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Originally published In Press as doi:10.1074/mcp.M500203-MCP200 on September 16, 2005.
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Molecular & Cellular Proteomics 4:1977-1989, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

High Throughput Quantitative Glycomics and Glycoform-focused Proteomics of Murine Dermis and Epidermis*

Rie Uematsu{ddagger},§, Jun-ichi Furukawa{ddagger}, Hiroaki Nakagawa{ddagger}, Yasuro Shinohara{ddagger},, Kisaburo Deguchi{ddagger}, Kenji Monde{ddagger} and Shin-Ichiro Nishimura{ddagger},||

From the {ddagger} Division of Biological Sciences, Graduate School of Science, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University, Sapporo 001-0021 and the § Basic Research Laboratory, Kanebo COSMETICS INC., Odawara 250-0002, Japan


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite recent advances in our understanding of the significance of the protein glycosylation, the throughput of protein glycosylation analysis is still too low to be applied to the exhaustive glycoproteomic analysis. Aiming to elucidate the N-glycosylation of murine epidermis and dermis glycoproteins, here we used a novel approach for focused proteomics. A gross N-glycan profiling (glycomics) of epidermis and dermis was first elucidated both qualitatively and quantitatively upon N-glycan derivatization with novel, stable isotope-coded derivatization reagents followed by MALDI-TOF(/TOF) analysis. This analysis revealed distinct features of the N-glycosylation profile of epidermis and dermis for the first time. A high abundance of high mannose type oligosaccharides was found to be characteristic of murine epidermis glycoproteins. Based on this observation, we performed high mannose type glycoform-focused proteomics by direct tryptic digestion of protein mixtures and affinity enrichment. We identified 15 glycoproteins with 19 N-glycosylation sites that carry high mannose type glycans by off-line LC-MALDI-TOF/TOF mass spectrometry. Moreover the relative quantity of microheterogeneity of different glycoforms present at each N-glycan binding site was determined. Glycoproteins identified were often contained in lysosomes (e.g. cathepsin L and {gamma}-glutamyl hydrolase), lamellar granules (e.g. glucosylceramidase and cathepsin D), and desmosomes (e.g. desmocollin 1, desmocollin 3, and desmoglein). Lamellar granules are organelles found in the terminally differentiating cells of keratinizing epithelia, and desmosomes are intercellular junctions in vertebrate epithelial cells, thus indicating that N-glycosylation of tissue-specific glycoproteins may contribute to increase the relative proportion of high mannose glycans. The striking roles of lysosomal enzymes in epidermis during lipid remodeling and desquamation may also reflect the observed high abundance of high mannose glycans.

The rapid progress in the sequence analysis of genomes of a variety of living organisms is accelerating the investigation of various related proteins involved in biological processes and disorders. Carbohydrate modifications can profoundly affect protein function. Their importance in disease is evident from a growing number of embryonic lethal phenotypes seen in knock-out mice with defects in glycoconjugate assembly or processing (1), and hence a large scale protein glycosylation analysis has become important.

The accurate identification of protein glycosylation is challenging because of the complex structures, labile nature, and microheterogeneity of glycoproteins. The presence of non-glycopeptides in a sample also limits the sensitivity for analysis of glycopeptides on mass spectrometry due to ion suppression. Although several new techniques enabling the large scale identification of glycoproteins have recently been developed (2–4), they cannot provide information about oligosaccharide moieties because the analysis is performed on peptides of which such moieties are enzymatically removed prior to the analysis. It is also well known that glycosylation is cell type-specific, so a single glycoprotein can have a different spectrum of glycan structures when expressed in different cells. Recent progress in mass spectrometry (e.g. electron capture-induced dissociation using Fourier transform mass spectrometry (5, 6) and MALDI-LIFT-TOF/TOF (7)) has demonstrated the ability to provide information both on peptide sequence and glycan structure for the analysis of glycopeptides. However, the throughput of these techniques is not high enough to apply to large scale protein glycosylation analyses. Therefore, unveiling the significance of protein glycosylation in an efficient manner requires further thought. One solution is to develop a focused approach based on function and information content.

In this study, we describe a glycomic approach to rationalize the focusing process using murine dermis and epidermis as models. A gross N-glycan profiling of the tissue(s) of interest was first elucidated both qualitatively and quantitatively to grasp the characteristics of N-glycosylation profiles of the tissue(s). A rapid and sensitive quantitative N-glycosylation profiling technique based on stable isotope-coded, glycan-selective derivatization was newly developed for this purpose. Glycoproteins carrying unique oligosaccharides were then selectively analyzed following affinity enrichment. Considering that glycoproteins are often involved in the adhesion of cells and their extracellular matrices, the glycoproteome of the epidermis, where living cells undergo desquamation, which comprises a major part of the epithelial barrier, and are continuously being renewed (8), may provide a new insight into the functional role of protein glycosylation.

Recently comparative proteomic profiling of murine epidermis and subepidermal tissues (9) and proteomic characterization of the plasma membrane of human epidermis (10) were successively carried out. These studies successfully identified a number of epidermal proteins but yielded no information on protein glycosylation. The structural elucidation of mammalian epidermal glycoconjugates has been studied mostly histochemically using lectins (11) or monoclonal antibodies (12). These studies revealed that cell surfaces of keratinocytes in the epidermis contain numerous glycoconjugates. However, in the histochemical approach, it is often difficult to differentiate whether the glycoconjugate of interest is a glycolipid or an N-glycosylated or an O-glycosylated glycoprotein. This approach does not provide detailed structural information of the oligosaccharides or their carrier proteins.

In this study, we reveal that the N-glycosylation of epidermal glycoproteins is characterized by markedly high levels of high mannose type oligosaccharides using a novel, stable isotope-assisted N-glycan profiling technique. Based on this observation, we identified glycoproteins that carry high mannose type glycans by MALDI-TOF/TOF mass spectrometry following direct tryptic digestion of protein mixtures and affinity enrichment of the glycopeptides of interest. Our approach allowed rapid and sensitive quantitative glycomic profiling, and it also provides not only the identification of glycoproteins carrying particular glycoforms but also the determination of the N-glycosylation sites and the relative quantities of the microheterogeneous glycoforms present at each N-glycan binding site. Finally the biological significance of the observed dominance of high mannose type N-glycans will be discussed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Male hairless mice (Hos/HR-1) were obtained from Nippon SLC (Shizuoka, Japan). They were fed a standard mouse diet and water ad libitum. 7–12-week-old animals were used in this study. Trypsin, chymotrypsin, and {alpha}-galactosidase were purchased from Sigma. {alpha}-Mannosidase, ß-N-acetylhexosaminidase, ß-galactosidase from jack bean, concanavalin A (ConA)1-agarose and 2-aminopyridine (PA)-isomalto-oligosaccharides were purchased from Seikagaku Co. (Tokyo, Japan). 2,5-Dihydroxybenzoic acid, human angiotensin II, bombesin, and adrenocorticotropic hormone-(18–39) were obtained from Bruker Daltonik (Bremen, Germany). Other materials were bought from the sources indicated: Sephadex G-15, Amersham Biosciences; Bio-Gel P-4, Bio-Rad; sodium cyanoborohydride, Aldrich; peptide-N-glycosidase F (PNGase F), Roche Applied Science; Pronase, Calbiochem. {alpha}-L-Fucosidase from bovine kidney and other chemical reagents were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Synthesis of Novel Labeling Reagents
N{alpha}-((Boc-aminooxy)acetyl)tryptophanylarginine Methyl Ester Hydrochloride—
To a solution of N{alpha}-(benzyloxycarbonyl)tryptophanyl-arginine methyl ester (ZWR-OCH3) (18) (10 mg, 20 µmol) in MeOH (5 ml) at room temperature was added 10% palladium/carbon (10 mg) and stirred under hydrogen atmosphere. After 2 h, the reaction mixture was filtered through a membrane to remove palladium/carbon, and the filtrate was concentrated to give WR-OCH3; positive ion mode MALDI-TOF m/z 376. To a solution of (Boc-aminooxy)acetic acid (172 mg, 0.9 mmol) in dry tetrahydrofurane (2 ml) were added N-methylmorpholine (110 µl, 1.0 mmol) and isobutyl chloroformate (131 µl, 1.0 mmol) at –20 °C. The mixture was heated to room temperature and stirred for 15 min followed by the addition of a solution of the WR-OCH3 (374 mg, 1.0 mmol) and NaHCO3 (84 mg, 1.0 mmol) in H2O (1 ml) at 0 °C. After stirring for 1 h, solvents were removed under reduced pressure. The residue was chromatographed on a column of silica gel using 6:1 CHCl3-MeOH to give the Boc-aoWR-OCH3 (360 mg, 73%).; 1H NMR (500 MHz, CD3OD) {delta} 7.60–6.98 (m, 5H, indole), 4.24 (s, 2H, CH2ONHBoc), 3.62 (s, 3H, CH3O); positive ion mode MALDI-TOF m/z 547.

N{alpha}-((Aminooxy)acetyl)tryptophanylarginine Methyl Ester (aoWR(H)) Hydrochloride—
BocNHOCH2COWR-OCH3 (18 mg, 33 µmol) was added to trifluoroacetic acid (2 ml) at –20 °C and stirred for 2 h. The reaction mixture was evaporated and co-evaporated with toluene three times to give aoWR(H).; positive ion mode MALDI-TOF m/z 447.

Arginine d3-Methyl Ester Dihydrochloride—
Thionyl chloride (1.4 ml, 20mmol) was added to CD3OD (25 ml) at –10 °C, and the solution was stirred for 10 min. Arginine (1.1 g, 5 mmol) was added to a solution at room temperature and stirred for 2 days. The reaction mixture was evaporated and co-evaporated with CD3OD three times. The residue was dissolved in CD3OD (3.5 ml), and diethyl ether (15 ml) was added. The crystalline product was collected, washed with diethyl ether, and dried to give Arg-OCD3 in quantitative yield; 1H NMR (500 MHz, CD3OD) {delta} 4.10 (t, 1H, J = 6.5 Hz, {alpha}-CH), 3.3 (s, 2H, CH2ONHBoc), 3.26 (t, 2H, J = 7.0 Hz, CH2NH).

Tissue Preparation
Preparation of epidermis and dermis was performed according to the procedure described previously (13) with minor modifications. Briefly full thickness skin samples were taken from the dorsal area of mice. The epidermis was peeled from dermis by heat separation at 60 °C for 30 s. Epidermis and dermis were minced and heated at 90 °C for 10 min in water, then defatted as described by Bligh and Dyer (14), and lyophilized. For the glycomic analysis, sialic acid residues were hydrolyzed with HCl prior to defatting.

N-Glycan Release and Chemical Derivatization
Each defatted and lyophilized tissue (equivalent to ~3 mg) was dissolved in 7 M guanidine hydrochloride, 0.5 M Tris-HCl (pH 8.5), 10 mM EDTA; reduced with dithiothreitol; S-carbamoylmethylated; and dialyzed against 10 mM ammonium bicarbonate. Following deglycosylation by PNGase F treatment, samples were deproteinated by precipitation with acetonitrile. The supernatant was evaporated to dryness and redissolved in 30 µl of H2O. An aliquot (10 µl) of dermis and epidermis samples was mixed with 10 µl of aoWR(D) and aoWR(H) (0.4 mM in 40 mM acetate buffer, pH 4.0), respectively, and were heated at 90 °C for 1 h to prepare aoWR derivatized oligosaccharides. A weakly acidic condition (e.g. pH 4.0) was found to be recommended for the derivatization because the methyl ester moieties of aoWRs could be partly deesterified under the weakly basic condition. Alternatively the same samples were derivatized with PA and sodium cyanoborohydride according to the procedure described previously (15–17). After removal of unreacted PA by Sephadex G-15, the PA-oligosaccharides were further purified by collecting the elution from 7 to 12 min from amide-80 (4.6 x 250 mm, Tosoh, Tokyo, Japan) using HPLC.

Gross N-Glycan Profiling by MALDI-TOF
Each aoWR derivatized sample (1 µl) was directly diluted with 2,5-dihydroxybenzoic acid (10 mg/ml in 30% acetonitrile) 100-fold without any further purification, and an aliquot (1 µl) was deposited on the stainless steel target plate. The aoWR(H) derivatized epidermis sample and aoWR(D) derivatized sample were first analyzed separately to elucidate the relative quantities of the different oligosaccharides present in each tissue. Each sample was then mixed in equal quantities and was subjected to MALDI-TOF analysis for the relative quantitation of dermis and epidermis oligosaccharides. MALDI-TOF data were obtained using an Ultraflex time-of-flight mass spectrometer (Bruker Daltonik) equipped with a LIFT-TOF/TOF unit controlled by the FlexControl 2.0 software package. All of the spectra were obtained using reflectron mode with an acceleration voltage of 25 kV, a reflector voltage of 26.3 kV, and a pulsed ion extraction of 160 ns in the positive ion mode. These were the results of the signal averaging of 1,000 laser shots. Signal intensity of each mass was automatically calculated by FlexAnalysis 2.0. Estimation of N-linked oligosaccharide structures was obtained by input of peak masses into the GlycoMod Tool (www.expasy.ch/cgibin/glycomod_form.html).

Gross N-Glycan Profiling by Two-dimensional Mapping Technique
The PA derivatized oligosaccharides were analyzed according to the procedure described previously (15). Briefly the mixture of PA-oligosaccharides was applied to an octadecylsilyl silica (ODS; 6 x 150 mm, Shimadzu, Kyoto, Japan) HPLC column, and the elution times of the individual peaks were normalized with reference to the PA derivatized isomalto-oligosaccharides of polymerization degrees 4–20 and represented by GU (ODS). Then individual fractions separated on the ODS column were applied to the amide-80 column. Similarly the retention time of the individual peaks on the amide-80 column were represented by GU (amide). Thus, a given compound from these two columns provided a set of GU (ODS) and GU (amide) values, which corresponded to coordinates of the two-dimensional sugar map. By comparison with the coordinates of reference PA-oligosaccharides, the N-glycans from skin were identified. Identification was confirmed by co-chromatography with a candidate reference on the columns and sequential exoglycosidase digestion. Molar ratios of N-glycans were calculated from the individual peak areas.

Preparation of Glycopeptides
Defatted and lyophilized epidermis (50 mg) was S-carbamoylmethylated. The alkylated proteins were dialyzed against 10 mM ammonium bicarbonate and were digested with trypsin. The digested proteins were applied to a ConA-agarose column (0.7 x 13 cm) equilibrated with 150 mM NaCl, 10 mM Tris-HCl buffer, pH 7.5. After washing the column with 5 mM methyl {alpha}-glucopyranoside in 150 mM NaCl, 10 mM Tris-HCl buffer, pH 7.5, the ConA-bound glycopeptides were eluted with 0.1 M methyl {alpha}-mannopyranoside in 150 mM NaCl, 10 mM Tris-HCl buffer, pH 7.5. The bound fraction was then separated by an ODS column using HPLC by a linear gradient of acetonitrile (0–32%) in 0.1% formic acid. Chromatography was carried out at a flow rate of 1 ml/min at room temperature and was monitored at 214 nm. The glycopeptide mixture was separated into 100 fractions and dried with a centrifugal vacuum concentrator. The fractionated glycopeptides were dissolved in 10 µl of 30% acetonitrile. A portion (1 µl) of each fraction was deglycosylated by PNGase F and dissolved in the matrix solution.

Glycopeptide Identification by MALDI-TOF/TOF
Each fraction with or without PNGase F treatment was mixed with 2,5-dihydroxybenzoic acid (10 mg/ml in 30% acetonitrile) or {alpha}-cyano-4-hydroxycinnamic acid (saturated solution in 0.1% trifluoroacetic acid) and was applied on the MALDI target plate. MALDI-TOF(/TOF) data were obtained using an Ultraflex time-of-flight mass spectrometer. In MALDI-TOF, spectra were obtained using reflectron mode with an acceleration voltage of 25 kV, a reflector voltage of 26.3 kV, and a pulsed ion extraction of 160 ns in the positive ion mode. For fragmentation ion analysis in the TOF/TOF mode, precursors were accelerated to 8 kV and selected in a timed ion gate. Fragment ions generated by laser-induced decomposition of the precursor were further accelerated by 19 kV in the LIFT cell, and their masses were analyzed after passing the ion reflector. Masses were automatically annotated by using FlexAnalysis 2.0. External calibration of MALDI-TOF mass was carried out using singly charged monoisotopic peaks and fragments of a mixture of human angiotensin II (m/z 1046.542), bombesin (m/z 1619.823), and adrenocorticotropic hormone-(18–39) (m/z 2465.199).

Protein Identification by Database Search
Peak lists were generated from the MS/MS spectra using Bruker FlexAnalysis and were processed by the MASCOT algorithm (Matrix Science Ltd.) to assign peptide based on the mouse genome sequence database. The database (Mass Spectrometry Protein Sequence Database (MSDB)) was searched for tryptic peptides with up to one miscleavage. All cysteine residues were treated as being carbamoylmethylated. Deamidation of asparagines caused by deglycosylation was considered. We first screened the candidate peptides with probability-based Mowse scores that exceeded their thresholds (p < 0.05) and with MS/MS signals for y- or b-ions >5. If the peptide did not contain the consensus tripeptide sequence for N-linked glycosylation (NX(S/T/C)), the data were eliminated regardless of the matching score.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Analytical Protocol—
The analytical protocol used is comprised of two steps (Scheme 1). First whole N-glycans present in the epidermis and dermis were liberated, and gross N-glycan profiles were elucidated both qualitatively and quantitatively by MALDI-TOF utilizing novel, stable isotope-coded derivatization reagents. Based on the observed N-glycan profile, an affinity reagent to enrich particular types of glycopeptides was chosen. We used a shotgun proteomic approach rather than a classical gel electrophoresis-based approach because many of the glycoproteins are known to be membrane-associated. For the identification of glycopeptides, off-line LC-MALDI-TOF/TOF was used. LC separation prior to MALDI-TOF/TOF was introduced with the aim to detect as many glycopeptides as possible by reducing the complexity and ion suppression effect. Off-line connection of LC and MALDI-TOF/TOF was used to allow separated glycopeptides to be individually digested by PNGase F. By introducing this process, one can be assured of the occurrence of N-glycosylation and the accurate size of N-glycan. It also improves the peptide identification efficiency.



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  		SCHEME 1. General scheme for glycoform-focused proteomics.

 
Gross N-Glycan Profiling of Murine Epidermis and Dermis—
Aiming to establish a rapid, sensitive, and quantitative N-glycosylation profiling technique, novel stable, isotope-coded glycan-selective derivatization reagents were developed. Based on our previous findings that incorporation of guanidino and hydrophobic functionalities significantly enhances the ionization efficiency of oligosaccharide (18), tryptophanyl-arginine was used as a scaffold. The N terminus of the dipeptide was used for the incorporation of the oxylamino functionality, and the C-terminus was methyl esterified to generate a deuterated (d3-methyl) analog. As indicated in Fig. 1, oligosaccharides preferentially react with aoWRs to give corresponding oximes. In this study, epidermis and dermis samples were labeled with aoWR(H) and aoWR(D), respectively. As shown in Fig. 2, a and b, the MALDI-TOF spectra obtained from each sample differed significantly. GlycoMod search revealed that the m/z values of most of the signals observed were found to be identical to those of oligosaccharides known to be present in mammal Table I). All the assigned signals in Table I were confirmed to be aoWR derivatives by comparing the m/z differences when the labeling reagents for epidermis and dermis samples were exchanged (data not shown). The MALDI-TOF/TOF analysis of aoWR(H) and aoWR(D) derivatized oligosaccharides gave very a intense fragment signal at m/z 431 and 434, respectively, that corresponds to the N–O bond cleavage of the oxime. Thus, this diagnostic signal could also be useful to distinguish between aoWR derivatives and contaminant signals. The same samples were also analyzed by a two-dimensional sugar mapping technique, and we confirmed that the N-glycan profiles derived from the two different techniques were almost the same both qualitatively and quantitatively except that MALDI-TOF analysis could detect far more N-glycans than the two-dimensional mapping technique as indicated in Table I. Detailed structures of major oligosaccharides were determined by sequential glycosidase digestions and are shown in Table I.



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  		FIG. 1. Structures of aoWRs and the chemistry of adduct formation with oligosaccharides.

 


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  		FIG. 2. MALDI-TOF spectra showing the N-glycan profiles of murine dermis and epidermis glycoproteins. N-Glycans derived from dermis and epidermis were derivatized with aoWR(D) and aoWR(H), respectively, and analyzed either independently (a, epidermis; b, dermis) or after mixing them in equal quantities (c). Magnified views of the mass spectrum (c) for the region of signals designated as 2, 8, and 20 are shown in d, e, and f, respectively. Black and red arrows indicate aoWR(H) and aoWR(D) derivatives, respectively. Structures of the ions with numbers are shown in Table I.

 

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  		TABLE I Observed signals of aoWR-labeled oligosaccharides released from dermis and epidermis glycoproteins

Oligosaccharides indicated with an asterisk were detected also by a two-dimensional mapping technique. Structures were determined by a two-dimensional mapping technique combined with sequential exoglycosidase digestion. {square}, GlcNAc; {circ}, Man; •, Gal; {triangledown}, Fuc. Hex, hexose; HexNAc, N-acetylhexosamine; dHex, deoxyhexose; theor, theoretical; obsd, observed.

 
Relative quantitation between dermal and epidermal oligosaccharide profiles was performed by mixing the aoWR(H) and aoWR(D) derivatized samples in equal quantities and subsequent MALDI-TOF analysis (Fig. 2c). By incorporating mass differences of 3 Da, the oligosaccharides from the isotope-encoded sample were easily distinguished from the corresponding natural isotopic abundance oligosaccharides in the mass spectrometer, allowing a direct quantitative measure of the tissue-specific N-glycosylation (Fig. 2, d–f). Based on the relative signal intensities between the aoWR(H)- and aoWR(D) derivatized oligosaccharides, the relative quantities of N-glycans present in dermis and epidermis were determined as shown in Fig. 3.



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  		FIG. 3. Relative quantitation of N-glycans present in dermis and epidermis glycoproteins.

 
The glycomic analysis of murine dermis and epidermis revealed distinct features of the N-glycosylation dermal and epidermal profiles. A noteworthy feature is that the murine epidermal glycoproteins are characterized by a high abundance of high mannose type oligosaccharides. On the contrary, in dermal glycoproteins, the large majority of N-glycans are the complex type. This appeared to be unique because in vertebrates high mannose type glycans are generally present at low levels, although they can normally be found (19).

In this study, glycomic analyses were performed after the removal of sialic acid residues (see "Experimental Procedures") to avoid any effects on the quantitative analysis of oligosaccharides due to the possible occurrence of the metastable fragmentation of sialic acid residue(s) during the flight time of the ions. This inconsistency can be solved for instance by methyl esterification of the sialic acid residue(s) to render sialylated oligosaccharides chemically equivalent to neutral oligosaccharides as reported previously (20). Development of a rapid chemical modification procedure to stabilize sialic acid residue is also currently in progress in our laboratory.

Identification of Glycoproteins Carrying High Mannose Type Oligosaccharides in Epidermis—
Driven by the observed unique N-glycan profile in epidermal tissues, identification of the proteins that carry high mannose type oligosaccharides was performed. ConA was used as an affinity reagent to selectively recover the glycopeptides that carry high mannose type oligosaccharides (21). As shown in Fig. 4, the lectin chromatography greatly reduced the complexity of the peptide mixture. Following the enrichment of glycopeptides of interest in a crude tryptic digest of epidermis, the ConA-bound fraction was further separated into ~100 fractions by reversed-phase chromatography.



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  		FIG. 4. Elution profiles of epidermal peptides digested with trypsin on the ODS column before (A) and after (B) enrichment by ConA affinity chromatography.

 
Simultaneous identification of peptide and N-glycan moieties was possible for some of the glycopeptides by MALDI-TOF/TOF mass spectrometry. An example is shown in Fig. 5. The most intense fragment signals of glycopeptides were a cleavage of the chitobiose core and an 0,2X ring cleavage of the innermost GlcNAc. By way of these signature signals, the signal for the deglycosylated peptide ion could be readily identified. Besides cleavages of glycan moieties, cleavages of peptide bonds were also observed. A database search based on the deglycosylated peptide ion mass, the fragment mass, and intensity values lower than the molecular weight of deglycosylated peptide ion identified this peptide to be YYHGELSYLNVTR of cathepsin D with high probability-based Mowse scores (p < 0.05). The TOF/TOF fragmentation of the glycan moiety further indicated that this peptide was modified with Man5GlcNAc2. To our knowledge, this is the first successful direct glycopeptide identification based on raw TOF/TOF data taken from one glycopeptide derived from a complex biological mixture.



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  		FIG. 5. Tandem mass spectrum derived from TOF/TOF of the [M + H]+ precursor, m/z 2831.6. The amino acids are represented in their single letter codes. The sequence identification of the peptide and database search showed that this glycopeptide was derived from cathepsin L with the sequence of YYHGELSYLNVTR. The signal for the deglycosylated peptide is marked with an asterisk. 0,2X, ring fragmentation of the innermost GlcNAc.

 
Despite the preferable nature of direct glycopeptide identification by MALDI-TOF/TOF, glycopeptide identification was performed after PNGase F digestion to improve the identification efficiency due to the lack of suitable software for the glycopeptide identification. An example is shown in Fig. 6. The mass spectra obtained from HPLC fractions often contained ions from multiple glycopeptides. The characteristic signal patterns separated by 162 Da (hexose) were often observed for those peptides carrying different high mannose type glycoforms on the same peptide backbone (e.g. m/z 2211.36, 2534.35, 2696.40, and 2858.45 in Fig. 6A). Following the PNGase F digestion, all those signals disappeared. Instead a new signal appeared (e.g. at m/z 1318.90 in Fig. 6B). This observation evidently revealed that the peptide was N-glycosylated with Man3GlcNAc2, Man5GlcNAc2, Man6GlcNAc2, and Man7GlcNAc2. The peptide was identified as TGEINITSIVDR of desmoglein by MALDI-TOF/TOF analysis as shown in Fig. 6C.



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  		FIG. 6. Identification of glycopeptide by MALDI-TOF/TOF following PNGase F digestion of purified glycopeptides. MALDI-TOF spectra obtained for the purified glycopeptide with (A) and without (B) PNGase F treatment are shown. C, tandem mass spectrum derived from TOF/TOF of the [M + H]+ precursor, m/z 1318.9. The sequence identification of the peptide and database search showed that this glycopeptide was derived from desmoglein with the sequence of TGEINITSIVDR.

 
We observed that glycopeptides of different glycoforms on the same peptide tended to elute in close proximity on the reversed-phase chromatography analyses; peptides modified with larger N-glycans eluted earlier, and those with smaller N-glycans eluted later. Therefore, the relative quantitation of the microheterogeneity of different glycoforms present at a particular N-glycan binding site could be determined by comparing the signal intensities upon mixing equivalent volumes from each successive fraction containing the same peptide backbone. As shown in Fig. 7, the microheterogeneity of TGEINITSIVDR of desmoglein was clearly elucidated.



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  		FIG. 7. Determination of N-glycosylation microheterogeneity present at TGEINITSIVDR of desmoglein. A, MALDI-TOF spectrum upon mixing the same volume from each successive HPLC fraction that contains same peptide backbone but with different glycoforms. B, relative quantitation of each glycoform present at TGEINITSIVDR of desmoglein. Values are shown as mean ± S.D. (n = 3). M5, Man5GlcNAc2; M6, Man6GlcNAc2; M7, Man7GlcNAc2; M8, Man8GlcNAc2; M9, Man9GlcNAc2.

 
Based on the described procedure, we identified and characterized the major glycopeptides carrying high mannose type oligosaccharides in the epidermis and listed them in Table II. Of 15 glycoproteins identified in this study, four were bona fide transmembrane proteins. The non-transmembrane proteins were mostly designated as either extracellular (two proteins) or lysosomal (six proteins), thus belonging to two cellular compartments known to be enriched for glycoproteins.


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  		TABLE II Summary of glycoproteins identified in this study

M5, Man5GlcNAc2; M6, Man6 GlcNAc2; M7, Man7GlcNAc2; M8, Man8GlcNAc2; M9, Man9GlcNAc2; ND, not determined.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We described a glycomic approach to rationalize the focused glycoproteome analysis of the murine epidermis. Upon qualitative and quantitative gross N-glycan profiling analysis, we observed distinct features of the N-glycosylation profile between the two tissues for the first time. The newly developed stable isotope-assisted derivatization reagents allowed a rapid and sensitive N-glycan profiling by MALDI-TOF analysis in a quantitative manner of both intra- and intersamples. Because of its high ionization efficiency of aoWR on MALDI-TOF analysis, aoWR derivatives could be highly selectively detected with minimum purification steps. We have recently reported that polymers displaying oxylamino groups provide a useful platform for "glycoblotting," a high throughput oligosaccharide purification procedure in a crude mixture (22, 23). Derivatization with aoWR is therefore regarded as an analogue of glycoblotting where chemoselective ligation is done using functional low molecular weight compounds instead of polymers.

Among many qualitative and quantitative differences in N-glycan profiling, we focused on the high mannose type N-glycans carrying glycoproteins in this study. Following the affinity enrichment, they were identified by off-line LC-MALDI-TOF/TOF mass spectrometry, which simultaneously provided information about the sugar binding site and the relative quantitation of microheterogeneity of different glycoforms present at each N-glycan binding site. Although the number of identified glycoproteins is so far limited, they are most likely to represent the major high mannose glycan-carrying proteins because the glycopeptide identification was carried out for those signals whose intensities were strongest.

Among glycoproteins identified in this study, almost half belong to lysosomal hydrolases. This observation appeared to be reasonable considering that the major sorting mechanism of those hydrolases to lysosome is by the mannose 6-phosphate pathway (24). In the epidermis, enzymes of lysosomes and lamellar granules are present also in the extracellular compartment and are responsible for the lipid remodeling required to generate the barrier lamellae as well as for the reactions that result in desquamation (25). Lamellar granules (LGs), which are considered to be lysosome-related organelles (8), are organelles present in the cytoplasm of cells in the granular layer and account for about 10% of the volume of the granular cell cytosol (26). LGs likely originate from the Golgi apparatus and are currently thought to be elements of the tubulovesicular trans-Golgi network (27, 28), although it is not known whether these cells use the same sorting system for LGs as that used for lysosomes. LGs are a major source of stratum corneum lipid precursors and various hydrolytic enzymes such as lipases, proteases, acid phosphatases, and glycosidases (27, 29, 30) and other proteins including corneodesmosin (31). Among lysosomal glycoproteins identified in this study, glucosylceramidase and cathepsin D have been reported to be present in LGs (29, 31). Although further investigation is required, the high abundance of high mannose type oligosaccharides observed in epidermis may be attributable to the striking roles of lysosomal enzymes and/or the high abundance of lamellar granule in epidermis.

Desmoglein along with the desmocollins comprise the desmosomal cadherins; the extracellular domains of these proteins make up the extracellular core domain of the desmosome (32). These proteins are expressed in a tissue-specific manner and provide the sticky adhesion of the desmosome required between adjacent cells. Desmogleins 1 and 3 and desmocollins 1 and 3 expression is restricted to certain specialized epithelia such as epidermal, tongue, tonsil, and esophagus tissues. Given that these tissue-specific glycoproteins are dominantly modified with high mannose glycans, it may also prove beneficial to increase the relative proportion of high mannose glycans in epidermis. Although desmogleins and desmocollins are reported to be glycoproteins containing mainly N-glycans, detailed information about oligosaccharide structures has been so far limited (33–35). The current study also raises an interesting query: why desmosomes require high mannose glycans particularly when it is possible that any type of oligosaccharides could protect desmosomal proteins from protease degradation and may prevent premature desquamation as suggested by Walsh and Chapman (36).

Other extracellular glycoproteins identified in this study are lymphocyte antigen 6 complex locus G6C protein and extracellular matrix protein-1 (ECM1). The Ly6g6c gene is predicted to encode members of the Ly-6 superfamily of proteins based on translations of the predicted gene sequences (37). Murine Ly-6 antigens were originally identified as markers for hematopoietic cells. Although the precise functions of the Ly-6 antigens have not been determined, several lines of evidence have suggested that they are involved in cell signaling and cell adhesion (38). ECM1 is a secreted glycoprotein first isolated from an osteogenic mouse cell line in concomitance with studies on bone matrix biology (39). Subsequently the human homologue has been found to regulate endochondral bone formation and to stimulate proliferation of endothelial cells and induce angiogenesis. The Ecm1 gene has two splice variants. The long isoform is expressed in a number of tissues including liver, heart, and lungs, whereas expression of the short isoform is confined to skin and cartilage-containing tissues such as tail and front paw. ECM1 may be involved in the control of keratinocyte differentiation, but its precise role within the epidermis is not clear (40). To our knowledge, structural elucidations of N-glycans of theses glycoproteins have been scarce. The evidence that extracellular glycoproteins are actually modified with high mannose type oligosaccharides indicates that epidermis cells project high mannose glycans on the cell surface and may be involved in molecular recognition events. For example, the trimeric extracellular domain of langerin, a cell surface receptor unique to Langerhans cells, is reported to bind mammalian high mannose oligosaccharides, although the intrinsic ligand is not known (41).

The described protocol allows for determination of the relative quantities of the microheterogeneous glycoforms present at each N-glycan binding site. It should be noted that relatively abundant Man3GlcNAc2 (M3), Man3GlcNAc2Fuc (M3F), and Man4GlcNAc2 (M4) were typically observed for lysosomal proteins. High mannose oligosaccharides are assembled in the endoplasmic reticulum and cis-Golgi and contain between five and nine mannose residues. Therefore, the observed M3, M3F, and M4 should be considered to be the degraded products. This can be explained by the presence of glycosidase (e.g. {alpha}-mannosidase) in lysosomes (42) and LGs (43). A peptide from desmoglein exhibited the unexpected presence of M3 and M4. The mechanism of the occurrence of such degradation on the plasma membrane surface may need to be further elucidated. Other glycopeptides from extracellular glycoproteins are commonly modified with high mannose oligosaccharides with between five and nine mannose residues in a distinct, microheterogeneic manner. In this study, two functionally unknown proteins were also identified, RIKEN cDNA 1100001H23 (44) and RIKEN cDNA 2310020A21 (45), as high mannose type oligosaccharide-carrying proteins. The N-glycosylation microheterogeneity analysis revealed that the former is modified with Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, and Man8GlcNAc2, whereas the latter is predominantly modified with M3 and M3F. These observations may indicate that the latter belongs to lysosomal proteins, whereas the former does not.

In summary, we proposed and demonstrated the feasibility of glycomic profiling in developing a focused approach for glycoproteomics. Protocols utilizing novel, stable isotope-coded derivatization reagents coupled with MALDI-TOF and off-line LC-MALDI-TOF/TOF were developed as key techniques for the glycomic and glycoproteomic analysis. This study demonstrates that the gross N-glycan profiling prior to glycoprotein identification makes the particular glycoform-focused approach functionally meaningful and greatly accelerates the data mining process.


   ACKNOWLEDGMENTS
 
We thank Joseph Justin Mulvey for correcting English.


  FOOTNOTES
 
Received, July 6, 2005, and in revised form, September 6, 2005.

Published, MCP Papers in Press, September 16, 2005, DOI 10.1074/mcp.M500203-MCP200

1 The abbreviations used are: ConA, concanavalin A; aoWR, N{alpha}-((aminooxy)acetyl)tryptophanylarginine methyl ester; GU, glucose unit; LG, lamellar granule; PA, 2-aminopyridine, PNGase F, peptide-N-glycosidase F; Boc, t-butoxycarbonyl; ODS, octadecylsilyl silica; Man, mannose; ECM1, extracellular matrix protein-1; M3, Man3GlcNAc2; M3F, Man3GlcNAc2Fuc; M4, Man4GlcNAc2. Back

* This work was supported in part by SENTAN, Japan Science and Technology Agency. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence may be addressed. Tel. and Fax: 81-11-706-9043; E-mail: shin{at}glyco.sci.hokudai.ac.jp Back

To whom correspondence may be addressed. Tel. and Fax: 81-11-706-9043; E-mail: yshinohara{at}glyco.sci.hokudai.ac.jp


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