested, agarose-bound hydrazide (Affi-Gel Hz) and hydrazide coupled to silica (H-CPG). The Affi-Gel is widely used in the enrichment of N-linked glycopep-tides.29,31 The H-CPG resin has been applied to the selectiveisolation of oxidative stress related 4-hydroxynonenal modifiedproteins.36 In the O-GlcNAc enrichment no significant differ-ence in the performance of the two resins was observed. Afterremoval of the nonglycosylated proteins, the resin-boundproteins were digested with trypsin and the nonglycosylatedpeptides were also removed. The modified peptides werereleased either by �-elimination or hydrazone bond cleavage.For the latter approach, three reactions were tested: periodate,40

acidic36,39 or hydroxylamine cleavage. The �-elimination strat-egy (coupled to Michael-addition) gave moderate yields. Thehydrazone bond was found to be cleaved by periodate insolution, however, this approach was inefficient when appliedto solid-phase bound Crystallin. The acidic cleavage was alsoomitted because a partial sugar loss was observed resulting indecreased sensitivity. For the cleavage of the hydrazone bondthe overnight hydroxylamine treatment under mildly acidicconditions (pH 5) was found to be the most efficient convertingthe O-GlcNAc modified peptides to oxime derivatives. TheMALDI-TOF mass spectrum of the enriched R-Crystallin pep-

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tides is shown in Figure 1. The oxime derivative of theR-Crystallin A glycopeptide AIPVSgREEKPSSAPSS is observedat m/z 1873.0 as the most abundant peak. Signals at m/z 1443.7and 1786.0 represent the truncated forms AIPVSgREEKPS andAIPVSgREEKPSSAPS, respectively. The AIPVSgREEKPSSAPS pep-tide represents the shorter isoform of R-Crystallin A, theAIPVSgREEKPS peptide is either a degradation product orbelongs to an even shorter isoform. The oxime derivative ofthe R-Crystallin B glycopeptide EEKPAVTgAAPK was observedonly in the LC-MS/MS analysis (Supplemental Figure 2,Supporting Information).

CID Fragmentation of the Oxime Derivatives of O-GlcNAcModified Peptides. In general, O-glycosylated peptides showcharacteristic fragmentation upon collisional activation. Sincethe glycosidic bond is much more susceptible to fragmentationthan the amide bond, extensive sugar loss from the precursorion is observed in the CID spectrum along with abundantoxonium ion at the corresponding m/z. Moreover, due to a gas-phase rearrangement the sugar is eliminated without “marking”the originally modified residue in the process. The oximederivatives of the oxidized O-GlcNAc modified peptides showslightly different fragmentation as demonstrated by the CIDspectrum of the R-Crystallin A peptide AIPVSgREEKPSSAPSS,m/z 936.98 acquired on a Q-TOF mass spectrometer (Figure2, left panel). Cleavage of the glycosidic bond results in productions observed at m/z 821.78 and 1641.87 corresponding toneutral loss and charged loss of the sugar derivative (-231 Da),respectively. As a new feature, an additional characteristic lossfrom the precursor ion is observed at m/z 884.48 correspondingto the neutral loss of 105 Da. No oxonium ion is detected atm/z 232.09, instead a sugar-related product ion is observed atm/z 127.05. Note that this product ion and the neutral loss of105 Da add up to the mass of the oxonium ion. Since theCrystallin peptide contains multiple prolines and the amidebond N-terminally to proline is also relatively facile, most ofthe sequence ions detected correspond to these cleavages,either retaining the modification or having lost the above-mentioned 105 or 231 Da. This distinct fragmentation of theoxime derivatives of the oxidized O-GlcNAc modified peptidesis summarized in the right panel of Figure 2. Since thecarbohydrate ring is split open in the periodate oxidation,cleavage of the C-O bond on either side of the carbonyl Cresults in two characteristic losses. In pathway A, the sugar is

eliminated in the same manner as intact carbohydrates fromO-glycosylated peptides in general, that is, without any indica-tion where the modification was originally located. Dependingon where the charge is retained, an oxonium ion is observedat m/z 232 and the “unmodified” peptide is detected of thesame charge as (constant neutral loss) or one charge less than(constant charged loss) the precursor. If the adjacent acetalbond is cleaved (pathway B), only half of the carbohydratederivative is released. The result is a neutral loss of 105 Da anda mass signature of 126 Da on the modified amino acid. Furtherfragmentation leads to a product ion of m/z 127, we hypoth-esize an isoxasolium structure for this product. The fragmenta-tion pattern of the oxime derivatives of the O-GlcNAc modifiedpeptides largely depends on the type of the activation. In iontraps, due to the single activation of the precursor ion mainlythe neutral loss of 231 and 105 Da from the precursor ion isobserved along with the oxonium ion at m/z 232 indicating apeptide being O-GlcNAc modified. As a consequence, ion trapCID spectra may not contain sufficient information for peptideidentification and/or site assignment. In Q-TOF instruments,multiple collisions in the collision cell lead to additional peptidebackbone cleavages and also to the splitting of the oxoniumion.

Proteasome Interacting Proteins are O-GlcNAc Modified.The O-GlcNAc enrichment protocol was applied to the sub-complexes of the proteasome purified from Drosophila mela-nogaster in two fractions. One of the fractions contained mainlythe subunits of the catalytic core particle while the other onethe subunits of the regulatory complex. Fractions were con-centrated by acetone treatment, then protein precipitate wassolubilized in SDS containing sodium acetate buffer at pH 7and the pH adjusted to pH 5-6 for the periodate oxidationand the subsequent coupling to the Affi-Gel. For the release ofthe O-GlcNAc modified peptides the hydroxylamine cleavagewas applied. Since oximes are acid labile, the desalted and driedsamples were dissolved in water prior to LC-MS/MS analysison a Q-TOF mass spectrometer and a linear ion trap-Orbitraptandem mass spectrometer equipped with ETD. Additionally,a portion of the samples was subjected to BEMAD experimentsto assist site assignment. The database searches were per-formed supplementing appropriate variable modifications onserine/threonine residues, that is, opening of the sugar ringand conversion to oxime in the hydroxylamine cleaved samplesor conversion of the modified serine/threonine to the corre-sponding cysteine derivative in BEMAD experiments. Addition-ally, several subunits of the proteasome start with serine orthreonine due to processing of the N-terminal methionine ora presequence. N-terminal oxidation and hydrazide captureof these proteins was also taken into account by conversion ofpeptide N-terminal serine/threonine to glyoxylyl oxime usedas variable modification.

Altogether, a total of 12 GlcNAc modified peptides wereobserved in the enriched fractions. The modified peptidesgenerally eluted in the low organic part of the chromatographypreceding the nonglycosylated peptides. This chromatographicseparation is due to the increased hydrophilic character of theO-GlcNAc modified peptides, a consequence of the sugarmodification as well as the amino acids surrounding themodification site.28 The nonspecific background mostly cor-responded to N-terminal oxidation and hydrazide capture ofsome proteasome subunits (Supplemental Figure 3, SupportingInformation). The O-GlcNAc modified peptides representedapproximately 4% of the total MS/MS spectra, which was less

Figure 1. MALDI-TOF mass spectrum of the isolated R-Crystallinglycopeptides. The peptides at m/z 1443.7, 1786.0, and 1873.0denoted as RAg point to the same glycosylation site at Ser-162in the R-Crystallin A chain and correspond to peptides AIPVSg-REEKPS, AIPVSgREEKPSSAPS, and AIPVSgREEKPSSAPSS,respectively.

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than the 8.5% obtained by lectin weak affinity chromatographyin a postsynaptic density preparation.28

Seven GlcNAc modified peptides detected could be identifiedunambiguously, representing six different glycosylation sitesas summarized in Table 1. The modification sites were deter-mined from CID, ETD, or BEMAD experiments. No O-GlcNAcmodification was located on proteasomal subunits. Instead,interacting partners of the proteasome were found GlcNAcmodified.

In the deubiquitinating enzyme Faf (CG1945) O-GlcNAcglycosylation at Thr-2617 was determined from the CIDspectrum of the precursor at m/z 881.43 (2+) (SupplementalFigure 4, Supporting Information). Faf belongs to the ubiquitinprocessing protease (UBP/USP) class of deubiquitinatingenzymes.41,42 It fulfills an editing role by hydrolyzing theubiquitin tag from target proteins thus preserving them fromproteasomal degradation.41,43 Faf is essential for proper devel-opment of the eye: by deubiquitination of its substrate liquidfacets it assists delta signaling and cell fate determination.44

Two O-GlcNAc sites were identified in CG7546, Thr-719 andSer-792. Modification site assignment in the 713ANTLPT-TATQTR724 peptide was accomplished from the ETD spectrumof the precursor at m/z 753.3821 (2+) (Figure 3), the massdifference between z5* and z6 unambiguously confirmed modi-fication at Thr-719. For Ser-792 only the BEMAD experimentprovided sufficient information for site assignment (Supple-

mental Figure 5, Supporting Information). CG7546 has aconserved ubiquitin-like domain (UBL) on its N-terminus. Ahomologous UBL domain is found in Xenopus laevis or humanScythe/Bat3 proteins. Moreover, in Xenopus Scythe tandemUBL domains (UBL1 and UBL2) were found that redundantly

Figure 2. Fragmentation of the oxime derivatives of oxidized O-GlcNAc modified peptides. (Left) CID spectrum of the oxime derivativeof the O-GlcNAc modified R-Crystallin peptide AIPVSgREEKPSSAPSS; precursor ion at m/z 936.98 (2+); acquired in a Q-TOF massspectrometer; g denotes a sugar-related product ion. (Right) Fragmentation scheme. Pathway A: The sugar is eliminated in the samemanner, as from O-glycosylated peptides in general. An oxonium ion (1) is observed at m/z 232 and the “unmodified” peptide isdetected of the same charge as or one charge less (2) than the precursor ion. Pathway B: Upon cleavage of the adjacent acetal bondonly half of the carbohydrate derivative is released as a neutral (3; 105 Da) and a mass signature of 126 Da is left behind on themodified amino acid (4). Further fragmentation leads to a product ion of m/z 127, we hypothesize an isoxasolium structure (5) for thisproduct.

Table 1. O-GlcNAc Modified Peptides Identified from Drosophila melanogaster Proteins

Flybasesymbol name function m/za sequence

O-GlcNAcsite

method forsite assignment

CG1945 Faf/fatfacets

ubiquitin-specificprotease

881.43 (2+) 2614TPTTqSSPSTAAWPAR2628 Thr-2617 CID

CG7546 CG7546 apoptosis 668.10 (4+) 776STSTAPAGGATVVPPTSq

AAVTRPVTTGR803Ser-792 BEMAD

753.3821 (2+)b 713ANTLPTTqATQTR724 Thr-719 ETDCG2918 CG2918 HSP70 chaperone 723.67 (3+) 613SEESTKQDTEAKNqETIK629 Asn-625 CID

745.8537 (2+) 619qDTEAKNqETIK629 Asn-625 CIDCG5462 scribbled polarization of

embryonic epithelia511.7723 (2+) 1209VTETITK1215 + GlcNAc Thr-1210 or

Thr-1212 or Thr-1214None

CG1483 MAP205 microtubulebinding

908.9448 (2+) 1026NTSSTTqTSTATATITK1041 Thr-1031 ETD

a m/z value is given for the precursor of the respective (either the oxime or the BEMAD) derivative used for the modification site assignment.b Precursor masses detected in the Orbitrap mass spectrometer were within 5 ppm of the calculated values. q Denotes the glycosylated residue. “q” standsfor pyroglutamic acid.

Figure 3. ETD spectrum of the “O-GlcNAc-oxime” modified713ANTLPTTgATQTR724 peptide; precursor ion at m/z 753.3821(2+); Thr-719 was identified as the site of modification. Asterisksindicate ions produced by hydrogen migration, that is, z+1fragments.

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interact with the embryonic Xrpn10c subunit of the protea-some.45 Scythe was also demonstrated to interact with theATPase domain of HSC70/HSP70 via its C-terminal BAGdomain inducing inhibition of the chaperone function ofHSP70.46 HSP70/HSC70 proteins are also known interactors ofthe proteasome.22,23

Interestingly, the protein CG2918 found glycosylated in thisstudy shows HSP70 homology. Manual validation of the CIDspectrum of the precursor at m/z 745.86 (2+) revealed that inthis protein the GlcNAc modification is attached to Asn-625,as confirmed by fragments y5 (m/z 730.37) and b8 (m/z 1025.43)retaining the 126 Da mass signature (Figure 4). The N-glycosidicbond is fairly stable upon collisional activation while the C-Obond on the other side of the carbonyl C is easily fragmented,hence the abundant 105 losses and the retention of the 126Da sugar fraction on the peptide fragments. This modificationsite fits to the Asn-X-Ser/Thr consensus sequence of N-glycosylation. Moreover, the protein has on the C-terminus aretention signal for the endoplasmic reticulum where N-glycosylation takes place. A single N-GlcNAc modification ontransmembrane proteins has already been demonstrated,28

however, there is no information about the role of thismodification and the enzymes involved. Additionally, anotherN-glycosylation site has previously been identified in CG2918at Asn-49747 which is conserved in its human ortholog (HYOU1)and is also glycosylated.29

Two other proteins not connected to the proteasomaldegradation pathway were also present in the mixture and werefound O-GlcNAc modified. Scribbled (CG5462) is a scaffolding,multi-PDZ domain protein required for the polarization ofembryonic epithelia48 and involved in synapse formation.49

In this protein the modification was located on the1209VTETITK1215 peptide, however, the exact site of the modi-fication could not be determined from either CID or ETD ofthe precursor at m/z 511.77 (2+). In the CID spectrum no masssignature (either 126 or 231 Da) was retained on the sequenceions (Supplemental Figure 6, Supporting Information), and theETD spectrum was low quality for site assignment. In the 205kDa microtubule-associated protein (MAP205, CG1483) theglycosylated peptide 1026NTSSTTTSTATATITK1041 contains amultitude of potential modification sites. Here, ETD spectrumof the precursor at m/z 908.9448 (2+) revealed modificationat Thr-1031 (Supplemental Figure 7, Supporting Information).High molecular weight MAPs isolated from rat brain havealready been described as O-GlcNAc modified.12,50

Conclusions

An enrichment protocol utilizing periodate oxidation, hy-drazide capture and release of the modified peptides byhydroxylamine cleavage was developed for the analysis of theO-GlcNAc modification. Using this approach and mass spec-trometry analysis, four Drosophila proteins copurified with the26S proteasome were identified as O-GlcNAc modified. Simi-larly to other glycosylation studies11,32,51 the combination ofdifferent fragmentation techniques, that is, CID and ETD, wasapplied for modification site assignment, but the use of furtherchemical derivatization (BEMAD) also proved beneficial. In-terestingly, N-linked glycosylation of the HSP70 chaperone likeCG2918 with a single GlcNAc in the proper consensus positionwas also detected, just like in a recently published study.28

At the present stage N-terminal oxidation of serine/threonineresidues compromises the use of this enrichment method inlarge scale analysis. However, N-terminal derivatization isexpected to overcome this shortcoming, which may also enableenrichment at the peptide level. Since the method also enrichesN-glycopeptides, prior PNGase F treatment has to be performedwhen applied to complex mixtures containing extracellular ormembrane proteins.

Our O-GlcNAc enrichment procedure provides an alternativeto the existing methods. As demonstrated earlier in a phos-phoproteomic study52 the different enrichment techniquesyield complementary information. Similarly, the different O-GlcNAc enrichment methods may identify different modifiedpeptide populations.

Acknowledgment. This work was supported by Hun-garian Science Foundation grant OTKA T60283, National Officefor Research and Technology grant OMFB-00467/2009 (toK.F.M.) and by NIH grant NCRR P41RR001614 to the UCSF MSFacility (director A.L. Burlingame). We thank Lajos Kovacs foruseful discussion.

Supporting Information Available: MALDI-TOF massspectra of the periodate oxidized O-GlcNAc peptide standard;base peak chromatogram of an enriched fraction of theproteasome; extracted ion chromatogram and full-scan massspectrum of a selected glycopeptide (m/z 745.85); CID and ETDspectra of the oxime or BEMAD derivatives of O-GlcNAcmodified peptides. This material is available free of charge viathe Internet at http://pubs.acs.org.

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