Reliable Determination of Site-Specific In Vivo Protein N

B American Society for Mass Spectrometry, 2014DOI: 10.1007/s13361-013-0823-6

J. Am. Soc. Mass Spectrom. (2014) 25:729Y741

RESEARCH ARTICLE

Reliable Determination of Site-Specific In Vivo ProteinN-Glycosylation Based on Collision-Induced MS/MSand Chromatographic Retention Time

Benlian Wang,1 Yaroslav Tsybovsky,2 Krzysztof Palczewski,1,2 Mark R. Chance1

1Center for Proteomics and Bioinformatics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA2Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA

Abstract. Site-specific glycopeptide mapping for simultaneous glycan and peptidecharacterization by MS is difficult because of the heterogeneity and diversity ofglycosylation in proteins and the lack of complete fragmentation information foreither peptides or glycans with current fragmentation technologies. Indeed,multiple peptide and glycan combinations can readily match the same mass ofglycopeptides even with mass errors less than 5 ppm providing considerablyambiguity and analysis of complex mixtures of glycopeptides becomes quitechallenging in the case of large proteins. Here we report a novel strategy toreliably determine site-specific N-glycosylation mapping by combining collision-induced dissociation (CID)-only fragmentation with chromatographic retention

times of glycopeptides. This approach leverages an experimental pipeline with parallel analysis of glyco- anddeglycopeptides. As the test case we chose ABCA4, a large integral membrane protein with 16 predictedsites for N-glycosylation. Taking advantage of CID features such as high scan speed and high intensity offragment ions together combined with the retention times of glycopeptides to conclusively identify the non-glycolytic peptide from which the glycopeptide was derived, we obtained virtually complete information aboutglycan compositions and peptide sequences, as well as the N-glycosylation site occupancy and relativeabundances of each glycoform at specific sites for ABCA4. The challenges provided by this example provideguidance in analyzing complex relatively pure glycoproteins and potentially even more complex glycoproteinmixtures.Key words: Site-specific N-glycosylation, Chromatographic retention time, CID, Glycopeptides

Received: 7 August 2013/Revised: 23 December 2013/Accepted: 23 December 2013/Published online: 19 February 2014

Introduction

Protein glycosylation, the covalent attachment of glycansto a protein, is one of the most common forms of post-

translational modification and is implicated in variouscellular processes, including protein folding and trafficking,cellular signaling, recognition, and adhesion [1]. Glycopro-teins with glycans attached to Asn (N-glycosylation), andSer or Thr residues (O-glycosylation) comprise over 50 % ofhuman proteins. Aberrant glycosylation has been reported toassociate with various inherited and acquired diseases [2].

Mass spectrometry (MS) coupled with advanced separationmethodology has emerged as the most powerful technologyfor glycosylation analyses [3–5]. The existing analyticpipelines focus on identifying glycans released from proteinsby deglycosylation and/or glycopeptides obtained by proteindigestion with proteases [6, 7]. Unlike the direct glycananalysis, glycopeptide analyses can provide informationabout glycan composition at specific sites, which is crucialfor detailed structural and functional information.

A number of studies have dealt with site-specific N-glycosylation mapping for simultaneous glycan and peptidecharacterization using MS [8–10]. Many analytical advanceshave been made in various steps including enzymaticproteolysis [11], glycopeptide enrichment [12–14], chro-matographic fractionation and tandem MS/MS fragmenta-tion [15–17]. In brief, high-resolution MS is essential foraccurate glycopeptide mass assignments, and tandem MS is

Electronic supplementary material The online version of this article(doi:10.1007/s13361-013-0823-6) contains supplementary material, whichis available to authorized users.

Correspondence to: Mark R. Chance; e-mail: [email protected]

http://dx.doi.org/10.1007/s13361-013-0823-6

required to verify glycan composition or glycopeptidesequences through different fragmentation methods such ascollision induced dissociation (CID), electron capture ortransfer dissociation (ECD or ETD), higher energy colli-sional C-trap dissociation (HCD), and infrared multi-photondissoc ia t ion ( IRMPD). A para l le l ana lys i s ofdeglycopeptides from PNGase F treatment in normal orO18 water can aid in the identification of N-glycosylationsites [13, 14] and determination of site occupancy [18, 19].

Basically, there are two critical unknowns for site-specificglycopeptide analysis that can be revealed by MS: thepeptide mass and the glycan mass. ECD and ETD lead tofragmentation of the peptide backbone but leave the sugarmoieties intact, whereas CID mainly results in fragmentationof the sugar moieties but not the peptide. HCD allowsdetection of diagnostic oxonium ions and some product ionscorresponding to the peptide backbone, which helps inglycosylation site identification, but is less effective fordetermination of glycan structure [20, 21]. None of thesemethods alone can achieve complete fragmentation andcoverage for both peptides and glycans. Multiple peptideand glycan combinations are very likely to match the sameglycopeptide mass, especially with complex mixtures ofglycopeptides because of the heterogeneous nature ofglycosylation and missing fragment ion information foreither peptides or glycans. Correct assignment of glycopep-tide composition and structure is still a key challenge forsite-specific glycopeptide analysis by MS.

Recently, attempts have been made to employ thecombination of CID and ETD to achieve in-depth charac-terization of glycopeptides by utilizing the advantages ofthese fragmentation techniques simultaneously, with CIDemployed for analyzing the glycans and ETD for sequencingthe glycopeptide [22, 23]. The key limitation of ETD is thatthis fragmentation method selectively favors higher charge-state precursor ions (plus three and above). This requirementis particularly problematic if the glycopeptides containcomplex glycans with negatively charged NeuAc (sialicacid) or phosphorylated residues driving charge-state neu-tralization. In addition, long glycopeptides with high chargestates needed for successful ETD fragmentation can hostmore than one glycosylation site, which will interfere withunambiguous site identification. More recently, a CID-HCDcombined fragmentation approach has been applied toglycopeptide mapping [24]. Similarly, CID serves to analyzeglycan composition and branching, whereas HCD primarilycontributes to the identification of glycosylation sites. Infact, HCD implemented in the Orbitrap generates fewerglycopeptide product ions for peptide sequencing. In arecent case confident assignment of glycosylation sites wasachieved by MS3 analysis of the peptide-HexNAc ion (Y1)following MS2 by HCD [21, 25]. Thus, in combinedapplications of either CID-ETD or CID-HCD, the ETD orHCD steps nicely complement CID.

In this study, we report a novel alternative strategy toreliably determine site-specific glycosylation based on the

combination of CID-only fragmentation and known glycopep-tide retention times. As a test case for the method, we chose theATP-binding cassette transporter 4 from subfamily A ofmammalian ABC transporters (ABCA4), an endogenousmembrane protein from bovine retina with the molecular massover 250 kDa (2281 amino acids) and 16 theoretical N-linkedglycosylation sites (N-X-S/T motif, where X can be any aminoacid except proline). Previously, we reported the first system-atic study of post-translational modifications including phos-phorylation and N-glycosylation in native ABCA4 [26]. Here,we focus on a strategy developed to achieve the correct andreliable assignments N-glycopeptide mapping of the proteinbased on collision-induced tandem MS in combination withchromatographic retention time. The results provide guidancefor determining virtually complete information about glycancompositions and peptide sequences, as well as theN-glycosylation site occupancy and relative abundances ofeach glycoform at specific sites for a complex glycoprotein andpossibly for moderately complex glycoprotein mixtures.

ExperimentalMaterials and Reagents

Dithiothreitol (DTT), iodoacetamide, and PNGase F werepurchased from Sigma-Aldrich (St. Louis, MO, USA).Sequencing grade modified trypsin and chymotrypsinwere from Promega (Madison, WI, USA) and Roche(Indianapolis, IN, USA), respectively. All others chemicalsand biochemical are from Sigma-Aldrich.

Preparation of ABCA4 Samples from Bovine RodOuter Segments

ABCA4 was prepared as described earlier [26]. Briefly,bovine rod outer segments (ROS) from frozen retinas wereisolated under dim red light and homogenized in thehypotonic buffer (5 mM Bis-trispropane (BTP), pH 7.5,1 mM DTT). ROS membranes were separated by centrifu-gation at 20,000 g for 30 min and solubilized with buffercomposed of 20 mM BTP, pH 7.5, 10 % glycerol, 30 mMNaCl, 1 mM DTT, and 20 mM n-dodecyl-β-D-maltopyranoside (DDM). Solubilized membranes wereapplied to a DE52 ion exchange column and washed withbuffer containing 20 mM BTP, pH 7.5, 10 % glycerol,30 mM NaCl, 1 mM DTT, and 1 mM DDM. The proteinfraction containing ABCA4 was eluted with 150 mM NaClin the same buffer and subjected to SDS-PAGE.

In-Gel Digestions and Mass Spectrometry

Bovine ABCA4 was deglycosylated with commerciallyobtained PNGase F from Elizabethkingia meningosepticaunder denaturing conditions according to the manufacturerprotocol. Cleavage of oligosaccharides was confirmed by aslight increase in ABCA4 mobility in SDS-PAGE gels (data

730 B. Wang et al.: N-Glycosylation by CID MS/MS & Retention Time

not shown). Coomassie-stained SDS-PAGE gel piecescontaining intact or PNGase F-treated ABCA4 weredestained with 50 % acetonitrile in 100 mM ammoniumbicarbonate followed by 100 % acetonitrile. Next, cysteineresidues were reduced by incubating the sample with 20 mMDTT at room temperature for 60 min, followed by alkylationwith 50 mM iodoacetamide for 30 min in the dark. Thesolution was removed and the gel pieces were washed with100 mM ammonium bicarbonate and dehydrated in acetoni-trile. Gel pieces were then dried in a SpeedVac centrifuge,rehydrated in 50 mM ammonium bicarbonate containingsequencing grade modified trypsin or chymotrypsin and leftfor overnight digestion at 37 °C. Proteolytic peptides wereextracted from the gel with 50 % acetonitrile in 5 % formicacid, dried, and reconstituted in 0.1 % formic acid for MSanalysis.

Liquid chromatography-tandem mass spectrometry analysesof the resulting peptides were performed with an LTQ OrbitrapXL linear ion trap mass spectrometer (Thermo Fisher Scientific,Waltham, MA, USA) coupled to an Ultimate 3000 HPLC system(Dionex, Sunnyvale, CA, USA). The digests were first trappedonto a C18 pre-column (PepMap100, 0.3 mm×5 mm, 5 μmparticle size, Dionex) and then equilibrated and desalted with0.1 % formic acid for 4 min. The peptides were eluted on C18Acclaim PepMap 100 column (0.075 mm×150 mm; Dionex)with a linear gradient of acetonitrile from 2 % to 60 % in 60 min-long run at a flow rate of 0.3 μL/min. Spectra were acquired in thepositive ionizationmode by data-dependentmethods consisting ofa full MS scan in the high mass accuracy FT-MS mode(resolution: 100,000) andMS/MS scans of the fivemost abundantprecursor ions in the CIDmode at the normalized collision energyof 30 %. The spray voltage was set at 2.4 kV. A dynamicexclusion function was applied with a repeat count of 2, repeatduration of 45 s, exclusion duration of 15 s, and exclusion size listof 350. The obtained data were submitted for a database searchagainst the ABCA4 amino acid sequence using Mascot Daemon(Matrix Science, Boston, MA, USA). Carbamidomethylation ofCyswas set as a fixedmodification, whereas oxidation ofMet andconversion of Asn to Asp were selected as variable modifications.The mass tolerance was set as 10 ppm for precursor ions and0.8 Da for product ions. Candidate N-linked-glycosylation siteswere initially identified in deglycosylated peptides based onconversion of Asn residues to Asp by PNGase F. Thecorresponding glycosylated peptides were then identified byCID tandem MS. Sugar compositions of glycopeptides initiallydetermined with GlycoMod software were further verified bymanual interpretation of their MS/MS spectra.

ResultsCID Tandem Mass Spectra Reveal the CompleteComposition and Some Sequence InformationAbout the Glycans in Native N-Glycopeptides

A typical CID tandem mass spectrum of an ABCA4glycopeptide with the initial m/z of 1353.2650(3+) illustrat-

ing sequential loss of sugar moieties is shown in Supple-mental Figure 1. In Supplemental Figure 1b, the fragmentions with m/z 1299.3 and 1867.7 can be easily recognized astriply and doubly charged parent glycopeptides with loss ofone and two hexoses, respectively. The subsequent losses ofhexoses followed by the dissociation of one HexNAc can beeasily traced based on the fragment ions of either the triplycharged form (1299.3 → 1245.4 → 1191.3 → 1137.6 →1083.5 → 1029.6 → 975.5 → 921.3 → 867.3 → 799.7) orthe doubly charged form (1867.7 → 1786.6 → 1706.0 →1624.5 → 1543.9 → 1463.0 → 1381.5 → 1300.8 →1198.9). Ions with m/z 799.7 and 1198.9 correspond to thetriply and doubly charged (peptide + HexNAc) ions,respectively. The complete glycan composit ion(Hex)9(HexNAc)2 thus could be resolved and confirmedfrom both triply and doubly charged series of ions. On theother hand, high resolution of the full mass allows accuratedetermination of the charge state and monoisotopic mass ofthe parent ion, and therefore the mass of the glycopeptide(i.e., the sum of the masses of the peptide and glycan. Themass value of 2193.145 for the peptide itself derived fromthe calculated glycan mass subtracted from the measuredmass of the glycopeptide matches ABCA4 peptideSTEILQDLTDR1527NVSDFLVK with a mass error of1.511 ppm.

A hybrid type of N-glycopeptide was also examined andthe CID tandem mass spectrum is shown in Figure 1. Thesingly charged fragment ion m/z of 1885.9 was identified toresult from losses of one NeuAc, one HexNAc, and threeHex residues from the parent ion. The peptide ion carrying asingle N-acetylglucosamine (m/z 1196.5), and also the mostabundant fragment, was derived from the m/z 1885.9fragment by losing three Hex and one HexNAc (m/z1885.9 → 1723.8 → 1561.7 → 1399.6 → 1196.5). Similarto Supplemental Figure 1b, the glycan composition alongwith the conjugated peptide could be determined as(NeuAc)(Hex)6(HexNAc)3 + DIF504NITDR based on thefragment ions from CID tandem MS and the accurate fullMS of the glycopeptide. Aside from these singly chargedfragment ions, there was more rich information on doublycharged fragment ions. Among these, two major fragmentions (m/z 1352.6 and 1287.8) were derived directly from thedoubly charged parent ion through the loss of Hex orNeuAc. Then, following the loss of either NeuAc or Hex byseparate pathways (m/z 1433.6 → 1352.6 → 1207.0 or m/z1433.6 → 1287.8 → 1207.0), they reached the samefragment ion m/z of 1207.0. After one subsequent Hex lossfrom m/z 1207.0 to 1126.0, there was a further loss of Hexand HexNAc (m/z 1126.0→ 1045.0→ 943.4) to arrive at m/z943.4 (m/z 1126.0 → 1024.3 → 943.4). This ion with m/z943.4 corresponds to the peptide conjugated to three Hexand two HexNAc groups, a core structure for N-linkedglycosylation. Thus, the entire sugar composition wasverified by a series of doubly charged fragment ions.Alternatively, fragment ions with m/z 819.1 → 657.1 →454.1 corresponding to NeuAc-Hex-HexNAc-Hex →

B. Wang et al.: N-Glycosylation by CID MS/MS & Retention Time 731

Figure 1. Mass spectra of hybrid type glycans attached to different ABCA4 peptides at 504Asn: (a) DIF504NITDR from trypsindigestion, (b) NWRDIF504NITDRAL, and (c) NGPREGQADDVQNFNWRDIF504NITDRAL from chymotrypsin digestion. Green,blue, and purple arrows represent the loss of Hex, HexNAc, and NeuAc residues, respectively


NeuAc-Hex-HexNAc → NeuAc-Hex confirmed the pres-ence of the NeuAc-Hex-HexNAc-Hex glycan, which ap-pears associated with glycan sequence. However, caution isadvisable because monosaccharide rearrangements mayoccur, and the situation will be more complex for largeand highly branched glycans. In general, one should trust theglycan composition derived from the CID data, but notnecessarily the structure assignment. Glycan structuraldetermination requires the release of glycans prior to theirLC/MS analysis.

Besides trypsin, we also performed glycoprotein digestionwith chymotrypsin to increase sequence converge and obtainmore information about each possible glycosylation site.Chymotrypsin mainly cuts at hydrophobic residues such asTrp, Tyr, and Phe, and also slowly at Leu and Met residues.Nonspecific cleavage by chymotrypsin produces peptides withextensive sequence overlaps. As an example, the glycanmoiety

(NeuAc)(Hex)6(HexNAc)3 was observed conjugated tothe same glycosylation site 504Asn of different peptides,in particular DIF504NITDR generated with trypsind i g e s t i o n a n d NWRD I F 5 0 4N I T DRAL a n dNGPREGQADDVQNFNWRDIF504NITDRAL producedwith chymotrypsin (Figure 1a–c). Mass errors for theseassignments were 1.301, 1.463, and −1.168 ppm, respectively.Such protease digests by different or nonspecific proteases thatproduce glycopeptides with varying lengths of the peptideportion but the same glycan portion can serve as a secondaryverification for site- specific glycosylation mapping [27].

CID Tandem Mass Spectra Help Minimizethe Occurrence of False Positives DuringAssignment

There are 16 potential N-glycosylation sites in ABCA4 withthe consensus sequence Asn-X-Ser/Thr(X can be any aminoacid except Pro) located at positions 14, 98, 415, 504, 950,1455, 1527, 1586, 1660, 1732, 1803, 1817, 1931, 2005,2050, and 2251. For complex digests of large proteins likeABCA4 carrying multiple NXS/T motifs, different combi-nations of glycans and peptides could match the sameglycopeptide mass even when a high resolution massspectrometer is employed. As an example, a glycopeptidewith m/z 1213.2280(3+) has two possible assignments, bothwith a mass tolerance under 2 ppm (Figure 2a and Table 1).One is peptide STEILQDLTDR1527NVSDFLVK +(Hex)3(deoxyhexose)1(HexNAc)4, and the other is peptideDKNPEEYGITVISQPL1660NLTK + (Hex)6(HexNAc)2.However, the CID tandem mass spectrum of this glycopep-tide shown in Figure 2b clearly shows six Hex losses fromthe parent ion (m/z 1213.2 → 1159.7 → 1105.5 → 1051.5→ 997.5 → 943.4 → 889.4) followed by another HexNAcresidue loss leaving a (peptide + HexNAc) ion. Thus, onlythe latter assignment is supported by the MS/MS data. Thisresult indicates that glycopeptide mapping cannot be solelybased on a high resolution full MS; tandem MS is requiredto minimize false positive assignments [28].

It is generally accepted that characterization of glycopro-teins by mass spectrometry is typically more difficult thanthe mass spectrometric analysis of proteins because of theextensive heterogeneity and lower ionization efficiency ofglycoproteins [29]. Thus, glycopeptides are usually presentin low abundance in MS data compared with theircorresponding non-glycosylated peptides. Many glycopep-tide peaks are skipped in data-dependent LC-MS/MS runs.A challenging problem arises if the MS/MS spectrum of anindividual glycopeptide lacks the quality to solve the

Table 1. Two ABCA4 Glycopeptides That Can be Assigned to the Same Parent Ion with m/z of 1213.2280(3+), Creating Ambiguity in GlycopeptideMapping. The Mass Error is Under 2 ppm in Both Cases

Observed GP mass Peptide sequence Glyco site Glycan composition Theoretical GP mass Error (ppm)

3637.669 STEILQDLTDRNVSDFLVK Asn 1527 (Hex)3(Deoxyhexose)1 (HexNAc)4 3637.673 −1.173DKNPEEYGITVISQPLNLTK Asn 1660 (Hex)6(HexNAc)2 3637.663 1.746

Figure 2. Full MS (a) and CID MS/MS (b) mass spectra ofan ABCA4 glycopeptide with the parent ion m/z of1213.2280(3+)


complete glycan composition. To address this problem, LC-MS profiles of ABCA4 glyco- and de-glycopeptides wereextracted as shown in Figure 3. A typical glycopeptidepattern appeared in the glycopeptide pool in which ions withm/z such as 1137.19(3+), 1191.21(3+), 1245.23(3+),1299.25(3+), and 1353.27(3+) all differed by a mass of162 Da, which corresponds to the mass of the Hex sugarresidue. After PNGase F treatment, these glycopeptidesdisappeared and new peaks with m/z of 1097.57(2+) and732.05(3+) (the latter is not shown in this m/z range) wereformed corresponding to the same peptide with Asnconverted to Asp by PNGase F. The deconvoluted tandemmass spectrum of ions with m/z 732.05(3+) or 1097.57(2+)confirmed the peptide sequence of STEILQDLTDR1527N/DVSDFLVK (Supplemental Figure 2). That is, the glyco-peptide ions listed above originated from the same peptideSTEILQDLTDR1527NVSDFLVK conjugated with differentglycans. There was no free peptide observed in samples with

or without PNGase F treatment, indicating 100 % occu-pancy of the 1527Asn glycosylation site. The higherabundance fractions in the glycopeptide pool that allowdetermination of the entire glycan composition, such as1353.27(3+) ion shown in Supplemental Figure 1, can helpin interpreting and verifying other less abundant glycopep-tides if their own MS/MS spectra cannot provide the entireglycan composition.

Reliable Determination of Site-Specific In VivoN-Glycosylation by CID Tandem MS and HPLCRetention Time

As noted for all the glycopeptides we observed in ABCA4and, previously, in the 5HT4R serotonin receptor, glyco-peptides containing the same peptide but differing in theirsugar chains almost always eluted as a single cluster duringHPLC, with elution time differences spanning a couple of

Figure 3. Full MS of the glycosylated (a) and deglycosylated (b) ABCA4 peptide STEILQDLTDR1527NVSDFLVK eluting at 38–41 min. The peak at m/z 1097.57(2+) corresponds to the deglycosylated peptide with a mass shift of 0.980 Da because of theconversion of 1527Asn to an Asp residue due to PNGase F treatment

Table 2. List of Potential ABCA4 Glycopeptides with the Parent Ion m/z of 1037.2098(4+) Matching the Same Theoretical Mass

Observed GP mass Peptide sequence Glyco site Glycan composition Theoretical GP mass Error (ppm)

4145.8158 LLLWKNWTLRK Asn 14 (Hex)14(HexNAc)2 4145.792 5.676LNITFYESQITAFLGHNGAGK Asn 950 (Hex)9(HexNAc)2 4145.795 4.952DKNPEEYGITVISQPLNLTK Asn 1660 Na(Hex)9(HexNAc)2 4145.803 3.143


minutes [30]. Thus, we decided to determine site-specific N-glycosylation mapping by combining CID tandem MS withthe HPLC retention times of glycopeptides. Table 2 displaysanother example of an ABCA4 glycopeptide with m/z of1037.2098(4+) with multiple possible assignments. Theextensive fragment ions observed in Figure 4 revealed theentire glycan composition of (Hex)9(HexNAc)2. For exam-ple, triply charged fragment ions with m/z from 1329.3 to829.2 account for the sequential loss of nine Hex residuesfrom the parent ion followed by a HexNAc residue loss (m/z1329.3 with one Hex residue loss from the parent ion, then1329.3 → 1274.9 → 1221.2 → 1167.2 → 1113.3 → 1058.8→ 1005.3 → 951.2 → 896.7 → 829.2), which results in thepeptide carrying a single N-acetylgucosamine (m/z 829.2).A s s i g n m e n t o f t h e L L LWK 1 4NWTLRK +(Hex)14(HexNAc)2 glycopeptide could be easily excludedbecause of the incorrect glycan composition. Again, ourresults showed that CID MS/MS can be very helpful inminimizing the assignment of false positives.

In another case, two candidate glycopeptides had thesame mass with an error less than 5 ppm and carried thesame glycan moiety attached to different peptides,L950NITFYESQITAFLGHNGAGK or DKNPEEYGITVISQPL

1660NLTK. The only difference in the sugar portion was thepresence of a sodium adduct (Figure 4). The fragment ion atm/z 509.3 in CID tandem MS corresponding to Na(Hex)3implied the existence of a sodium adduct, but the ionintensity was low, and no other sodium adducts wereobserved. Thus, it was very difficult to distinguish betweenthese two possibilities based only on CID mass spectra.However, when we took the retention time into account(Figure 5), it was noted that the non-glycosylatedL950NITFYESQITAFLGHNGAGK peptide with the m/z of761.06 (3+) eluted at 43.82 min in the same LC-MS run,whereas the glycopeptide ion in question [m/z of1037.2098(4+)] had a retention time around 36.11 min.Although the addition of a high mannose type sugar moietyand a Na adduct could result in a slight retention timechange, a 7-min difference between the same peptide withand without such a polysaccharide in the same LC/MS run isfar too great. On the other hand, a glycopeptide with the m/zo f 1375 . 2804 (3+ ) un i que l y r ep r e s en t i ng th eDKNPEEYGITVISQPL1660NLTK + (Hex)9(HexNAc)2 con-jugate was observed with a retention time around 36.05 min,only 0.06 min different from that of the 1037.2098(4+) ion.Furthermore, the corresponding DKNPEEYGITVISQPL

Figure 4. Full MS (a) and CID MS/MS (b) spectra of an ABCA4 glycopeptide with parent ion m/z 1037.2098(4+)


1660N/DLTK peptide deglycosylated with PNGase F elutedat 38.84 min, only 2.73 min apart from its glycopeptide in aseparate LC/MS run. This small difference could beattributed to the conversion of Asn to Asp [31]. The peptidesequence and the N/D conversion were confirmed by thetandem MS shown in Supplemental Figure 3. Hence, bycombining the information from CID tandemMSwith retentiontimes of glycopeptides, we were able to confidently assign1037.2098(4+) ion to the DKNPEEYGITVISQPL1660NLTK +Na (Hex)9(HexNAc)2 glycopeptide.

Another important result provided in Figure 3 is that thedeglycosylated peptide with the Asn converted to an Asp by

PNGase F actually appears at the same retention time rangeof 38–41 min with its clustered glycopeptides even in aseparate LC-MS run. Deconvolution of the CID tandemmass spectrum of this deglycosylated peptide, which isusually easier to obtain compared with its glycoforms, canclearly reveal the peptide sequence. Moreover, if a glyco-sylation site is only partially occupied as we previouslyobserved in the 5HT4R serotonin receptor [30], the samepeptide with and without attached saccharides can beidentified in a single LC-MS run. In such a case,determination of the unglycosylated peptide is simplebecause the retention time of the root peptide is close tothat of its glycoform without the delay introduced by theAsn to Asp conversion [30]. Identification of the glycosyl-ation site and glycopeptide can then be confirmed based onthe retention times of un- or deglycosylated root peptide.Thus, the addition of HPLC retention time data couldprovide a solution to the problem of CID tandem MS oflacking sufficient peptide information for accurate glyco-peptide mapping.

DiscussionAmong the now-used fragmentation modes, only CID canprovide complete glycan composition, whereas alternativefragmentation modes like HCD, ETD, and ECD functionprimarily to identify glycopeptide or glycosylation sites.Moreover, CID is the most routinely used method with themost rapid acquisition speed and the highest intensity ofproduct ions. This mode can provide an average scan speedover 2 times faster than HCD [32] and a signal intensity 10times higher than ETD spectra for the same peptidefragmentation [33]. Thus, the CID-retention time approachcan be beneficial for characterization of low-abundanceglycopeptides. In addition, LC-MS analysis of correspond-ing deglycosylated peptides from PNGase F treatment innormal or O18water is already included in the standardexperimental pipeline for site-specific glycosylation map-ping to screen N-glycosylation sites and calculate theiroccupancies. No additional experimental steps are requiredto determine the retention times of deglycosylated peptides.

Factors Influencing the Retention Timeof Glycopeptides

Obviously, the HPLC retention time of a glycopeptidedepends on the properties of its components, the glycanand the peptide. However, these components differentiallyaffect the retention time. As shown in Figure 6, addition ofhexose and HexNAc sugar residues causes a glycopeptide toelute earlier, a phenomenon attributable to the hydrophiliccharacter of the sugar moieties. In contrast, the presence ofNeuAc or a phospho- group on the glycan chain results in anincrease in retention time, as the negatively-charged NeuAcand phospho groups counteract the positive charge of theglycopeptide proton, resulting in a tighter binding to the

Figure 5. Comparison of retention time of (a) the1037.2098(4+) ion introduced in Figure 4; (b) peptideL950N ITFYESQITAFLGHNGAGK; (c ) glycopept ideDKNPEEYGITVISQPL1660NLTK+(Hex)9(HexNAc)2; (d) pep-tide DKNPEEYGITVISQPL1660N/DLTK from PNGase F treat-ed sample in a separate LC/MS run


reversed phase HPLC matrix. For example, addition of oneNeuAc or phospho group shifts the retention time ofglycopeptide DIF504NITDR from 32.02 min to 36.86 or37.68 min, respectively. The larger the glycan moiety, thegreater the effect it has on the retention time of a glycopeptide.However, this generality does not seem to apply to Hex andHexNAc saccharides. For instance, the retention time ofglycopeptide STEILQDLTDR1527NVSDFLVK changes from39.36 only slightly to 39.09 min because of an increase in Hexresidues from five to nine. The peptide component also has amajor influence on the retention time of a glycopeptide.Examples of a strong peptide effect on the retention time areshown in Figure 6 and Table 3. Retention times of 25.41,36.27, 39.30, and 50.91 min correspond to the same glycan(Hex)6(HexNAc)2 conjugated to peptides NA

415NSTFEELER,DKNPEEYGITVISQPL1527NLTK, STEILQDLTDR1527

NVSDFLVK, and LPAPPFTGEALVGFLSDLGQLM1586

NVSGGPMTR, respectively. The retention time of a glyco-peptide strongly depends on its size and composition as well asthe physical and chemical properties of its amino acids. Thus, itis the peptide component rather than the attached glycans (withan exception for those altering the charge, such as NeuAc andphosphor- groups) that primarily contributes to the retentiontime of the corresponding glycopeptide. This fact enablesglycopeptide identification based on comparison with retentiontime of the corresponding un- or deglycosylated peptide.

Retention time-based prediction of peptides has beenapplied in proteomics to improve confidence in peptideidentification in shotgun experiments [34, 35]. Attempts alsohave been made to use retention time predictions forpeptides with some post-translational modifications, suchas acetylation, methylation, oxidation, and phosphorylation[36–39]. Of course, the more hydrophobic the peptide, themore organic solvent is required to elute it from a reversed-phase column. A linear dependence of peptide retentiontimes on peptide mass is often observed when lineargradients are used in reversed-phase chromatography [40].For post-translational modifications that do not alter charge,it is reasonable to expect modest changes in retention time[41], with the exception of Met oxidation [42]. This is inagreement with our observation of retention time changes forglycopeptides.

In addition to its retention time, the apparent relativeabundance of each glycoform at a specific site can also becalculated from the area under the corresponding HPLCpeak (with an assumption of modest variation in ionizationefficiencies), which is essential to understanding the struc-ture and function of glycoproteins. Since no glycopeptideenrichment was performed in our study, there were noassociated shifts in the glycopeptide yields. The resultingsite-specific glycopeptide mapping of ABCA4 includesidentification of N-glycosylation sites, glycan compositions,

Figure 6. Retention time of glycopeptides (a) STEILQDLTDR1527NVSDFLVK with high mannose type of glycosylation andDIF504NITDR with hybrid type glycosylation (b)


Tab

le3.

Sum

maryof

N-G

lycosylatio

nMapping

ofABCA4by

MassSpectrometry

Using

CID

-OnlyFragm

entatio

nandHPLCRetentio

nTim

e(RT)of

Glycopepides(G

P)

ObservedGPmass

RT(m

in)

Glycancompo

sitio

nRelativeAbu

ndance

Sequence

Glyco

site*

Theoretical

GPmass

Error

(ppm

)

2688.080

25.41

Hex

6(H

exNAc)

26%

NANSTFEELER

N41

526

88.078

0.64

428

50.131

25.36

Hex

7(H

exNAc)

252

%28

50.130

0.25

632

12.185

25.22

Hex

8(H

exNAc)

285

%30

12.183

0.57

431

74.238

25.17

Hex

9(H

exNAc)

210

0%

3174.236

0.54

5

2575.059

32.02

Hex

6(H

exNAc)

310

%DIFNITDR

N50

425

75.055

1.44

927

04.102

37.08

Hex

5(H

exNAc)

3(N

euAc)

110

%27

04.098

1.37

928

66.154

36.86

Hex

6(H

exNAc)

3(N

euAc)

110

0%

2866.150

1.30

126

55.015

37.68

Hex

6(H

exNAc)

3(phos)1

14%

2655.021

−2.361

3459.473

36.81

Hex

3(H

exNAc)

310

0%

CLKEEWLPEFPCGNSSPWK

N14

5534

59.476

0.66

836

62.559

36.70

Hex

3(H

exNAc)

416

%36

62.555

1.12

2

3409.566

39.36

Hex

5(H

exNAc)

27%

STEILQDLTDRNVSDFLVK

N15

2734

09.563

0.96

835

71.620

39.30

Hex

6(H

exNAc)

246

%35

71.615

1.32

437

33.673

39.25

Hex

7(H

exNAc)

270

%37

33.667

1.53

538

95.725

39.14

Hex

8(H

exNAc)

210

0%

3895.720

1.21

440

57.778

39.09

Hex

9(H

exNAc)

242

%40

57.773

1.16

6

4259.006

51.20

Hex

4(H

exNAc)

23%

LPAPPFTGEALVGFLSDLGQLMNVSGGPMTR

N15

8642

58.977

6.88

044

21.034

51.08

Hex

5(H

exNAc)

210

0%

4421.030

0.97

345

83.113

50.91

Hex

6(H

exNAc)

224

%45

83.082

6.70

5

3637.669

36.27

Hex

6(H

exNAc)

229

%DKNPEEYGITVISQPLNLTK

N16

6036

37.662

1.76

837

99.718

36.22

Hex

7(H

exNAc)

211

%37

99.714

0.99

539

61.772

36.16

Hex

8(H

exNAc)

210

0%

3961.767

1.09

341

23.826

36.05

Hex

9(H

exNAc)

255

%41

23.820

1.29

2

*Glycancompo

sitio

nson

N98

wereno

tavailable.


apparent N-glycosylation site occupancies, and apparentrelative abundances of each polysaccharide detected for asingle site (Table 3). The corresponding experimentalpipeline is presented in Scheme 1. Here we focus especiallyon the strategy to achieve reliable N-glycosylation mapping,since the standard approaches to determine site-specificglycosylation have been well reviewed [43]. In our case,glycoproteins and their corresponding deglycosylated pro-teins were simply separated by SDS PAGE, the targetprotein band was ‘in-gel’ digested, and MS analysis basedon CID-only fragmentation and chromatographic retentiontime was performed. First, a high-resolution full MSprovides a highly accurate glycopeptide mass with an errorwithin 5 ppm. Then, a complete glycan fragmentation byCID tandem MS allows the correct assignment of glycancomposition. Thus, the peptide mass is calculated bysubtracting the entire glycan mass from the glycopeptidemass. Candidate glycosylated peptides then were initiallyproposed based on this peptide mass and the known protein

sequence. Finally, identification of the peptide confirmedbased on the retention time of either the unglycosylatedpeptide in the same LC-MS/MS run in the case of partialoccupancy of the corresponding site, or that of thedeglycosylated peptide in a separate LC-MS/MS run ifthe site is fully glycosylated. Furthermore, in addition toproteolysis with a specific protease (usually trypsin), anonspecific protease such as chymotrypsin can beemployed. Verification of the glycopeptide assignmentthen is accomplished by utilizing different proteases(which can have varying specificity), which generatepeptides of varying lengths with extensive sequenceoverlaps. The use of different enzymes for digestion isnecessary for our analytical strategy, not only to covermost of the potential glycosylation sites, but also toassist in assigning each glycosylation site to differentglycopeptides.

Notably, these unequivocal assignments were madewithout the input of protein structure or topological model

Scheme 1. The experimental pipeline for site-specific N-glycopeptide mapping using CID-retention time approach. See maintext for details


information, which can be very advantageous for studies ofpoorly characterized proteins. In the case of ABCA4,however, the known membrane topology [26] was usedto validate our N-glycosylation mapping. Among 16potential N-glycosylated sites in ABCA4, seven arefound N-glycosylated (with further glycan compositionssolved for six of them) and eight are unglycosylated. Incomplete agreement with this topological model, all thedetected N-glycosylation sites were found situated in theexo-cytoplasmic protein regions, whereas none of thetheoretical N-glycosylation sites in the cytoplasm wereshown to be glycosylated. The only undetected site isAsn1732 located in a short loop that connects twotransmembrane helices. Such a location makes it diffi-cult to obtain the corresponding peptide by enzymaticdigestion.

Since no glycopeptide enrichment was performed here,all unglycosylated peptides were also available for analysis.Thus, protein identification should be easily achieved by asimple database search without the input of any glycosyla-tion information. Thus, our CID-retention time approachworks well for known and unknown proteins and is notlimited to a single protein. It could face a greater challengefor glycoproteomic studies with large number of glycopep-tides in the mixture. However, with the assistance ofincreasing structural data about investigated proteins avail-able in the literature and the development of software forretention time predictions, coupled to continuing improve-ments in instrumentation, our approach is expected toprovide valuable information for analysis of protein mixturesas well.

ConclusionsWe present a comprehensive approach to site-specific N-glycopeptide mapping that allows simultaneous glycanand peptide characterization by combining CID tandemmass spectrometry with the HPLC retention times ofglycopeptides. The former can provide the entire glycancomposition, and the latter can assist in identifying theroot peptide from which the glycopeptide is derived.Detailed information about N-glycosylation sites, glycancompositions and occupancies as well as relative abun-dances of each glycoform at a specific site was obtainedbased on just two parallel analyses of glycosylated anddeglycosylated peptides.

AcknowledgmentsThe authors are grateful to Dr. Leslie T. Webster, Jr., forcritical comments. This publication was made possible inpart through support by the National Institute for Allergyand Infectious Diseases (Center for AIDS Research Proteo-mics Core, P30-AI-036219), the National Cancer Institute(Cancer Center Proteomics Core, P30-CA-043703), and theNational Institute for Biomedical Imaging and Bioengineer-

ing (R01-EB-009688) (to M.R.C.) and the National EyeInstitute of the National Institutes of Health under awardnumbers of R01EY009339 and P30EY11373 (to K.P.). K.P.is John H. Hord Professor of Pharmacology. M.R.C. isCharles W. and Iona A. Mathias Professor of CancerResearch.

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