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Low Abundant N‑linked Glycosylation in Hen Egg White Lysozyme Is Localized at Nonconsensus Sites Arndt Asperger,*,† Kristina Marx,† Christian Albers,† Laura Molin,‡ and Odra Pinato‡ †

Bruker Daltonik GmbH, Fahrenheitstrasse 4, 28359 Bremen, Germany Chelab Silliker, Via Fratta 25, 31023 Resana, Italy

‡

S Supporting Information *

ABSTRACT: Although wild-type hen egg white lysozyme (HEL) is lacking the consensus sequence motif NX(S/T), in 1995 Trudel et al. (Biochem. Cell Biol. 1995, 73, 307−309) proposed the existence of a low abundant N-glycosylated form of HEL; however, the identity of active glycosylation sites in HEL remained a matter of speculation. For the first time since Trudel’s initial work, we report here a comprehensive characterization by means of mass spectrometry of N-glycosylation in wild-type HEL. Our analytical approach comprised ZIC-HILIC enrichment of N-glycopeptides from HEL trypsin digest, deglycosylation by 18 O/PNGase F as well as by various endoglycosidases, and LC−MS/MS analysis of both intact and deglycosylated N-glycopeptides engaging multiple techniques of ionization and fragmentation. A novel data interpretation workflow based on MS/MS spectra classification and glycan database searching enabled the straightforward identification of the asparagine-rich N-glycopeptide [34−45] FESNFNTQATNR and allowed for compositional profiling of its modifying N-glycans. The overall heterogeneity profile of N-glycans in HEL comprised at least 26 different compositions. Results obtained from deglycosylation experiments provided clear evidence of asparagine residues N44 and N39 representing active glycosylation sites in HEL. Both of these sites do not fall into any known N-glycosylation-specific sequence motif but are localized in rarely observed nonconsensus sequons (NXN, NXQ). KEYWORDS: hen egg white lysozyme, HEL, N-glycosylation, nonconsensus sequon, glycan heterogeneity, MALDI-TOF/TOF, LC−MS/MS, CID, ETD, bioinformatics

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INTRODUCTION Glycosylation represents one of the most frequently appearing modifications in proteins. At the same time, it is the most complex type of modification due to its tremendous site-specific structural heterogeneity, which adds to the experimental challenges faced in the process of comprehensive characterization of glycoproteins. Commonly, the amino acid sequence motif NX(S/T), where X may be any amino acid with the exception of proline, is considered as a consensus sequon required for biosynthetic Nglycosylation to occur.2,3 Hen egg white lysozyme (HEL) is one of the most extensively studied glycoside hydrolase enzymes and was the first enzyme for which a crystal structure had been published.4 It exhibits strong antibacterial functionality by attacking peptidoglycans found in the cell walls of bacteria.5 The protein sequence of HEL consists of 129 amino acid residues (excluding the signal peptide), resulting in a molecular weight of 14.4 kDa. The sequence does not contain the recognized glycosylation motif NX(S/T). Nevertheless, in 1995, Trudel et al.1 proposed the existence of a low abundant N-glycosylated form of wild-type HEL based on results obtained from Concanavalin A binding, SDS-PAGE separation, Edman © 2015 American Chemical Society

sequencing, and deglycosylation by peptide-N-glycosidase F (PNGase F); however, the actual sites of glycosylation in HEL remained a matter of speculation. Trudel suggested asparagine residues N74 and N113, respectively, as potential glycosylation sites. This assumption was based on the fact that both of these sites are at least part of the sequon NXC, which had been previously reported to represent a minor N-glycosylation motif;6−8 however, this hypothesis was never sustained by experimental data, such as data obtained from mass spectrometry (MS). Surprisingly, until today further references regarding Nglycosylation in wild-type HEL do not exist. This may be due to the fact that most of the analytical strategies applied to screening of N-glycosylation take into account only those modification sites that are related to the commonly accepted consensus sequons NX(S/T/C), which are considered essential for N-glycosylation in proteins; however, cases of nonconsensus N-linked glycosylation have been reported for several proteins from different origins. For example, Valliere-Douglass et al. described in detail the presence of nonconsensus NReceived: February 23, 2015 Published: May 12, 2015 2633

DOI: 10.1021/acs.jproteome.5b00175 J. Proteome Res. 2015, 14, 2633−2641

Article

Journal of Proteome Research glycosylation at low abundance levels on four asparagine residues in IgG1 and IgG2 antibodies.2,3 Recently, Chandler et al. documented the occurrence of nonconsensus N-linked glycosylation in interalpha-trypsin inhibitor derived from both serum and recombinant expression.9 Nearly 20 years after Trudel’s initial findings, we revisited the proposed low abundant N-glycosylation in wild-type HEL with the aim of its in-depth characterization by means of MS-based methods. In the resulting work presented here we applied an analytical approach combining ZIC-HILIC enrichment of Nglycopeptides with deglycosylation by PNGaseF and multiple endoglycosidases, respectively, followed by LC−MS/MS analysis of both the intact and deglycosylated N-glycopeptides employing various MS techniques (MALDI-TOF/TOF, ESI− CID−MS/MS, ESI−ETD−MS/MS). A novel bioinformatics workflow, based on classification of MS/MS spectra and subsequent glycan database searching, enabled the automated detection and characterization of intact N-glycopeptides from LC−MS/MS data sets. This workflow facilitated the initial identification of an asparagine-rich HEL peptide carrying the proposed N-linked glycosylation and allowed for profiling the compositional heterogeneity of the modifying N-glycans. Results obtained from deglycosylation experiments, in addition, provided final evidence about the identity of the previously unknown sites of N-linked glycosylation in HEL, which are localized in rare nonconsensus sequons that have been observed in only very few cases in eukaryotes before.

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Figure 1. Methodologies applied to the analysis of low abundant Nglycosylation in HEL.

mM IAA dissolved in 50 mM AmBic was added, followed by 30 min of incubation at room temperature in the dark. Following incubation, the remaining IAA was destroyed by the addition of another 6.2 μL of DTT solution and incubation at 50 °C for 15 min. Finally, 20 μL of a 100 ng/μL solution of trypsin was added and incubated at 37 °C overnight. Digestion was terminated by the addition of 35 μL 10% TFA in water. ZIC-HILIC Enrichment of N-Glycopeptides. The protocol applied for the enrichment of the N-glycopeptides was derived from the one reported by Neue et al.10 In brief, 600 pmol of HEL trypsin digest was dried down in a speedvac and reconstituted in 50 μL of buffer A (ACN/H2O/FA 80/20/2 v/ v/v). Prior to glycopeptide binding the ZIC-HILIC tips were equilibrated by pipetting 10 times 50 μL of buffer A. The glycopeptides were then bound by aspirating and dispensing 20 times the 50 μL aliquot of HEL digest reconstituted in buffer A. Trapped glycopeptides were washed by pipetting 5 times with 50 μL buffer A and were eluted in 30 μL of buffer B (H2O/FA 98/2 v/v). The eluate was dried down in a speedvac. Deglycosylation by PNGaseF in 18O Water. The dried sample resulting from ZIC-HILIC enrichment of 600 pmol HEL trypsin digest was reconstituted in 30 μL of 50 mM AmBic solution prepared in 18O water, to which 2 μL of PNGaseF solution (10 units/μL) was added. The solution was incubated at 37 °C overnight. After incubation, the solution was dried down in a speedvac and reconstituted in 20 μL of 0.1% TFA for further analysis by MS. Deglycosylation by Endoglycosidases. In addition to PNGase F, N-glycopeptides enriched from 600pmol of HEL trypsin digest were subjected to deglycosylation by a variety of endoglycosidases. All of the endoglycosidases were used as per the suppliers’ protocols. In a first step, Endo F2 and F3 were applied as a mixture (0.004 and 0.006 units, respectively). Second, Endo F1 was applied (0.005 units), followed by Endo H (500 units). Deglycosylations by Endo F were performed in 50 μL reaction volume at 37 °C for 1 h, whereas Endo H treatment was performed in a reaction volume of 20 μL at 37 °C overnight. Between the individual deglycosylation steps, the sample was dried down in a speedvac and reconstituted in the reaction buffer supplied with the enzyme to be applied next. The readily deglycosylated samples were dried down and finally reconstituted in 20 μL of 0.1% TFA.

EXPERIMENTAL SECTION

Materials

Wild-type HEL (UniProt accession P00698) was obtained from Chelab Silliker (Resana, Italy). Sequencing-grade trypsin (porcine), peptide-N-glycosidase F (PNGaseF, Elizabethkingia miricola overexpressed in E. coli), and endoglycosidase H (Endo H, Streptomyces plicatus overexpressed in E. coli) were purchased from Promega (Madison, USA). Endoglycosidases F1, F2, and F3 (Endo F1−3, Elizabethkingia miricola) were purchased from Sigma-Aldrich (Buchs, Switzerland) in the form of the native protein deglycosylation kit. Dithiothreitol (DTT), iodoacetamide (IAA), ammoniumbicarbonate (AmBic), trifluoroacetic acid (TFA), formic acid (FA), and ammonium phosphate monobasic (AmPhos) also came from Sigma-Aldrich. MALDI matrix alpha-cyano-4-hydroxycinnamic acid (HCCA) was used from Bruker (Billerica, USA).18O water (>95% isotopic purity) was purchased from Campro Scientific (Berlin, Germany). UPLC−MS-grade acetonitrile (ACN) was purchased from Biosolve BV (Valkenswaard, Netherlands). Deionized water (conductivity 0.055 μS/cm) was prepared in the lab using an ELGA LabWater system (Veolia, Celle, Germany). Protea ZIC-HILIC tips, 10−200 μL, were purchased from Dichrom (Marl, Germany). Methods

A schematic outline of the analytical approaches utilized in the analysis of low abundant N-glycosylation in wild-type HEL is depicted in Figure 1. Sample Preparation

Trypsin in-Solution Digestion of HEL. 100 μg of HEL was dissolved in 659 μL of 50 mM AmBic buffer. After the addition of 6.2 μL of 45 mM DTT dissolved in 50 mM AmBic, the solution was incubated at 50 °C for 30 min. 3.1 μL of 100 2634


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Journal of Proteome Research Mass Spectrometry Analysis

spotted onto a Bruker MTP Anchorchip 1536 TF plate. All other parameters used for MALDI spotting and automated MALDI-MS data acquisition were identical to those previously described for the analysis of intact glycopeptides. MALDI-MS/ MS spectra of the deglycosylated peptides of interest were acquired manually from the spot positions representing the LC peak maxima of the individual deglycosylated peptide isoforms. Depending on the precursor intensity, between 7000 and 12 000 laser shots were accumulated per MS/MS spectrum. LC−ESI−MS/MS Analysis. The LC setup used for ESI− MS/MS coupling employed the same UPLC system and identical eluents as previously described for UPLC−MALDIMS analysis. All separations were performed at 50 °C, with a flow rate of 300nL/min. For the LC−ESI−MS/MS analysis of intact N-glycopeptides, the following UPLC gradient was applied: 0 min, 2% B; 10 min, 2% B; 220 min, 15% B; 225 min, 40% B. The same UPLC conditions were used for the separation of N-glycopeptides deglycosylated with 18O/PNGaseF. N-Glycopeptides deglycosylated with Endo F and H were separated using a slightly different gradient: 10 min, 2% B; 180 min, 20% B; 250 min, 40% B. The UPLC system was interfaced to a Bruker amaZon speed ETD high-capacity ion trap mass spectrometer equipped with a CaptiveSpray nanoBooster ion source (Bruker) operated under Compass for amaZon series 1.7 software. All experiments were performed in positive ion mode using the following ion source parameters: capillary voltage, 1.3 kV; nebulizer gas pressure of acetonitrile-enriched sheath gas, 0.2 bar; dry gas flow, 3 L/min; dry gas temperature, 150 °C. The iontrap instrument was operated at scan speeds of either 8100 amu/s (enhanced resolution scan) or 32 500 amu/s (UltraScan). For LC−ESI−ETD−MS/MS analysis of the intact N-glycopeptides, an inclusion list was used containing the 3+ and 4+ charged molecular ions of the most abundant Nglycopeptide identified by previous MALDI-TOF/TOF experiments. In the analyses of the deglycosylated N-glycopeptides, up to three most abundant precursors per MS scan were selected for fragmentation. For ETD, a reaction time of 100 ms was applied. A complete list of further MS and MS/MS parameters used in the individual LC−ESI−MS/MS analyses is given in Supplementary Figure S1 in the Supporting Information (SI).

Direct MALDI-MS/MS Analysis. 0.5 μL of the reconstituted samples resulting from ZIC-HILIC enrichment and deglycosylation of HEL trypsin digest was spotted on a Bruker MTP Anchorchip 384 TF plate and dried down under a mild stream of air at a slightly elevated temperature. 0.5 μL of HCCA matrix solution (1.4 mg/mL HCCA dissolved in 85% ACN, 15% H2O, 0.1% TFA, 1 mM AmPhos) was added to the dried sample and dried down under room conditions. Bruker Peptide Calibration Standard II was spiked into the matrix solution spotted on the nearest neighbor calibrant spot position for external mass calibration. MALDI spectra were acquired in positive ion mode using a Bruker ultraflextreme MALDI-TOF/ TOF instrument equipped with smartbeam-II laser11 operated under Compass for flex series 1.4 software. MS spectra were acquired at 2 kHz laser repetition rate in reflector mode using an acceleration voltage of 25 kV and covering a detection m/z range of 700−4000. MS/MS spectra were acquired in LIFT mode12 at 1 kHz laser repetition rate applying 7 kV for initial acceleration of ions and 19 kV for reacceleration of fragments in the LIFT cell. Depending on signal intensity, between 4000 and 10 000 laser shots were accumulated per spectrum. LC−MALDI−MS/MS Analysis. LC−MALDI−MS/MS analysis of the ZIC-HILIC enriched intact N-glycopeptides was performed on a Bruker EASY nano LC II operated in twocolumn mode. A trap column NS-MP-10 (Nanoseparations, Nieuwkoop, Netherlands, 20 mm length × 100 μm inner diameter, 5 μm particle size) was coupled with an analytical column Acclaim PepMap100 (Thermo Fisher, 150 mm length × 75 μm inner diameter, 3 μm particle size). The system was operated at room temperature at an analytical flow rate of 300nL/min. A 24 min linear gradient was applied ranging from 2 to 45% eluent B using 0.05% TFA in water as eluent A and 0.05% TFA in 90/10 ACN/H2O as eluent B. 96 LC fractions were collected (15 s each; fraction collection started 10 min after the LC gradient had started) and were automatically spotted onto a Bruker MTP Anchorchip 384 TF plate using a PROTEINEER fc II fraction collector (Bruker). HCCA matrix solution (420 nL per spot, 1.5 mg/mL dissolved in 95% ACN, 5% H2O, 0.1% TFA, 1 mM AmPhos) was added during fraction collection via an integrated syringe pump and a microtee mixing chamber. MALDI-MS and -MS/MS data were acquired automatically in positive ion mode, accumulating 4000 laser shots per spectrum. All other parameters of data acquisition were identical to those used in direct MALDI-TOF/TOF analysis. From the LC fractions containing the main portion of the N-glycopeptides, additional MALDI-TOF-MS spectra were acquired in linear operation mode using both positive and negative ion polarities. When operating the instrument in negative ion mode, an acceleration voltage of 20 kV was used. For the separation of the enzymatically deglycosylated Nglycopeptides, an Ultimate 3000 RSLC nano UPLC (Thermo Fisher) was used equipped with an Acclaim PepMap100 nanoViper trap column (Thermo Fisher, 20 mm length, 100 μm inner diameter, 5 μm particle size) and an Acclaim PepMap RSLC C18 (Thermo Fisher, 500 mm length, 75 μm inner diameter, 2 μm particle size) as analytical column. Deglycosylated peptides were separated at 50 °C at a flow rate of 300 nL/ min in a 237 min multistep gradient: 0 min, 2% B; 10 min, 2% B; 20 min, 5% B; 227 min, 32% B; 237 min, 40% B (eluent A: 0.1% FA in water, eluent B: 0.1% FA in ACN). 1360 LC fractions were collected (fraction width 10 s, fraction collection started 18 min after LC gradient had started) and automatically

Data Analysis

For further analysis, all MS and MS/MS data were imported into the ProteinScape 3.1 database software (Bruker). LC−MS/MS data of the intact HEL N-glycopeptides were analyzed using an automated interpretation workflow in ProteinScape 3.1. Figure 2 displays a schematic of this data analysis workflow when applied to N-glycopeptide data obtained from LC−MALDI−MS/MS analysis. The software, in an initial step called “Classification”, detects and groups MS/ MS spectra of N-glycopeptide candidates by means of the recognition of a N-glycopeptide-specific, diagnostic fragment pattern and derives from this pattern the masses of the peptide and glycan moieties linked to each other. Supplementary Figure S2 in the SI contains the complete list of Classification parameters used in the analysis of LC−MALDI-TOF/TOF data obtained from the ZIC-HILIC-enriched HEL trypsin digest. The classification algorithm used here has been described in more detail by Hufnagel et al.13 The successful application of this algorithm to the characterization of N2635


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detected in step 1 (Classification) is identified by matching the MS/MS spectra against the theoretical masses and calculated fragments of tryptic peptides obtained from in silico digestion of HEL.

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RESULTS

Analysis of Intact N-Glycopeptides Localizes the HEL Sequence Region Carrying N-Glycosylation

Because of their expected low abundance (0.3% glycosylation rate previously determined by comparative SDS-PAGE analysis1), the N-glycopeptides were enriched from HEL tryptic digest by ZIC-HILIC pretreatment. Comparison of the MALDI-TOF-MS spectra prior and after ZIC-HILIC enrichment indicates a significant enhancement of glycopeptide signals after the ZIC-HILIC treatment (Figure 3). An intense pattern of MS peaks appearing at glyco-specific distances (162 mass units representing a difference of one hexose residue, 203 mass units representing a difference of one N-acetylhexosamine residue) is observed in the m/z range above 2000. For a more in-depth analysis, the ZIC-HILIC-enriched HEL digest was subjected to LC−MALDI−MS/MS analysis. Figure 4 displays an example MS/MS spectrum acquired from precursor m/z 3051.2 representing the most abundant Nglycopeptide candidate in the LC−MALDI−MS/MS data set, as detected by the Classification algorithm. MALDI-MS/MS spectra of N-glycopeptides are unique in that they provide rich information on both the sequence of the peptide moiety and the composition of the modifying glycan simultaneously. In particular, interpretation of the N-glycopeptide specific fragment pattern17 shown in the inset of Figure 4 enabled the reliable determination of a peptide moiety of MH+ = 1428.7 Da. This protonated peptide mass matches HEL tryptic peptide [34−45] FESNFNTQATNR, which was further confirmed by aligning its theoretical fragments to the MS/MS spectrum. (See annotation of peptide b,y- and y-17 fragment ions in Figure 4.) LC−ESI−ETD−MS/MS data also confirmed the identity of this amino acid sequence. The annotated ETD-MS/MS spectrum acquired from the most abundant N-glycopeptide FESNFNTQATNR/(Hex)5(HexNAc)4 is shown in Supplementary Figure S3 in the SI.

Figure 2. Data interpretation workflow featured in the ProteinScape software for the detection and characterization of N-glycopeptides from LC−MALDI−MS/MS data.

glycopeptides from a monoclonal antibody has been reported by Ayoub et al.14 As a further proof of principle, utilization of this algorithm contributed to the identification of a previously unknown N-glycosylation site in human prostate specific antigen.13,15 The glycopeptide candidate MS/MS spectra yielded from the Classification, in the second step, are submitted to a glycan database search against Carbbank16 using the GlycoQuest search engine (Bruker).13 When searching MS/MS data of glycopeptides, the GlycoQuest algorithm handles the peptide moiety determined in the prior Classification step as a mass offset attached to the glycan’s reducing end. Matching MS/MS spectra are being scored according the spectral coverage of theoretical glycan fragments calculated by GlycoQuest according to the fragmentation profile defined in the search method. Supplementary Figure S2 in the SI provides the complete list of parameters utilized in the GlycoQuest search of the LC−MALDI-TOF/TOF data obtained from the ZICHILIC-enriched HEL trypsin digest. In the final step of the data analysis workflow, the amino acid sequence of the peptide moiety present in the N-glycopeptides

Figure 3. MALDI-TOF-MS spectra of HEL tryptic digest. 1: Without ZIC-HILIC enrichment of N-glycopeptides; 2: After ZIC-HILIC enrichment of N-glycopeptides. 2636


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Figure 4. MALDI−MS/MS spectrum of the most abundant HEL N-glycopeptide [34−45] FESNFNTQATNR modified with N-glycan (Hex)5(HexNAc)4. Annotated b- and y-type ions represent free peptide fragments. Fragments annotated with [...-Pep] represent glycan fragments with the intact peptide moiety attached.

Table 1. List of N-Glycan Compositions Resulting from GlycoQuest Database Search of LC−MALDI−MS/MS Data Obtained from ZIC-HILIC-Enriched HEL N-Glycopeptide [34−45] FESNFNTQATNRa 1 2 3 4 5 6 7 S 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 a

peptide sequence

glycan composition

m/z measured

mass error [ppm]

GlycoQuest score

FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR FESNFNTQATNR

(Hex)3(HexNAc)2 (Hex)4(HexNAc)2 (Hex)5(HexNAc)2 (Hex)6(HexNAc)2 (Hex)7(HexNAc)2 (Hex)3(HexNAc)3 (Hex)4(HexNAc)3 (Hex)5(HexNAc)3 (Hex)3(HexNAc)4 (Hex)4(HexNAc)4 (Hex)5(HexNAc)4 (Hex)6(HexNAc)4 (Hex)3(HexNAc)5 (Hex)4(HexNAc)5 (Hex)5(HexNAc)5 (Hex)6(HexNAc)5 (Hex)3(HexNAc)6 (Hex)4(HexNAc)6 (Hex)5(HexNAc)6 (Hex)6(HexNAc)6 (Hex)3(HexNAc)7 (Hex)4(HexNAc)7 (Hex)5(HexNAc)7 (Hex)3(HexNAc)8 (Hex)4(HexNAc)8 (Hex)3(HexNAc)9

2320.962 2433.019 2645.074 2807.126 2969.174 2524.043 2686.099 2848.152 2727.126 2889.181 3051.234 3213.283 2930.208 3092.259 3254.308 3416.358 3133.285 3295.332 3457.381 3619.417 3336.364 3498.401 3660.416 3539.430 3701.541 3742.567

−2.31 −0.56 0.29 0.03 −1.66 −1.44 −0.13 0.04 −0.03 0.82 0.79 −0.58 0.67 0.06 −0.93 −1.88 −0.12 −1.89 −2.86 −7.47 −0.23 −4.62 −14.81 −4.01 11.88 11.78

17.1 20.4 25.5 24.2 19.3 33.1 30.6 23.2 38.9 43.8 41.3 24.3 39.4 33.4 32.0 30.5 34.1 31.3 23.0 37.0 30.1 25.6

Glycan compositions lacking GlycoQuest score values were not confirmed by MS/MS but only detected in MS.

Profiling the Heterogeneity of the N-Glycans in HEL

search returned 26 different glycan compositions modifying HEL peptide [34−45] FESNFNTQATNR. Twenty-two of them were confirmed by MS/MS data. (See Table 1.) As previously stated, (Hex)5(HexNAc)4 was found to represent the most abundant glycan form. Figure 4 shows the MALDIMS/MS spectrum of the respective glycopeptide with the

To characterize the composition of the N-glycans modifying HEL, the LC−MALDI−MS/MS data obtained from the ZICHILIC-enriched glycopeptides, after preprocessing with the Classification algorithm previously mentioned, were searched against Carbbank using the GlycoQuest search engine. The 2637


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Figure 5. N-Glycan heterogeneity profile derived from LC−MS analysis of ZIC-HILIC-enriched HEL N-glycopeptide [34−45] FESNFNTQATNR. Error bars represent standard deviations resulting from three-fold replicate measurements. Glycan compositions marked with * were detected in MS but have not been confirmed by MS/MS data.

Figure 6. Result obtained from 18O/PNGaseF assisted LC−ESI−CID−MS/MS analysis of ZIC-HILIC enriched HEL N-glycopeptides [34−45] FESNFNTQATNR. *N indicates conversion of Asn to (18O)Asp resulting from 18O/PNGaseF treatment.

glycan fragments annotated. All glycan fragments denoted with [Pep-...] contain the intact peptide moiety. The LC−MS data obtained from the ZIC-HILIC-enriched N-glycopeptides were further explored to profile the compositional heterogeneity of the N-glycans found in HEL (Figure 5). For comparison, MALDI and ESI results, which are in good agreement with each other, are displayed side by side. The profiles were generated by integrating the extracted ion chromatograms (EICs) of three replicate analyses each. While the MALDI results are based on the EIC traces of the singly charged glycopeptide molecular ions, the ESI profile represents the sum of the three major glycopeptide charge states (2+, 3+, 4+). As ionization efficiencies of N-glycopeptides are known to vary depending on the composition of the modifying glycans,18

the glycan heterogeneity profile given in Figure 5 has to be considered semiquantitative. Identification of Active N-Glycosylation Sites in HEL by Means of Deglycosylation Experiments

While the analysis of intact N-glycopeptides provided clear evidence of N-glycosylation occurring in HEL sequence range [34−45] FESNFNTQATNR, the exact identification of active glycosylation sites was not directly possible. The HEL glycopeptide identified by us comprises three asparagine residues in very close proximity to each other (N37, N39, N44). Assuming all of these three sites are possibly glycosylated, a mixture of up to three isobaric N-glycopeptide isomers could be generated, which would be difficult to separate in intact form. Thus, deglycosylation experiments were performed to eliminate the glycan heterogeneity and to enable 2638


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Figure 7. Result obtained from LC−ESI−ETD−MS/MS analysis of ZIC-HILIC enriched HEL N-glycopeptides [34−45] FESNFNTQATNR after deglycosylation with endo F1−F3 and endo H. ■ indicates N-linked HexNAc modification resulting from endoglycosidase treatment.

a more efficient separation of the isobaric N-glycopeptide species on the reversed-phase chromatography setup in use.

glycosylation motif; however, as clearly shown by the data we obtained from both intact and deglycosylated N-glycopeptides, asparagine residues N44 and N39 have to be considered as active glycosylation sites in wild-type HEL. Both of these sites are not part of any known glyco-specific sequence motif but fall into the unusual sequons NXN and NXQ, respectively. So far, these sequons have only been reported in very few cases for glycoproteins of eukaryotic origin.20 According to our LC− MS/MS data, only one of the identified N-glycosylation sites is occupied at a time. Neither the intact N-glycopeptide data nor the results obtained from enzymatic deglycosylation provided evidence of N-glycosylation occurring at both sites in parallel. The glycan heterogeneity profile given in Figure 5 shows an obvious lack of N-glycans containing sialic acid residues. Acidic N-glycopeptides are known to undergo extensive metastable decay in MALDI-TOF-MS due to neutral loss of sialic acids, which makes them “invisible” in reflector TOF-MS analysis; however, comparative LC−MALDI-TOF-MS experiments performed in linear TOF-MS mode in both positive and negative ion polarities (data not shown) yielded highly consistent glycan profiles when compared with the results obtained from the previous reflector TOF-MS measurements. Furthermore, LC−ESI−MS analysis did not reveal sialylated glycopeptide species either. This suggests the true absence of significant sialylation in HEL. While the glycopeptide MS/MS data presented here allowed us to determine the compositions of the N-glycans modifying wild-type HEL, further conclusions regarding their detailed structure were not drawn from that data; however, a comparison of our results with literature data available for glycan structures identified in other chicken egg white glycoproteins provides more insight into the potential structures of the HEL carbohydrates. Harvey et al.21 studied in detail the N-glycans present in ovalbumin and further glycoproteins copurified from hen egg white and identified 28 glycan compositions representing at least 37 structures of highmannose, hybrid and complex type, respectively. Twenty-four out of the 26 HEL glycan compositions observed by us were also reported by Harvey. Because glycoproteins of the same

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O/PNGaseF-Assisted LC−MS/MS Analysis

Figure 6 shows the results obtained from LC−ESI−CID−MS/ MS analysis of the ZIC-HILIC-enriched HEL N-glycopeptides after 18O/PNGaseF treatment. The data clearly pinpoint N44 and N39 as active N-glycosylation sites in HEL. The EIC of m/ z 716.3 and m/z 477.9, representing the doubly and triply charged molecular ion of the 18O-deamidated peptide [34−45] FESNFNTQATNR, shows separation of two major LC peaks. Interrogation of the underlying MS/MS spectra unambiguously indicates conversion of Asn to (18O)Asp at positions N44 and N39, respectively. LC−MALDI−MS/MS data acquired in parallel (given in Supplementary Figure S4) confirm these results. Endoglycosidase-Assisted LC−MS/MS Analysis

To supplement and confirm the findings derived from the 18O/ PNGaseF data and to exclude the possibility of any potential interferences resulting from chemical deamidation artifacts produced by the 18O/PNGaseF method,19 we performed additional experiments treating the ZIC-HILIC-enriched HEL N-glycopeptides with endo F1−F3 and endo H prior to LC− MS/MS analysis (Figure 7). Again, the EICs of 2+ and 3+ molecular ions of the deglycosylated peptide FESNFNTQATNR, together with the underlying MS/MS spectra, clearly point toward N44 and N39 as the active sites of Nglycosylation in HEL. The respective LC−MALDI−MS/MS data (given in Supplementary Figure S5 in the SI) are fully consistent with the results obtained from ESI−ETD−MS/MS analysis.

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DISCUSSION Our finding of N-glycosylation being present on HEL peptide [34−45] FESNFNTQATNR disproves previous assumptions proposing asparagine residues N74 and N113 as potential glycosylation sites.1 Trudel’s proposal was purely based on the fact that both of these suggested sites are part of the sequon NXC, which had been known to represent a minor N2639



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origin, that is, hen egg white, may be expected to be glycosylated by the same oligosaccharyltransferases, it seems to be reasonable to adopt the structures published by Harvey21 to the N-glycans found by us in wild-type HEL, at least to the most abundant ones. According to this, the two major glycans we detected in HEL, (Hex)5(HexNAc)4,and (Hex)4(HexNAc)4, would correspond to hybrid structures comprising a beta-1,4 linked bisecting GlcNAc residue. Furthermore, the occurrence of N-glycans in HEL that are particularly rich in GlcNAc but lack further galactose residues terminating the antennae (in our data we detected glycans ranging up to (Hex)3(HexNAc)9) is fully consistent with previously published work reporting on the composition of Nlinked carbohydrates identified in other glycoproteins from hen egg white.21 The novel GlycoQuest data analysis workflow used in this work proved to be extremely useful in facilitating the automated detection and characterization of low-abundant N-glycosylation in HEL. This workflow, performed at the level of intact glycopeptides, based on MS/MS spectra classification in combination with glycan database searching, enabled the straightforward, unambiguous identification of the HEL sequence region carrying N-linked glycosylation and allowed for efficient profiling of the compositional heterogeneity of the modifying N-glycans. As shown in this work for HEL, the strategy provides improved access to completely unknown or highly unexpected N-glycosylations, including those linked to rarely observed nonconsensus sequons. Compared with the manual way of interpretation of glycopeptide MS/MS data, which usually had to be followed in the past due to the lack of suitable bioinformatics tools, the described GlycoQuest workflow contributes to a significantly improved time efficiency achieved in the detailed characterization of glycoproteins.

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ACKNOWLEDGMENTS

We thank Yvonne Jahn for excellent lab work.

REFERENCES

(1) Trudel, J.; Asselin, A. Detection of a glycosylated form of hen egg white lysozyme. Biochem. Cell Biol. 1995, 73, 307−309. (2) Valliere-Douglass, J. F.; Kodama, P.; Mujacic, M.; Brady, L. J.; Wang, W.; Wallace, A.; Yan, B.; Reddy, P.; Treuheit, M. J.; Balland, A. Asparagine-linked oligosaccharides present on a non-consensus amino acid sequence in the CH1 domain of human antibodies. J. Biol. Chem. 2009, 284 (47), 32493−32506. (3) Valliere-Douglass, J. F.; Eakin, C. M.; Wallace, A.; Ketchem, R. R.; Wang, W.; Treuheit, M. J.; Balland, A. Glutamine-linked and nonconsensus asparagine-linked oligosaccharides present in human recombinant antibodies define novel protein glycosylation motifs. J. Biol. Chem. 2010, 285 (21), 16012−16022. (4) Blake, C. C. F.; König, D. F.; Mair, G. A.; North, A. C. T.; Phillips, D. C.; Sarma, V. R. Structure of hen egg-white lysozyme: A three-dimensional fourier synthesis at 2 Å resolution. Nature 1965, 206, 757−776. (5) Vocadlo, D. J.; Davies, G. J.; Raine, R.; Withers, S. G. Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature 2001, 412, 835−838. (6) Grinnell, B. W.; Walls, J. D.; Gerlitz, B. Glycosylation of human protein C affects its secretion, processing, functional activities, and activation by thrombin. J. Biol. Chem. 1991, 266 (15), 9778−9785. (7) Stenflo, J.; Fernlund, P. Amino acid sequence of the heavy chain of bovine protein C. J. Biol. Chem. 1982, 257, 12180−12190. (8) Titani, K.; Kumar, S.; Takio, K.; Ericsson, L. H.; Wade, R. D.; Ashida, K.; Walsh, K. A.; Chopek, M. W.; Sadler, J.; Fujikawa, K. Amino acid sequence of human von Willebrand factor. Biochemistry 1986, 25 (11), 3171−3184. (9) Chandler, K. B.; Brnakova, Z.; Sanda, M.; Wang, S.; Stalnaker, S. H.; Bridger, R.; Zhao, P.; Wells, L.; Edwards, N. J.; Goldman, R. SiteSpecific Glycan Microheterogeneity of Inter-Alpha-Trypsin Inhibitor Heavy Chain H4. J. Proteome Res. 2014, 13 (7), 3314−3329. (10) Neue, K.; Mormann, M.; Peter-Katalinic, J.; Pohlentz, G. Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides. J. Proteome Res. 2011, 10 (5), 2248−2260. (11) Holle, A.; Haase, A.; Kayser, M.; Höhndorf, J. Optimizing UV laser focus profiles for improved MALDI performance. J. Mass Spectrom. 2006, 41, 705−716. (12) Suckau, D.; Resemann, A.; Schuerenberg, M.; Hufnagel, P.; Franzen, J.; Holle, A. A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics. Anal. Bioanal. Chem. 2003, 376 (7), 952−965. (13) Hufnagel, P.; Resemann, A.; Jabs, W.; Marx, K.; SchweigerHufnagel, U. Automated Detection and Identification of N- and OGlycopeptides. Proceedings of the 3rd Beilstein Glyco-Bioinformatics Symposium, Discovering the Subtleties of Sugars, 10−14 June 2013, Potsdam, Germany. www.proceedings.beilstein-symposia.org. (14) Ayoub, D.; Jabs, W.; Resemann, A.; Evers, W.; Evans, C.; Main, L.; Baessmann, C.; Wagner-Rousset, E.; Suckau, D.; Beck, A. Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques. mAbs 2013, 5 (5), 699−710. (15) Leymarie, N.; Griffin, P. J.; Jonscher, K.; Kolarich, D.; Orlando, R.; McComb, M.; Aquilan, J.; Alley, W. R.; Altmann, F.; Ball, L. E.; Basumallick, L.; Bazemore-Walker, C. R.; Behnken, H.; Blank, M. A.; Brown, K. J.; Bunz, S. C.; Cairo, C. W.; Cipollo, J. F.; Daneshfar, R.; Desaire, H.; Drake, R. R.; Go, E. P.; Goldman, R.; Gruber, C.; Halim, A.; Hathout, Y.; Hensbergen, P. J.; Horn, D. M.; Hurum, D.; Jabs, W.; Larson, G.; Ly, M.; Mann, B. F.; Marx, K.; Mechref, Y.; Meyer, B.; Möginger, U.; Neusüß, C.; Nilsson, J.; Novotny, M. V.; Nyalwidhe, J. O.; Packer, N. H.; Pompach, P.; Reiz, B.; Resemann, A.; Rohrer, J. S.; Ruthenbeck, A.; Sanda, M.; Schulz, J. M.; Schweiger-Hufnagel, U.;

ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figure S1: Detailed list of MS and MS/MS parameters used on the amaZon speed ETD iontrap instrument. Supplementary Figure S2: List of parameters used in the GlycoQuest data analysis workflow when applied to the LC− MALDI−TOF/TOF data obtained from the ZIC-HILIC enriched trypsin digest of HEL. Supplementary Figure S3: Annotated ETD-MS/MS spectrum obtained from most abundant HEL N-glycopeptide [34−45] FESNFNTQATNR. Supplementary Figure S4: Result obtained from 18O/PNGaseF assisted LC−MALDI−MS/MS analysis of ZIC-HILIC enriched HEL N-glycopeptides [34−45] FESNFNTQATNR. Supplementary Figure S5: Result obtained from LC−MALDI−MS/ MS analysis of ZIC-HILIC enriched HEL N-glycopeptides [34−45] FESNFNTQATNR after deglycosylation with endo F1−F3 and endo H. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b00175.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 421 2205 402. Fax: +49 421 2205 104. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 2640


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Journal of Proteome Research Sihlbom, C.; Song, E.; Staples, G. O.; Suckau, D.; Tang, H.; ThaysenAndersen, M.; Viner, R. I.; An, Y.; Valmu, L.; Wada, Y.; Watson, M.; Windwarder, M.; Whittal, R.; Wuhrer, M.; Zhu, Y.; Zou, C. Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multiinstitutional study 2012. Mol. Cell. Proteomics 2013, 12 (10), 2935− 2951. (16) Doubet, S.; Albersheim, P. CarbBank. Glycobiology 1992, 2 (6), 505. (17) Wuhrer, M.; Hokke, C. H.; Deelder, A. M. Glycopeptide analysis by matrix-assisted laser desorption/ionization tandem time-offlight mass spectrometry reveals novel features of horseradish peroxidase glycosylation. Rapid Commun. Mass Spectrom. 2004, 18, 1741−1748. (18) Stavenhagen, K.; Hinneburg, H.; Thaysen-Andersen, M.; Hartmann, L.; Varon Silva, D.; Fuchser, J.; Kaspar, S.; Rapp, E.; Seeberger, P. H.; Kolarich, D. Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides. J. Mass Spectrom. 2013, 48, 627−639. (19) Palmisano, G.; Melo-Braga, M. N.; Engholm-Keller, K.; Parker, B. L.; Larsen, M. R. Chemical deamidation: A common pitfall in largescale N-linked glycoproteomic mass spectrometry-based analyses. J. Proteome Res. 2012, 11 (3), 1949−1957. (20) Zielinska, D. F.; Gnad, F.; Wisniewski, J. R.; Mann, M. Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell 2010, 141, 897−907. (21) Harvey, D. J.; Wing, D. R.; Küster, B.; Wilson, I. B. H. Composition of N-linked carbohydrates from ovalbumin and copurified glycoproteins. J. Am. Soc. Mass Spectrom. 2000, 11, 564−571.

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