Analysis of Folate Binding Protein N-linked Glycans by Mass

Article pubs.acs.org/jpr

Analysis of Folate Binding Protein N-linked Glycans by Mass Spectrometry Nidhi Jaiswal,† Suraj Saraswat,† Manohar Ratnam,‡ and Dragan Isailovic*,† †

Department of Chemistry, University of Toledo, Toledo, Ohio 43606, United States Department of Biochemistry and Cancer Biology, Medical University of Ohio, Toledo, Ohio 43614, United States

‡

S Supporting Information *

ABSTRACT: The folate binding protein (FBP), also known as the folate receptor (FR), is a glycoprotein which binds the vitamin folic acid and its analogues. FBP contains multiple N-glycosilation sites, is selectively expressed in tissues and body fluids, and mediates targeted therapies in cancer and inflammatory diseases. Much remains to be understood about the structure, composition, and the tissue specificities of N-glycans bound to FBP. Here, we performed structural characterization of N-linked glycans originating from bovine and human milk FBPs. The N-linked glycans were enzymatically released from FBPs, purified, and permethylated. Native and permethylated glycans were further analyzed by matrix-assisted laser desorption/ ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS), while tandem MS (MS/MS) was used for their structural characterization. The assignment of putative glycan structures from MS and MS/MS data was achieved using Functional Glycomics glycan database and SimGlycan software, respectively. It was found that FBP from human milk contains putative structures that have composition consistent with high-mannose (Hex5−6HexNAc2) as well as hybrid and complex N-linked glycans (NeuAc0−1Fuc0−3Hex3−6HexNAc3−5). The FBP from bovine milk contains putative structures corresponding to high-mannose (Hex4−9HexNAc2) as well as hybrid and complex N-linked glycans (Hex3−6HexNAc3−6), but these glycans mostly do not contain fucose and sialic acid. Glycomic characterization of FBP provides valuable insight into the structure of this pharmacologically important glycoprotein and may have utility in tissue-selective drug targeting and as a biomarker. KEYWORDS: folate binding protein, folate receptor, N-linked glycans, mass spectrometry, MALDI-MS, ESI−MS, MS/MS

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INTRODUCTION The folate binding protein (FBP) is a glycopolypeptide which binds with high affinity (Kd < 10−9 M) to the vitamin folic acid and its analogues (collectively termed folates).1 FBP is found in cells, tissues, and bodily fluids in both soluble and cell membrane-associated forms, which are structurally similar but have different functional characteristics.1 Soluble FBP acts as an efficient folate binder. The membrane-associated FBP is clustered on the cell surface and can transport folate into the cells through nonclathrin mediated endocytosis. The FBP is present as several isoforms (α, β, γ/γ′, and δ) in human, bovine, and murine tissues.2 Human FBP α and β isoforms are bound to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor but can be also shed from the cell surface in a soluble form into the medium. FBP γ is a constitutively secreted soluble protein and FBP γ′ corresponds to a truncated form of it. The α isoform of FBP is expressed on the luminal surface of several types of normal epithelial cells where it is inaccessible through the bloodstream. On the other hand, this protein is overexpressed in several cancerous tissues where it is accessible through the bloodstream, making it an intensively investigated target for tumor selective drug delivery and as a serum biomarker.3 The FBP α is also secreted by © 2011 American Chemical Society

ductal epithelial cells in mammary glands into milk, from which it may be isolated using folate affinity chromatography.1,4 FBP isolated from bovine milk has 222 amino acids,5 and the molecular weight of the deglycosylated protein is ∼26.5 kDa.6 FBP α has also been identified in several human cell lines such as HeLa, KB, L1210, and M109.7−10 Different FBP isoforms are encoded by separate genes, while heterogeneity within each isoform is due to tissue-specific variability in glycosylation. For example, FBP α and β isoforms contain three and two N-linked glycosylation sites, respectively.2 It was shown that core glycosylation is important for proper folding, stability, intracellular trafficking, sorting, and functional expression of the FBP.2,10,11 However, the effect of glycosylation heterogeneity on biological characteristics of the mature protein needs to be further understood. Although previous studies reported the presence of N-linked glycans bound to the FBP,2,10−12 their detailed structure and composition remain to be elucidated. In last two decades, mass spectrometry (MS) techniques such as matrix-assisted laser desorption ionization (MALDI)-MS and electrospray Received: June 21, 2011 Published: December 22, 2011 1551

dx.doi.org/10.1021/pr2006044 | J. Proteome Res. 2012, 11, 1551−1560

Journal of Proteome Research

Article

analysis, the FBP separated on gel was transferred to PVDF membrane using Bio-Rad mini gel apparatus. The PVDF membrane was incubated with nonfat milk for an hour, and further incubated with primary antibody (rabbit anti-FBP antibody) in a cold room overnight. After washing with TBST solution (mixture of Tris buffer saline and Tween 20), secondary antibody (Anti-Rabbit IgG) was used for detection of the FBP bands with a chemiluminescent reagent (Amersham Biosciences, Upssala, Sweden).

ionization (ESI)-MS have been implemented for the analysis of both native and derivatized glycans. The MS is commonly used for the analysis of O- and N-linked glycans that are enzymatically cleaved and purified from the glycoproteins. Purified N-glycans are often permethylated because the permethylation improves the stability of sialic acid, which is present in glycans originating from human glycoproteins.13 The MS is used to putatively assign glycan structures based on the measurements of their molecular weights, while tandem mass spectrometry (MS/MS) is a prerequisite for their structural characterization.13−17 Recently, there was much interest in the MS analysis of glycans and glycoproteins present in milk and other bodily fluids.6,12,18,19 Besides physiological importance of glycans found in milk, it was shown that aberrant glycoprotein expression and altered glycosylation of serum proteins are associated with human diseases such as cancer.20,21 Most of currently used biomarkers of cancer are glycoproteins and their glycosylation was studied by MS previously. For example, glycosylation studies of biomarkers of ovarian (CA125), pancreatic (MUC1), and breast cancer (epidermal growth factor receptor) were accomplished by mass spectrometry.22−24 MS has been also extensively used to analyze N- and O-linked glycans isolated from serum of cancer patients.19,25 Besides its folate binding function in milk, FBP is overexpressed in various types of cancer cells and tissues and can transfer folate-based drugs into a cancer cell.3,26,27 Therefore, FBP has been extensively explored as both a putative biomarker of cancer and target of folate-based anticancer drugs, and the knowledge of the glycomic structure of FBP originating from biological fluids and tissues is biomedically relevant. In this study, we performed structural characterization of N-linked glycans isolated from FBPs originating from bovine and human milk by mass spectrometry. N-linked glycans were enzymatically cleaved from FBPs, purified, and permethylated. Native and permethylated N-glycans were further analyzed by MALDI-MS and ESI−MS, while tandem mass spectrometry (MS/MS) was used in combination with glycan databases for their structural elucidation. To the best of our knowledge, this is the first report on the structures of N-glycans associated with folate binding proteins.

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Enzymatic Release and Purification of N-Glycans from FBP

The FBP (1 mg of bovine and 0.1 mg of human milk protein, respectively) was resuspended in 1 mL of 20 mM NH4HCO3, pH 8.5. The protein was then thermally denatured by incubation at 90 °C for 5 min. After the sample was cooled down to room temperature, 1 μL of the enzyme PNGase F was added. The digestion mixture was incubated at 37 °C for 24 h, and solid phase extraction (SPE) was performed to separate glycans from deglycosylated protein. The SPE of N-glycans was performed similarly to a previously reported procedure.13 Briefly, C18 Sep-Pak cartridge was equilibrated with ethanol and water. Sample was further applied multiple times through the SPE cartridge. Deglycosylated protein was retained on and eluted from the SPE cartridge with 1 mL of acetonitrile. The eluent containing N-glycans was loaded on an activated charcoal cartridge, and this cartridge was spun down at 2000 rpm for 2 min. Upon washing with several solvents, glycans were eluted with 1 mL of 50:50% (v/v) acetonitrile/water solution containing 0.1% of TFA. Both Nglycans and deglycosylated FBP were dried in a vacuum concentrator (Eppendorf, Hauppauge, NY) and stored at −20 °C until further use. Permethylation of N-glycans

Permethylation of native N-glycans was also performed using a previously published procedure.13 Briefly, dried N-glycan samples obtained after purification on activated charcoal were dissolved in a solution consisting of 100 μL of DMSO, 3 μL of H2O, and 84 μL of iodomethane. Dissolved N-glycans were reloaded multiple times into a microspin column filled with NaOH beads. Then, the column was washed with 50 μL of ACN and spun at 10 kRPM to pull down permethylated glycans. The permethylated N-glycans were further subjected to liquid phase extraction using chloroform and water. The glycan sample was mixed with these solvents and centrifuged at 2000 rpm for 2 min, and chloroform layer was washed 3 times with water. Afterward, organic phase containing permethylated N-glycans was dried in a vacuum concentrator and stored at −20 °C until further use.

EXPERIMENTAL PROCEDURES

Materials

FBP from bovine milk (purity: 20−40% by Warburg-Christian assay), NaOH beads, enzyme peptide-N-glycosidase F (PNGase F), 2,5-dihydroxybenzoic acid (DHB), trifluoroacetic acid (TFA), and formic acid were purchased from Sigma (St. Louis, MO). FBP from human milk (purity: nominally >95% by SDSPAGE) was purchased from SunnyLab (SCIPAC Ltd., Sittingbourne, England). HPLC grade water, chloroforom, methanol, and acetonitrile were purchased from Fisher Scientific (Waltham, MA). C18 Sep-Pak solid phase extraction (SPE) cartridges were purchased from Waters (Franklin, MA). Activated charcoal columns and empty spin columns were purchased from Harvard Apparatus (Holliston, MA). SDSPAGE and Western blotting reagents were purchased from BioRad (Hercules, CA).

MALDI-MS Analysis of N-linked Glycans

N-linked glycans were analyzed by MALDI-MS in the presence of 2,5-dihydroxybenzoic acid (DHB) as the matrix. DHB solution was prepared by suspending 10 mg of DHB in 1 mL of 1 mM sodium acetate to produce matrix with concentration of 10 mg/mL. The dried native and permethylated N-Glycan samples were dissolved in the mixture consisting of 50:50% (v/v) methanol/water. 0.5 μL of analyte and 0.5 μL of freshly prepared DHB matrix were mixed on a MALDI sample plate. The spots were dried in a vacuum desiccator. The MALDI TOF/TOF instrument UltrafleXtreme (Bruker Daltonics, Bremen, Germany) was utilized for the analysis of glycans. This instrument is equipped with a Smartbeam-II laser emitting at 355 nm with 1 kHz firing rate. MALDI-MS profiling of both

Electrophoretic and Western Blot Analyses of FBP

The SDS-PAGE of FBP was performed on 12% separating and 4% stacking PAGE gels. After SDS-PAGE, gels were stained with Coomassie blue to detect FBP. For Western blotting 1552



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Figure 1. MALDI-TOF mass spectrum of native N-linked glycans from bovine milk FBP. Glycan structures were assigned by comparison of measured m/z values with m/z values calculated based on putative composition of native glycans. H represents hexose (galactose or mannose), and N represents N-acetyl glucosamine.

ESI−MS of N-linked Glycans

native and permethylated glycans was performed in reflectron positive ion mode. MALDI-TOF/TOF-MS analysis for structural characterization of glycans was performed by using both laser-induced dissociation (LID) and collision-induced dissociation (CID) in combination with a LIFT device.28 The LIFT device provides velocity focusing of fragment ions enabling high mass resolution and sensitivity of LID and high-energy CID MS/MS experiments.

The permethylated N-glycans originating from bovine milk were dissolved in 50:50% (v/v) methanol/water mixture containing 1 mM sodium acetate. The dissolved sample was electrosprayed at the flow rate of 2.5 μL/min into a quadrupoletime-of-flight (Q-TOF) mass spectrometer (Q-TOF Micro, Waters), using nebulizer gas (nitrogen) flow rate of 500 L/h and desolvation temperature of 200 °C. Tandem mass spectrometry experiments were performed for characterization of glycan structures using collision energy in the range of 30−40 eV depending on the size of the selected glycan. Argon was used as the collision gas.

Assignment of Glycan Structures

Putative glycan structures were assigned initially by comparison of measured molecular weights of glycans to those found in Functional Glycomics Glycan Structure Database.29 This database was searched for bovine and human glycan structures. MALDI MS/MS spectra were analyzed using SimGlycan software (Premier Biosoft, Palo Alto, CA) to assign glycan fragments and putative structures.30 The experimental data were searched after setting precursor ion m/z for sodium adduct, precursor ion tolerance of 1 Da, spectrum peak tolerance of 0.5 Da, positive ion mode, and free reducing terminal. Glycans were analyzed as either native or permethylated, and their structures were searched against a database of theoretical MS/MS spectra corresponding to bovine or human glycans.

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RESULTS AND DISCUSSION

Electrophoretic and Western Blot Analyses of FBPs

SDS-PAGE and Western blotting were employed to confirm the purity of FBP samples originating from bovine and human milk. These analyses indicated heterogeneity of FBPs due to glycosylation and aggregation, with latter being especially noticed in the sample of human milk FBP (Figure S1, Supporting Information). Several bands corresponding to glycosylated FBP from bovine milk showed apparent molecular weight of ∼30 kDa as expected from the previously determined molecular weight of deglycosylated protein.6 The bands were detected by Western blotting using an anti-FBP antibody (Figure S1a) and by Coomassie staining (Figure S1b). Additional high molecular weight bands were detected in FBP from human milk. According to the manufacturer and previously published literature,1 high molecular weight bands are due to aggregates of human milk FBP.

MALDI-MS Analysis of Deglycosylated FBP from Bovine Milk

The study of deglycosylated protein was performed using Bruker’s Omniflex MALDI-MS instrument at Bowling Green State University. The dried protein sample was dissolved in 10 μL of TA solution (0.1% TFA in water: acetonitrile in the volume ratio of 1:2). The sinapinic acid (SA) matrix was prepared by suspending 20 mg of SA in 1 mL of TA to produce matrix with concentration of 20 mg/mL. One microliter of analyte and 1 μL of freshly prepared SA matrix were mixed in a microcentrifuge tube, and 1 μL of prepared mixture was spotted on a MALDI target plate. The samples were air-dried. MALDIMS spectrum of FBP was obtained using positive ionization in linear mode.

Analysis of N-linked Glycans from Bovine Milk FBP by MALDI-MS

Native and permethylated N-glycans released from bovine milk FBP were examined by MALDI-MS using DHB as the matrix. As expected, glycan structures were detected as singly charged ions. Most of glycan ions detected by MALDI-MS of native N-glycans (Figure 1 and Table 1), were also found upon MALDI-MS of permethylated N-glycans (Figure 2 and Table 2). 1553



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Table 1. Assignments of Native Glycans Found in FBP from Bovine Milka

Table 2. Assignments of Permethylated Glycans Found in FBP from Bovine Milka

a

H represents hexose (mannose open circles, galactose solid circles); N represents N-acetyl glycosamine (solid squares) and N-acetyl galactosamine (open squares).

a

S represents sialic acid (diamonds); F represents fucose (triangles); H represents hexose (mannose open circles, galactose solid circles); N represents N-acetyl glycosamine (solid squares) and N-acetyl galactosamine (open squares).

FBP from bovine milk contains high-mannose (e.g., H5N2, H6N2, H7N2, H9N2) and hybrid/complex (e.g., H4N4, H5N4, H6N4)

Figure 2. MALDI-TOF mass spectrum of permethylated N-linked glycans from bovine milk FBP. Glycan structures were assigned by comparison of measured m/z values with m/z values calculated based on putative composition of permethylated glycans. S represents sialic acid, F represents fucose, H represents hexose (galactose or mannose), and N represents N-acetyl glucosamine. 1554



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Figure 3. LID-MALDI-TOF/TOF mass spectrum of native N-linked glycan H5N4 from bovine milk FBP [(M + Na)+, m/z = 1663.6]. The structure and linkages of H5N4 shown in the figure were identified via Functional Glycomics and SimGlycan database searches.

was observed (B2α, m/z 508.468; B3, m/z 832.764; B4, m/z 1036.140). 0,4X2 fragment (m/z = 711.649) suggests the presence of α1−6 linkage for α antennae and 1,5X3β ion (m/z = 1124.611) suggests the presence of α1−3 linkage for the β antennae. D ion (B3/Y3α) (m/z = 670.634) results from the cleavage of 3-linked antenna and two reducing end GlcNAc residues32 and confirms the sequence of 6-linked antenna found in H5N2 structure shown in Figure S2. SimGlycan search of the MS/MS spectrum of the same ion yielded three structures with the highest ranked structure being indeed the structure of highmannose glycan H5N2 (Figure S3, Supporting Information), and the other two structures not corresponding to N-linked glycans. In the case of isomeric glycans, SimGlycan assignment of glycan structures is ambiguous. For example, SimGlycan analysis of MS/MS spectrum of native [H5N4 + Na]+ ion (m/z 1663.592) gave hits corresponding to four structures (Figure S4, Supporting Information). The MS/MS spectrum shows that this ion yields various daughter ions: the most intense B-ions (e.g., m/z 1442.578, 1239.332, 550.002, 387.834), less intense Y-ions (e.g., m/z 1501.463, 1298.359, 446.920, 243.851), C4 and C5 ions (m/z 1257.385 and 1460.549, respectively), and several other daughter ions resulting from cross-ring fragmentation (Figure 3). Considering the putative structure of H5N4 shown in Figure 3, the presence of α1−6 linkage for β antennae was suggested by detection of 0,4 A4 fragment ion (m/z = 609.033), while the presence of α1− 3 linkage for α antennae was suggested by 2,4X2 ion (m/z = 1077.276). 0,2X3α/B3β fragment ion (m/z = 428.960) indicates the presence of β1−2 linkage. Other cross-ring fragment ions were also observed in the MALDI spectrum as shown in the inset of Figure 3. One of the most intense fragment ions, which results from the cleavage of 3-linked antenna and two reducingend GlcNAc residues (i.e., B 4 /Y 3α , m/z = 712.057), corresponds to D ion. The presence of D ion confirms the

glycans. Only two structures were found to possibly contain fucose and sialic acid (F1H5N4 and S1H3N6, respectively). Putative structures of N-linked glycans shown in the tables were initially assigned by comparison of measured molecular weights of glycans to those of bovine glycans found in Functional Glycomics glycan database.29 Some of the structures found in this database are present as multiple positional or linkage isomers (e.g., H4N4, H7N2, and H5N4) and could not be distinguished by our MS measurements. Analysis of N-glycans from Bovine Milk FBP by MALDI MS/MS

To further investigate glycan structures, several ions observed by MALDI-MS were selected and subjected to fragmentation using LID and CID. These two modes of MS fragmentation yielded similar type of glycosidic bond and cross-ring fragment ions in our experiments. The nomenclature of Domon and Costello was used to assign fragment ions.16 Structural glycan rearrangements were not noticed since both native and permethylated glycans were analyzed as sodium adducts, which commonly do not undergo rearrangement reactions.31 MS/MS spectra were both manually inspected and searched using SimGlycan software.30 SimGlycan predicts the structure of a glycan by comparison of experimentally obtained MS/MS spectra with the theoretical fragmentation spectra of glycans in a reference database. Glycans are further ranked based on composition and branching pattern scores with the most probable structure(s) being ranked the highest. Hence, the tandem mass spectrometry in combination with SimGlycan predicted putative structures of bovine milk FBP glycans, and most of the structures found correspond to those shown in Tables 1 and 2. For example, the abundance of glycosidic bond and crossring fragments was detected in the LID MS/MS spectrum of native H5N2 glycan ion (m/z = 1257.442) (Figure S2, Supporting Information). The spectrum exhibits B and Y ions as well as cross-ring fragment ions. B series of fragment ions 1555



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Figure 4. LID-MALDI-TOF/TOF mass spectrum of permethylated N-linked glycan H5N4 from bovine milk FBP [(M + Na)+, m/z = 2070.3]. Fragmentaions were assigned using SimGlycan and H5N4 structure shown in Figure 3.

Figure 5. MALDI-TOF mass spectrum of native N-linked glycans from human milk FBP. Glycan structures were assigned by comparison of measured m/z values with m/z values calculated based on putative composition of native glycans. F represents fucose, H represents hexose (galactose or mannose), and N represents N-acetyl glucosamine.

SimGlycan analysis of MS/MS spectrum of permethylated [H5N4 + Na]+ ion (m/z = 2070.304) yields the same structures found upon analysis of native glycan ion (Figure S4, Supporting Information). Upon LID, H5N4 parent ion yields various daughter ions characteristic of this oligosaccharide: the most intense C-ions (m/z 1811.269, 1565.725, 708.971, 504.864, 259.846), less intense Y-ions (m/z 1852.317, 1606.709,

identity of the 6-linked antenna of native H5N4 and indicates glycan structure shown in Figure 3. However, other structures shown in Figure S4 cannot be excluded since glycan fragmentation patterns and SimGlycan matching scores are similar. Therefore, it is possible that H5N4 is present in bovine milk FBP as a mixture of isomers. 1556



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Table 3. Assignments of Native Glycans Found in FBP from Human Milka

Table 4. Assignments of Permethylated Glycans Found in FBP from Human Milka

a

F represents fucose (triangles); H represents hexose (mannose open circles, galactose solid circles); N represents N-acetyl glycosamine (solid squares) and N-acetyl galactosamine (open squares).

1402.657, 544.971, 299.847), B-ions (m/z 1793.219 and 485.866), and several other daughter ions resulting from crossring fragmentation (Figure 4). The presence of 0,4X2/C5 (m/z = 1084.023) ion indicates α1−6 linkage for the β antennae and 2,4 X2 ion (m/z = 1339.482) indicates α1−3 linkage for α antennae. Again, MS/MS data suggest the H5N4 structure shown in Figure 3, but other isomeric structures shown in Figure S4 cannot be excluded. Analysis of Permethylated FBP Glycans Originating from Bovine Milk using ESI−MS

a

S represents sialic acid (diamonds); F represents fucose (triangles); H represents hexose (mannose open circles, galactose solid circles); N represents N-acetyl glycosamine (solid squares) and N-acetyl galactosamine (open squares).

The profiling of FBP glycans from bovine milk was also performed using an ESI−Q-TOF-MS instrument confirming that FBP contains high-mannose, hybrid, and complex N-linked glycans (Figure S5, Supporting Information). Triply and doubly charged sodiated ions were assigned in the ESI mass spectrum of N-glycans, and it was found again that the majority of Nglycans from bovine milk FBP do not contain fucose and sialic acid. To assist sequence assignment, several N-glycan ions observed by ESI−MS were further selected and fragmented using CID. While MALDI-MS/MS LID and high-energy CID experiments yielded both glycosidic bond and cross-ring fragment ions, the low-energy CID experiments performed by ESI−MS/MS produced mostly fragments formed by cleavages of glycosidic bonds. For example, parent ion (H4N4 + 2Na)2+ (m/z = 944.450) was subjected to MS/MS producing a doubly charged (C52+) fragment ion at m/z 814.556 (H4N3 + 2Na)2+ and singly charged Y1+, C4+, Z2+, and B1α+ fragment ions at m/z 282.131 (N+Na)+, m/z 1361.647 (H4N2 + Na)+, m/z 527.242 (N2 + Na)+, and m/z 268.115 (H + Na)+ (Figure S6, Supporting Information). The presence of these fragment ions indicates the structure of parent H4N4 shown in Table 2. N-glycan ion (H5N4 + 2Na)2+ (m/z = 1046.986) produced doubly charged (C42+ and C52+) fragment ions at m/z 794.307 (H5N2 + 2Na)2+

and m/z 916.929 (H5N3 + 2Na)2+ and singly charged (C4+ and Z2+) fragment ions at m/z 1565.831 (H5N2 + Na)+ and m/z 527.253 (N2 + Na)+ (Figure S7, Supporting Information). These fragments correlate well with H5N4 structure shown in Figure 3. MALDI-MS Analysis of Deglycosylated FBP Originating from Bovine Milk

After deglycosylation by PNGase F and SPE purification, FBP was analyzed by MALDI-MS. Molecular weight of deglysosylated bovine milk FBP was found to be approximately 26.4 kDa as shown in Figure S8 (Supporting Information). Besides singly charged peak for deglycosylated protein, peaks that correspond to singly charged multimers of deglycosylated FBP (dimer and trimer) were also found by MALDI-MS. The MW of deglycosylated FBP is less than the MW of FBP found by SDSPAGE showing that glycans were successfully released from the protein. The measured MW of FBP also correlates well with the MW of deglycosylated protein, which was measured by ESI−MS previously.6 1557



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Figure 6. MALDI-TOF mass spectrum of permethylated N-linked glycans from human milk FBP. Glycan structures were assigned by comparison of measured m/z values with m/z values calculated based on putative composition of permethylated glycans. S represents sialic acid, F represents fucose, H represents hexose (galactose or mannose), and N represents N-acetyl glucosamine.

Analysis of Native and Permethylated N-linked Glycans from FBP Originating from Human Milk using MALDI-MS

711.272) corresponds to D ion. The glycosidic bond and crossring fragments indicate the sequence and structure of parent ion with m/z 1809.7 (F1H5N4) shown in the inset of Figure S9. The SimGlycan analysis, however, yielded four F 1H 5N4 structures that have either different structure or glycosidic bond linkages (Figure S10, Supporting Information). Similarly, permethylated F1H5N4 ion (m/z = 2244.231) was fragmented by LID and analyzed by SimGlycan to confirm the structure of this glycan. The same structures were obtained by SimGlycan as in the case of native glycan ion. Considering the structure shown in Figure S9 (Supporting Information), the Y-series of ions (m/z values of 2056.822, 1781.764, 718.874, and 474.375) was observed along with a C-ion (m/z = 260.219) and a B-ion (m/z = 486.343) (Figure S11, Supporting Information). The presence of Y1γ ion (2056.822) indicates that fucose is present at the terminal GlcNac residue. A number of cross-ring fragmentation ions were also observed that were important to confirm the linkages of terminal fucose and 3linked antennae. For example, 3,5A6/Y4α (m/z = 1593.074), 0,4 X1γ/Y2 (m/z = 676.421), and 1,3X1γ/Y2 (m/z = 631.473) ions indicate α1−6 linkage for the terminal fucose. Similarly, 0,4X2/ B4 (m/z = 821.778) ion suggests the presence of α1−6 linkage for the 6-linked antennae. Additionally, we have analyzed MS/MS spectra of H5N4 and S1F1H5N4 originating from human milk FBP (data not shown). The structures found by SimGlycan correspond to those reported in data tables. Therefore, the analysis of MS/MS spectra by SimGlycan provided high throughput and a confidence level necessary for the assignment of glycan structures found in human milk FBP.

Due to much lower amount of available FBP from human milk, the analysis of glycans from this protein sample was done only by MALDI-MS using 2,5-DHB as the matrix. As shown in Figure 5 and Table 3, native FBP N-glycans are bi- and triantennary structures containing one to three fucose residues (Fuc0−3Hex3−6HexNAc2−5). Permethylated N-glycans showed masses that are compatible with high mannose structures (Hex5−6 HexNAc2) and complex-type N-glycan structures (NeuAc0−1Fuc0−1Hex3−6HexNAc3−5), as shown in Figure 6 and Table 4. Permethylated N-glycan profile shows the presence of sialylated glycans which were not present in the native N-glycan profile due to sialic acid loss during MALDI-MS analysis. Putative structures of Nlinked glycans were assigned based on measured MS data and by using Functional Glycomics database.29 Analysis of N-glycans from Human Milk FBP by MALDI MS/MS

Several ions observed in MALDI-MS of native and permethylated N-glycans from human milk FBP were further selected and fragmented. As in the case of N-linked glycans from bovine milk FBP, fragmentation spectra were inspected manually and by using SimGlycan. For example, the singly charged F1H5N4 ion (m/z 1809.650) revealed upon fragmentation Y-ions (m/z 1444.127, 1282.247, 592.894), B-ions (m/z 1442.309, 1238.761, 549.237, 387.210), and several other daughter ions resulting from cross-ring fragmentation (Figure S9, Supporting Information). 0,4A4 fragment ion (m/z = 609.236) indicates the α1−6 linkage of 6-linked antennae and 1,3X2/C5 fragment ion (m/z = 873.430) confirms the presence of α1−3 linkage for the 3-linked antennae. The intense Y1γ/Y5α ion at m/z 1501.421 indicates loss of terminal fucose. The ion that results from the cleavage of 3-linked antenna and two reducing end GlcNAc (B4/Y3α, m/z

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CONCLUSIONS The results of this study showed that FBPs originating from bovine and human milk contain abundance of high mannose and hybrid/complex type glycans. Glycan profiling of FBPs 1558



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(Tables 1−4) shows that 17 and 19 N-glycan structures were specific to FBP from bovine and human milk, respectively, while seven structures were found in both samples. A total of 11 N-linked glycans originating from human milk FBP are not reported in the Functional Glycomics glycan database if human milk is selected as the source of protein. N-glycans originating from bovine milk FBP mostly do not contain fucose and sialic acid, but many glycans containing fucose and sialic acid are present in human milk FBP. The increased fucosylation and syalylation of human proteins is common.21 However, the influence of glycosylation heterogeniety on folding and function of FBPs from various body fluids and tissues, including those associated with human cancers, remains to be resolved.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 419-530-5532. Fax: 419-530-4033. E-mail: Dragan.Isailovic@ utoledo.edu.

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ACKNOWLEDGMENTS This work was supported by research funds from the University of Toledo (DI). The acquisition of MALDI-TOF/TOF instrument and SimGlycan software was funded by an NSF-MRI grant (Award #0923184). We thank Dr. Jedrzej Romanovicz (Bowling Green State University) for help with initial MALDITOF analysis, Dr. Yong Wah Kim (University of Toledo) for initial help with using an ESI−MS instrument, and Venkantesh Chari (Medical University of Ohio) for help with Western blotting analysis.

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REFERENCES

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