Article pubs.acs.org/JAFC

Comparison of Chicken and Pheasant Ovotransferrin N‑Glycoforms via Electrospray Ionization Mass Spectrometry and Liquid Chromatography Coupled with Mass Spectrometry Kuan Jiang, Chengjian Wang, Yujiao Sun, Yang Liu, Ying Zhang, Linjuan Huang,* and Zhongfu Wang* Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi’an 710069, PR China S Supporting Information *

ABSTRACT: Species-specific ovotransferrin features a highly conservative protein sequence, but it varies in the structure of the attached oligosaccharides, which may contribute to the differences observed in its bioactivity and nutritional value. Herein, chicken ovotransferrin (COT) and pheasant ovotransferrin (POT) isolated by repeated ethanol precipitation of egg white were digested with peptide N-glycosidase F to release N-glycans. The obtained N-glyans were isotopically labeled with aniline and analyzed via electrospray ionization mass spectrometry and online hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC−MS/MS). Relative quantitation based on isotopic aniline labeling and HILIC−MS/MS analysis revealed in detail the conspicuous difference between COT and POT in the abundance of their N-glycan compositions and isomers. In total, 16 COT N-glycans were observed, including 1 core structure (3.18%), 3 hybrid type (5.42%), and 12 complex type (91.40%), whereas 21 POT N-glycans were found, including 1 truncated structure (1.88%), 1 core structure (6.26%), 3 high mannose type (5.20%), 6 hybrid type (19.14%), and 10 complex type (67.52%). To our knowledge, this study is the first qualitative and quantitative comparison of COT and POT N-glycosylation patterns. These results suggest that POT has a different glycosylation pattern compared to that of COT and thus the effect of its glycosylation pattern on its bioactivity is worthy of further exploration. KEYWORDS: Chicken, pheasant, ovotransferrin, N-glycans, isotopic labeling, ESI−MS, HILIC−MS, MS/MS



in their attached N-glycan structures,14 which may affect their biofunctions and even contribute to the specific egg quality of various avian species. Previous studies have investigated the Nglycan structures of chicken ovotransferrin (COT),15−18 but little is known about the N-glycan repertoire of pheasant ovotransferrin (POT). Hence, the glycoform characterization of POT and a glycoform comparison between COT and POT are important to reveal the roles of glycan moieties in various biological functions as well as to understand the quality difference between chicken and pheasant eggs. N-Glycans attached to proteins are highly complex because of their numerous structural possibilities resulting from different monosaccharide compositions, sequences, and linkages. Depending on their monosaccharide composition and branching, N-glycans are mainly classified as high mannose, complex, and hybrid types. Unlike various precisely constructed nucleic acids and proteins, glycans have no templates during biosynthesis, resulting in highly complex branches and isomers. Therefore, glycan structure analysis is rather complicated and requires more powerful techniques. Methods to promote total N-glycan release using peptide N-glycosidase F (PNGase F) followed by characterization via modern techniques including high-performance liquid chromatography (HPLC) and mass

INTRODUCTION Ovotransferrin, an N-glycosylated monomeric protein, accounts for 12 to 13% of total egg white proteins.1 Ovotransferrin features the ability to bind iron ions and can be used as a nutritional ingredient in iron-fortified products such as iron supplements, protein supplements, and iron-fortified beverages. 2 Moreover, ovotransferrin has been reported to demonstrate a wide range of biofunctions including antibacterial, antifungal, antioxidative, and antiviral and thus provides a natural defense system for the avian egg.3−7 Because of its antibacterial property, ovotransferrin has already been used as a prophylactic in artificially fed babies to prevent enteritic infections.8 In addition, ovotransferrin has also been suggested for use as a functional food ingredient to enhance antioxidant capacity due to its antioxidative activity.9 In short, ovotransferrin is attractive for its application as an ingredient in functional foods to improve human or animal health. Qualitative and quantitative differences in egg white proteins have been observed between chicken and pheasant.10 Egg white proteins from pheasant have proven to have advantages in their nutritive value over that of chicken egg white proteins in some cases.10 However, as one of the main components of egg white proteins, ovotransferrin is still poorly understand in terms of its functional mechanisms, either in chicken or pheasant. Glycosylation is a significant post-translational modification. It has been proven that glycan moieties of many glycoproteins affect their biofunctions, such as antibacterial, antifungal, and antioxidative.11−13 Different types of ovotransferrin differ only © 2014 American Chemical Society

Received: Revised: Accepted: Published: 7245

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were precipitated with 43% ethanol, leaving iron-bound ovotransferrin in the supernatant. Crude ovotransferrin was obtained when the ethanol concentration was increased to 59% (v/v). To obtain highly purified products, the crude was dissolved in 43% ethanol and treated again by an increase of the ethanol concentration to 59% (v/v). Finally, the highly purified ovotransferrin products were lyophilized for further use. Identification of Ovotransferrin by Sodium Dodecyl Sulfate Polyacrylmide Gel Electrophoresis (SDS-PAGE). SDS-PAGE was performed according to the procedure developed by Laemmli,33 using 4% acrylamide in stacking gels and 12% acrylamide in separating gels. Protein samples were dissolved in loading buffer (1% SDS, 40% glycerol, 0.1% dithiothreitol, and 0.05% bromophenol blue in 10 mM Tris-HCl buffer, pH 8.0) at the concentration of 2 mg/mL and then heated at 100 °C for 10 min. For a maximum visualization of egg white proteins, the amount of protein loaded was 10 μg for all samples. SDSPAGE molecular weight standards ranging from of 3.4 to 250 kDa were used. Electrophoresis was carried out at 80 V in the stacking gel and 120 V in the separating gel using a Tris-Glycine electrophoretic buffer containing 0.1% SDS (pH 8.3). The gel was stained for 30 min with 1% Coomassie Blue R250 dissolved in a mixture containing 45% methanol, 45% water, and 10% acetic acid and destained with a mixture containing 87.5% water, 5% methanol, and 7.5% acetic acid. The gels were scanned using ImageQuant 350 (GE Healthcare, Milwaukee, WI, USA) and assessed by Gel-Pro Analyzer. MALDI-TOF-MS Analysis. MALDI-TOF-MS was performed on an AXIMA Performance instrument (Shimadzu, Tokyo, Japan). The MS data acquisition was carried out in positive ion linear mode, using a nitrogen laser with the power level set at 98 and a matrix of 10 mg/mL α-cyano-4-hydroxy-cinnamic acid (CHCA) prepared in 50% acetonitrile containing 0.1% TFA. Two shots accumulated for each profile were acquired for each sample spot. Two milligrams of purified ovotransferrin was dissolved in 100 μL of deionized water, and 1 μL of the sample solution was mixed with 1 μL of matrix solution prior to spotting on a stainless steel target plate and drying at room temperature for analysis. Release and Clean Up of N-Glycans from Ovotransferrin. Ovotransferrin (2 mg) was dissolved in 200 μL of protein denaturing solution (5% SDS, 0.4 M DTT) and denatured at 100 °C for 10 min. After the sample cooled, 20 μL of sodium phosphate buffer (0.5 M, pH 7.5), 20 μL of 10% NP-40, and 1 μL of PNGase F (500 units) were added prior to incubation at 37 °C for 24 h. The sample was boiled for 5 min to stop the reaction and then loaded onto a SepPak C18 SPE column. The released N-glycans were not retained on the column and eluted with 10 mL of water. The water fraction containing N-glycans was collected and desalted using a graphitized carbon SPE column. The salts were removed by washing with water, and the target Nglycans were eluted with 25% acetonitrile containing 0.01% TFA. The eluates were dried under a stream of nitrogen gas for further use. Derivatization of N-Glycans with d0-Aniline and d5-Aniline. This procedure was based on a previously reported method.31 Fifty microliters of d0- or d5-aniline, 50 μL of a 1 M NaCNBH3 solution prepared in 30% acetic acid (v/v), and 50 μL of free N-glycan solution were mixed and sealed in a screw-capped tube, followed by incubation at 70 °C for 20 min. The resulting sample was purified using a graphitized carbon SPE column. After washing the column with 10−15 mL of distilled water, the target N-glycan derivatives were eluted with 30% acetonitrile, dried under a stream of nitrogen, and dissolved in 80% acetonitrile for chromatographic and MS analysis. ESI−MS Conditions. MS analysis was carried out using an LTQ XL linear ion trap electrospray ionization mass spectrometer coupled with a HPLC system (Thermo Scientific, USA). The samples were injected via a Rheodyne loop with a volume of 2 μL and subsequently brought into the electrospray ion source by a stream of 50% methanol (v/v) at a flow rate of 200 μL/min. The spray voltage was set at 4 kV, with a sheath gas (nitrogen gas) flow rate of 30 arb., an auxiliary gas (nitrogen gas) flow rate of 5.0 arb., a capillary voltage of 37 V, a tube lens voltage of 250 V, and a capillary temperature of 375 °C. For MS/ MS analysis, N-glycans were subjected to fragmentation by collision induced decomposition (CID), with helium (He) as the collision gas.

spectrometry (MS) have been established, facilitating our understanding of glycoconjugate structures.19 Electrospray ionization mass spectrometry (ESI-MS) is an ideal tool for structural characterization of various oligosaccharides, especially when coupled with ion trap instruments.20 Ion trap mass spectrometry (IT-MSn) allows selective fragmentation of target pseudoglycan ions and detailed elucidation of their sequence and branching and linkage types, but it is impractical for quantitative analysis of isomeric glycans. LC−MS and MS/MS, however, are available for glycan isomer separation and online identification. Hence, the LC− MS technique has been extensively employed as one of the most universal and successful methods for qualitative and quantitative glycan qualitation to date, although its resolving ability is not as powerful as that of MSn in the analysis of complicated glycan isomer linkages.21,22 Many researchers prefer online LC−ESI-MS, as it provides the possibility of peak integration, precise determination of retention times, and the principal ability to detect all types of glycans.23−26 Stable isotope reagents can be employed to assess rapidly and directly the qualitative and quantitative changes in different samples and to eliminate the variation in instrument response and ionization efficiency of the molecules with the same structures but from different resources. Accordingly, in comparative glycomic analysis, the stable isotopic labeling of glycans via reductive amination27−29 or hydrazone formation30 prior to HPLC separation and MS detection is usually involved. A glycan isotopic labeling approach using nondeuterated (d0-) and deuterated (d5-) aniline has been developed in our lab. This method is quite suitable for comparative glycomic analysis based on ultraviolet (UV) absorbance and relative ion abundance.31 In this article, ovotransferrin was purified from egg whites of chicken (domestic species) and pheasant (wild species), followed by composition profiling of their N-glycans released by PNGase F. Aniline stable isotopic labeling combined with ESI−MS analysis were utilized to compare quantitatively the Nglycan compositions of COT with those of POT. Online HILIC−MS and MS/MS were employed to identify N-glycan isomers and to quantify their relative abundance in COT and POT. This study is the first to reveal, comprehensively and in detail, the N-glycan patterns of COT and POT and to give a qualitative and quantitative comparison of COT and POT Nglycans.



MATERIALS AND METHODS

Materials. Chicken (Gallus domesticus) and pheasant (Phasianus colchicus) eggs were purchased from Luozhuang Farm (Xi’an, China). Molecular weight protein markers were purchased from Fermentas (Burlington, Canada). PNGase F was obtained from New England BioLabs (Ipswich, MA, USA). Nonporous graphitized carbon (Carbograph) solid-phase extraction (SPE) columns (150 mg/4 mL) were purchased from Alltech Associates (Deerfield, IL, USA). SepPak C18 SPE columns (100 mg/1 mL) were purchased from Waters (Milford, MA, USA). HPLC-grade methanol and acetonitrile were from Fisher Scientific (Fairlawn, NJ, USA). Other reagents used were of analytical grade. Preparation of Ovotransferrin. On the basis of the work of Ko and Ahn,32 the method of Fe3+ saturation and two-time ethanol precipitation was utilized to purify ovotransferrin from chicken and pheasant egg whites. Briefly, egg white was diluted with a 1-fold volume of distilled water, and then 2.5 mL of a 20 mM FeCl3·6H2O solution was added per 100 mL of diluted egg white solution to saturate the Fe3+-bound sites. The obtained mixture was left standing for 1 h after homogenization. The majority of the egg white proteins 7246

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Collision parameters were left at default values with a normalized collision energy degree of 60 and an isotope width of m/z 3.00. Activation Q was set at 0.25, and activation time, at 30 ms. The MS and MS/MS data were recorded using LTQ Tune software (Thermo). Online LC−MS Analysis. Online LC−MS analysis was performed using a Hypersil APS2 (NH2) column (2.1 mm × 150 mm, 3 μm) (Thermo scientific, USA) on an HPLC system (Thermo) equipped with a photo diode array (PDA) detector and an LTQ XL linear ion trap ESI-MS system. The sample injection volume was 10 μL, and the mobile phase flow rate was 200 μL/min. The elution gradient was as follows: solvent A, acetonitrile; solvent B, water; solvent C, 10 mM ammonium acetate (pH 4.5); time = 0 min (t = 0), 80% A, 16% B, 4% C; t = 45, 27.5% A, 34% B, 38.5% C. The eluates from the chromatographic separation were directly introduced into the ESI-MS system for detection. The MS and MS/MS data were recorded using Xcalibur software (Thermo). The parameters used for LC−MS/MS analysis were the same as those described for ESI−MS/MS analysis in the above section. The obtained date were manually interpreted, and the proposed N-glycan compositions and sequences were checked using the software GlycoWorkbench.34

more stable to chemicals such as ethanol than its iron-free form, the iron-free ovotransferrin in an egg white solution was converted to the iron-bound form by treatment with FeCl3 solution. Almost all egg white proteins except for iron-bound ovotransferrin were denatured and precipitated in 43% ethanol. Iron-saturated ovotransferrin in the 43% ethanol solution started to precipitate as the ethanol concentration increased from 43%, and the optimal conditions for precipitating ovotransferrin occurred when the ethanol concentration reached 59%.30 Therefore, using 43% ethanol to remove other egg white proteins and 59% ethanol to precipitate ovotransferrin, we obtained COT (lane 2 of Figure 1A) and POT (lane 6 of Figure 1A) with minor impurities. To obtain ovotransferrin that is pure enough for glycosylation analysis, the ethanol (increasing ethanol concentration from 43 to 59%) precipitation process was performed again using the crude COT and POT. As a result, a high purity of COT (lane 3 of Figure 1A) and POT (lane 7 of Figure 1A) was achieved. MALDI-TOF MS (Figure 1B) gave ion signals of COT at m/z 39 016 ([M + 2H]2+) and m/z 77 285 ([M + H]+) (blue mass spectrum in Figure 1B) and POT at m/z 38 743 ([M + 2H]2+) and m/z 76 775 ([M + H]+) (red mass spectrum in Figure 1B), indicating that the molecular weights of COT and POT are around 77 284 and 78 774 Da, respectively. These results are consistent with those of SDS-PAGE analysis as well as previous literature reports.37−39 Moreover, the results from the Gel-Pro Analyzer concerning the SDS-PAGE experiment suggested that the purities of COT and POT were both over 95%. Therefore, this approach proved to be an efficient and simple method for the purification of ovotransferrin. Considering that the variation between speciesspecific ovotransferrin proteins is related only to the attached glycan moieties,14 the difference in molecular weights between COT and POT may validate this fact to a certain extent as well as facilitate an understanding of the following comparative glycomic analysis of COT and POT. Composition Profiling of COT and POT N-Glycans. The N-glycan structures of COT have been investigated to a certain degree in previous reports.15−18 However, there is no Nglycomic information on POT up to now. In this work, Nglycan compositions of COT and POT were analyzed by ESI− MS, where sodiated ions were predominantly produced. Figure 2 presents the MS profiles of the N-glycan compositions of COT (Figure 2A) and POT (Figure 2B). The double-sodiated COT glycan ions ([M + 2Na] 2+ ) at m/z 782.73 (Man 3 GlcNAc 5 ), 884.27 (Man 3 GlcNAc 6 ), and 1087.27 (Man3GlcNAc7) and POT ion of m/z 782.73 (Man3GlcNAc5) were not annotated here because the corresponding singly charged ions ([M + Na]+) of these glycans were all observed. In total, 12 COT N-glycan compositions were observed as the [M + Na]+ type ions at m/z 933.27 (Man3GlcNAc2), 1136.27 (Man3GlcNAc 3), 1298.27 (Hex 1Man3GlcNAc3), 1339.27 (Man3GlcNAc 4), 1501.18 (Hex 1Man3GlcNAc4), 1542.27 (Man3GlcNAc 5), 1663.09 (Hex 2Man3GlcNAc4), 1704.27 (Hex 1Man3GlcNAc5), 1745.27 (Man3GlcNAc6), 1907.27 (Hex1Man3GlcNAc6), 1948.09 (Man3GlcNAc7), and 2151.18 (Man3GlcNAc8). In contrast, 15 N-glycan compositions were found as [M + Na]+ type ions at m/z 771.27 (Man2GlcNAc2), 933.27 (Man3GlcNAc2), 1095.27 (Man4GlcNAc2), 1136.27 (Man 3 GlcNAc 3 ), 1257.27 (Man 5 GlcNAc 2 ), 1298.27 (Hex 1Man3GlcNAc3), 1339.27 (Man3GlcNAc4), 1460.09 (Man5GlcNAc 3), 1501.18 (Hex 1Man3GlcNAc4), 1542.27 (Man3GlcNAc 5), 1663.09 (Hex 2Man3GlcNAc4), 1704.27



RESULTS AND DISCUSSION Purification and Identification of Ovotransferrin. Highly purified glycoprotein is essential for glycomic analysis.

Figure 1. Profiling ovotransferrin (OTf) by SDS-PAGE and MALDITOF MS. (A) SDS-PAGE pattern. Lane 1: standard marker; lane 2: COT obtained after the first ethanol precipitation process; lane 3: COT obtained after the second ethanol precipitation process; lane 4: total protein of chicken egg white; lane 5: blank; lane 6: POT obtained after the first ethanol precipitation process; lane 7: POT prepared with a second ethanol precipitation; lane 8: total protein of pheasant egg white. (B) MALDI-TOF MS profiles of highly purified COT (blue mass spectrum) and POT (red mass spectrum).

However, some commercially available samples of egg white proteins such as ovotransferrin and ovalbumin are usually copurified with some other proteins, resulting in difficulties for precisely analyzing their glycosylation.10,35,36 Thus, to compare the N-glycan patterns of COT and POT, a method for the efficient isolation of highly purified ovotransferrin from chicken and pheasant egg whites is needed. The ethanol method for ovotransferrin purification is simple in its operation and produces a high yield. Hence, in this article, we developed a method termed ethanol repeated precipitation to obtain highly purified ovotransferrin. Because iron-saturated ovotransferrin is 7247

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Figure 2. ESI−MS profiles of the N-glycans released from (A) COT and (B) POT. Sodiated ions are predominately produced. Putative glycan compositions are presented on the basis of the MS/MS analysis. Structural formulas: blue square, N-acetylglucosamine; green circle, mannose; gray circle, hexose.

Figure 3. Quantitative comparison of COT and POT N-glycan compositions based on stable isotopic labeling using d0- and d5-aniline. (A) ESI−MS profile of an equimolar mixture of d0-aniline derivatives of COT N-glycans (d0-An-COT N-glycans) and d5-aniline derivatives of POT N-glycans (d5An-POT N-glycans). (B) ESI−MS profile of an equimolar mixture of d0-aniline derivatives of POT N-glycans (d0-An-POT N-glycans) and d5-aniline derivatives of COT N-glycans (d5-An-COT N-glycans). The blue and red peaks are the signals of d0-aniline and d5-aniline derivatives, respectively. The intensity ratio between the d0-aniline derivative of each glycan and its corresponding d5-aniline derivative is shown as R, which consists of the mean value and standard deviation obtained from three repeated experiments. Sodiated ions are predominately produced. Structural formulas: blue square, N-acetylglucosamine; green circle, mannose; gray circle, hexose.

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Figure 4. Qualitative and quantitative comparison of the COT and POT N-glycan isomers derivatized with d0- or d5-aniline via online HILIC−MS. (A) Total ion chromatograms (TICs) showing the elution profiles of (i) the equimolar mixture of d0-An-COT N-glycans and d5-An-POT N-glycans and (ii) the equimolar mixture of d0-An-POT N-glycans and d5-An-COT N-glycans. (B) Online MS spectra of an equimolar mixture of d0-An-COT N-glycans and d5-An-POT N-glycans. The blue and red ion peaks are the signals of d0- and d5-aniline derivatives, respectively. The intensity ratio of the d0-aniline derivative of each glycan to its corresponding d5-aniline derivative is shown as R, which consists of the mean value and standard deviation obtained from three repeated experiments. Protonated ions are predominately produced. Structural formulas: blue square, Nacetylglucosamine; green circle, mannose; yellow circle, galactose.

composition profile of ovotransferrin derived from wild bird species. Quantitative Comparison of N-Glycan Compositions between COT and POT. For quantitative comparison of the N-glycan compositions between COT and POT, two aliquots of N-glycans released from COT or POT were separately derivatized with d0- and d5-aniline. The equimolar mixture of d0-aniline-derivatized N-glycans of COT (d0-An-glycans of COT) and d5-aniline-derivatized N-glycans of POT (d5-Anglycans of POT), as well as the equimolar mixture of d0-anilinederivatized N-glycans of POT (d0-An-glycans of POT) and d5aniline-derivatized N-glycans of COT (d5-An-glycans of COT), were analyzed by ESI−MS, and the compositions of these glycan derivatives were identified by MS/MS (Supporting Information, Figures S1 and S2). As shown in Figure 3A, the equal-ratio mixture of d0-Anglycans of COT and d5-An-glycans of POT exhibits 12 peaks of d0-An-glycans of COT and 15 peaks of d5-An-glycans of POT

(Hex1Man3GlcNAc 5), 1745.27 (Man3GlcNAc 6), 1866.27 (Hex2Man3GlcNAc5), and 1907.27 (Hex1Man3GlcNAc6). Obviously, 10 of these N-glycans, at m/z 993.27, 1136.37, 1298.27, 1339.27, 1501.27, 1542.27, 1663.27, 1704.27, 1745.27, and 1907.27, exist in both COT and POT. All of these N-glycan compositions were identified via MS/MS (Supporting Information, Figures S1 and S2) and are supported by the literature.18 These results revealed that all of the N-glycans of COT and POT are neutral oligosaccharides and that no acidic oligosaccharide or fucosylated oligosaccharide was found in either sample. The N-glycan of Hex2Man3GlcNAc4 corresponding to m/z 1663.27 was newly found in COT; however, the COT N-glycans including Man2GlcNAc2, Man1GlcNAc3, and Hex2Man3GlcNAc5 reported in previous work18 were not detected in our study, which may originate from other chicken egg white glycoproteins mixed in commercially obtained COT. Additionally, we are the first to demonstrate the N-glycan 7249

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Figure 5. Extracted ion chromatograms (EICs) of d0-aniline derivatives of COT N-glycans (d0-An-COT N-glycans) and d5-aniline derivatives of POT N-glycans (d5-An-POT N-glycans). The blue and red chromatograms represent the EICs of d0- and d5-aniline derivatives, respectively. The EICs that share the same glycan compositions were covered with gray shadow. The paired blue (d0-An-COT N-glycan) and red (d5-An-POT Nglycan) EIC peaks that share the same glycan sequences were simultaneously covered with green or blue shadow. Structural formulas: blue square, N-acetylglucosamine; green circle, mannose; yellow circle, galactose.

Additionally, although POT possesses more N-glycan compositions than COT (15 vs 12), as Figure 3 demonstrates, the average size of oligosaccharides from COT is larger than the oligosaccharides from POT, consistent with the MALDI-TOFMS analysis result (Figure 1) that demonstrated that the molecular weight of COT (77285 Da) is higher than that of POT (76774 Da). Qualitative and Quantitative Comparison of COT and POT N-Glycan Isomers. Isomers exist extensively within the glycans released from glycoprotein. ESI−MS analysis of a mixture of glycans provides a rapid composition profile but gives little information regarding the isomers. HILIC affords a suitable means for the separation of aniline-labeled N-glycan isomers, and online MS/MS allows extensive fragmentation of separated glycan isomers, which provides rich information for differentiation and sequencing of diverse glycan isomers.29 For the quantitative comparison of N-glycan isomers between COT and POT, an equimolar mixture of d0-An-glycans of COT and d5-An-glycans of POT and an equimolar mixture of d0-Anglycans of POT and d5-An-glycans of COT were separately analyzed by online HILIC−MS and online HILIC−MS/MS (Supporting Information, Figure S2). The total ion chromatograms (TICs) (Figure 4A) of the two equal-ratio sample mixtures show parallel elution profiles, which exhibit of the derivatives of specific N-glycans. The equimolar mixture of d0-An-glycans of COT and d5-An-glycans of POT was selected as the example for quantitative comparison analysis. Figure 4B shows the online MS spectra corresponding to the major peaks of the TIC of the equimolar mixture of d0-An-COT N-glycans and d5-An-POT N-glycans. The isomeric sequences of the N-glycan derivatives presented in the spectra were proposed on the basis of the data in the extracted ion chromatogram (EIC) (Figure 5) and HILIC−

in the ESI−MS profile, giving results that are consistent with those of the N-glycan composition profiling. All of those signals are assigned as [M + Na]+ type ions, and their corresponding compositions are all displayed in the figure. In total, 10 pairs of peaks (m/z 1010.36/1015.36, 1213.36/1218.36, 1375.27/ 1380.27, 1416.18/1421.18, 1578.36/1583.36, 1619.27/ 1624.27, 1740.27/1745.27, 1781.27/1786.27, 1822.27/ 1827.27, and 1985.27/1990.27) and seven single peaks (m/z 853.45, 1177.36, 1339.36, 1543.36, and 1948.27 of d5-aniline derivatives and m/z 2026.36 and 2229.27 of d0-aniline derivatives) were clearly observed. The peak pairs indicate the coexistence of the corresponding N-glycans in both COT and POT, and the MS signal intensity ratio of d0- to d5-aniline derivatives of each glycan represents its abundance ratio, namely, the molar ratio between COT and POT. Meanwhile, the single peaks of the d0- or d5-aniline derivative indicates the existence of the corresponding N-glycans only in COT or POT, respectively. Clearly, 10 N-glycans exist in both COT and POT, of which Man3GlcNAc2, Man3GlcNAc3, Hex1Man3GlcNAc3, Man3GlcNAc4, Hex1Man3GlcNAc4, Hex2Man3GlcNAc4, and Hex1Man3GlcNAc5 are less abundant (by 0.51, 0.13, 0.10, 0.49, 0.15, 0.50, and 0.46 times, respectively) in COT than in POT, whereas Man 3 GlcNAc 5 , Man 3 GlcNAc 6 , and Hex1Man3GlcNAc6 are more abundant (by 2.19, 19.69, and 2.87 times, respectively) in COT than in POT. Figure 3B shows the MS profile of the equal-ratio mixture of d0-Anglycans of POT and d5-An-glycans of COT. The results are well-consistent with the results of Figure 3A, confirming the correctness and excellent reproducibility of these results. Therefore, we conclude that the N-glycans of POT and COT exhibit conspicuous differences in both composition and abundance, although the two glycoproteins are encoded by identical genes and have the same amino acid sequence. 7250

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Table 1. Comprehensive List of COT and POT N-Glycans Detailing Monosaccharide Compositions, Proposed Structures, Glycan Types, Relative Quantity (%), and Abundance Ratios Found in Both COT and POT N-Glycan Compositions and Isomers

a

H, hexose; N, N-acetylhexosamine. bStructural formulas: blue square, N-acetylglucosamine; green circle, mannose; yellow circle, galactose. SD, standard deviation. 7251

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1358.09 is an exception, as it shares the same composition but different glycan sequences. The molar ratio between the two peaks is 0.49, consistent with the ratio shown in Figure 3. The singly occurring N-glycan derivatives from COT or POT were also annotated in the spectra. Some of these N-glycans are the isomers of paired N-glycans, whereas the others that exist in either COT or POT have no corresponding isomeric structures in POT or COT. The results of HILIC−MS analysis are highly consistent with the data presented in Figure 3. On the basis of ESI−MS and online HILIC−MS analysis, comprehensive analytical information on COT and POT Nglycans is summarized in Table 1, including monosaccharide compositions, proposed structures, glycan types, relative quantity (%) and abundance ratios of N-glycan compositions, and isomers found in both COT and POT. In total, 16 Nglycans of COT and 21 N-glycans of POT were found when glycan isomers were taken into account. Ten of these glycan structures exist in both COT and POT. Additionally, there is no acidic or fucosylated glycans observed in either COT or POT. The COT N-glycans with compositions of Man 3 GlcNAc 4 , Man 3 GlcNAc 5 , Gal 1 Man 3 GlcNAc 5 , and Man3GlcNAc5 and POT N-glycans with compositions of Man 4 GlcNAc 2 , Man 3 GlcNAc 5 , Hex 2 Man 3 GlcNAc 4 , and Gal1Man3GlcNAc5 each have two isomers, and the COT Nglycan composition of Hex1Man3GlcNAc4 has three isomers. The other N-glycan compositions have only single structures. As summarized in Table 2 and Figure 6, for COT N-glycans, 1 core structure (3.18%), 12 complex type (91.4%), 3 hybrid type (5.42%), and no high mannose type glycan were observed. In contrast, for POT N-glycan types, 1 core structure (6.26%), 3 high mannose type (5.20%), 10 complex type (67.52%), 6 hybrid type (19.14%), and 1 truncated structure (1.88%) (Man2GlcNAc2) were observed. COT contains five types of antennary complex-type structures described as follows: monoantennary (8.4%), biantennary (32.37%), triantennary (44.47%), tetra-antennary (3.56%), and penta-antennary (1.10%). However, POT contains four types of antennary structure including monoantennary (12.24%), biantennary (45.82%), triantennary (7.74%), and tetra-antennary (1.19%); no penta-antennary type structure was observed.

Table 2. Comparison of General Features in Glycosylation between COT and POT comparison features

COT

POT

total N-glycans truncated core high mannose hybrid complex

16 0 1 0 3 12

21 1 1 3 6 10

1 2 4 2 1

1 5 3 1 0

antennary monobitritetrapenta-

MS/MS (Suppporting Information, Figure S3). Protonated ions are predominately produced, and very few sodiated and potassiated ions are observed in the mass spectra. Twelve pairs of peaks (m/z 988.18/993.18, 1191.09/1196.09, 1353.09/ 1358.09, 1394.09/1399.09, 1556.09/1561.09, 1597.09/ 1602.09, 1597.09/1602.09, 1718.09/1723.09, 1759.09/ 1764.09, 1800.09/1805.09, 1759.09/1764.09, and 1962.09/ 1967.09) were clearly observed. The abundance ratios of the paired d0-An-COT derivatives and d5-An-POT derivatives at m/ z 988.18/993.18, 1191.09/1196.09, 1353.09/1358.09, and 1962.09/1967.09 (by 0.51, 0.13, 0.10, and 2.87 times, respectively) are consistent with the results of the glycan composition comparison, and EICs (Figure 5) indicate that there is no isomer for these glycan compositions. Meanwhile, due to the existence of glycan isomers, several pairs of peaks give new molar ratios when compared with those obtained through the above-described N-glycan composition comparison analysis. Here, seven pairs of d0-aniline derivatives and d5aniline derivatives at m/z 1556.09/1561.09, 1597.09/1602.09, 1597.09/1602.09, 1718.09/1723.09, 1759.09/1764.09, 1800.09/1805.09, and 1759.09/1764.09 give corresponding molar ratios of 0.86, 2.22, 0.28, 1.54, 0.56, 1.14, and 0.24, respectively, and each pair of peaks shares the same N-glycan sequence. Nevertheless, the pair of peaks at m/z 1353.09/

Figure 6. Charts showing the relative quantities of all N-glycan types in COT (A) and POT (B) as well as branched patterns of complex type Nglycans from COT and POT (C). 7252

dx.doi.org/10.1021/jf501352j | J. Agric. Food Chem. 2014, 62, 7245−7254

Journal of Agricultural and Food Chemistry

Article

This study, to our knowledge, is the first work that comparatively details COT and POT N-glycans, especially using qualitative and quantitative analysis of their N-glycan isomers through the combination of glycan isotopic aniline labeling, ESI−MS, HILIC−MS, and MS/MS. As the only variable component, attached N-glycans contribute to the primary structural differences between COT and POT and may influence the bioactivity of the two glycoproteins. This study provides a foundation for the further elucidation of the specific biofunctions of COT and POT, thereby stimulating, perhaps, their application in the food industry as a novel nutraceutical and functional food ingredient in the future.



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ASSOCIATED CONTENT

* Supporting Information S

Figures S1 and S2: ESI-MS/MS spectra for the ions of all free N-glycan compositions, d0-aniline derivatized N-glycan compositions, and d5-aniline derivatized N-glycan compositions from COT and POT. Figure S3: Online MS/MS spectra for all d0and d5-aniline derivatives of the glycans from COT or POT. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Authors

*(L.H.) E-mail: [email protected]. *(Z.W.) E-mail: [email protected]; Tel.: +86-02988305853; Fax: +86-029-88303534. Funding

This work was funded by the National Natural Science Foundation of China (nos. 31170773, 21375103, and 31370804). Notes

The authors declare no competing financial interest.



ABBREVIATIONS USED COT, chicken ovotransferrin; POT, pheasant ovotransferrin; ESI-MS, electrospray ionization-mass spectrometry; PNGase F, peptide N-glycosidase F; UV, ultraviolet; HILIC, hydrophilic interaction liquid chromatography; MS/MS, tandem mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ ionization-time-of-flight; DTT, dithiothreitol; SPE, solid-phase extraction; CID, collision induced decomposition; Hex, hexose; GlcNAc, N-acetylglucosamine; Man, mannose; Gal, galactose; Glc, glucose; An, aniline; TIC, total ion chromatogram; EIC, extracted ion chromatograms



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dx.doi.org/10.1021/jf501352j | J. Agric. Food Chem. 2014, 62, 7245−7254

Comparison of chicken and pheasant ovotransferrin N-glycoforms via electrospray ionization mass spectrometry and liquid chromatography coupled with mass spectrometry.

Species-specific ovotransferrin features a highly conservative protein sequence, but it varies in the structure of the attached oligosaccharides, whic...
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