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Keywords:

  • fucosylated haptoglobin;
  • pancreatic cancer;
  • tumor marker;
  • Lewis X;
  • Lewis Y;
  • LC-ESI MS;
  • site-specific analysis

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Glossary
  7. References
  8. Supporting Information

It was found in our previous studies that the concentration of fucosylated haptoglobin had increased in the sera of patients with pancreatic cancer (PC) compared to those of other types of cancer and normal controls. Haptoglobin, an acute phase protein, has four potential N-glycosylation sites, although it remains unknown which site is responsible for the change in fucosylated N-glycans. In the present study, site-specific N-glycan structures of haptoglobin in sera obtained from patients with PC or chronic pancreatitis (CP) were analyzed using liquid chromatography-electrospray ionization mass spectrometry. Mass spectrometry analyses demonstrated that concentrations of total fucosylated di-, tri- and tetra-branched glycans of haptoglobin increased in the sera of PC patients. Tri-antennary N-glycans containing a Lewis X-type fucose markedly increased at the Asn211 site of haptoglobin N-glycans. While fucosylated N-glycans derived from serum haptoglobin of patients with CP slightly increased, di-fucosylated tetra-antennary N-glycans were observed only at this site in PC patients, and were absent in the haptoglobin of normal controls and individuals with CP. Thus, the present study provides evidence that site-specific analyses of N-glycans may be useful as novel tumor markers for PC. © 2008 Wiley-Liss, Inc.

Pancreatic cancer (PC) is one of the leading causes of cancer-related deaths with an overall 5-year survival rate of less than 5%.1, 2 One of the reasons for the poor prognosis is that early diagnosis is quite difficult and risk factors for PC have not yet been identified. Carbohydrate antigen 19-9 (CA19-9) and the carcino embryonic antigen (CEA) are commonly used as markers of PC, but do not allow for early diagnosis.3 Diagnostic specificity could be increased through a combination of pancreatic tumor markers. To discover novel markers for PC, which have characteristics different from those of CA19-9 or CEA, we used glycomics (see glossary) to identify fucosylated haptoglobin as a potential marker.4 The addition of glycans to proteins is one of the most important posttranslational modifications carried out, and many studies have shown that changes in glycan structures occur during inflammation and tumorigenesis.5 Haptoglobin is an acute-phase protein, which is produced in the liver. Since a normal liver expresses low levels of fucosyltransferases and GDP-Fuc (guanosine diphospho fucose, a common donor substrate for fucosyltransferases),6 most haptoglobin is not fucosylated in healthy individuals.7, 8 Many researchers have reported that fucosylated proteins in serum increase in patients with cancer and/or inflammation.9–11 Recently, Zhao et al., also reported that several kinds of serum glycoproteins, which were mainly produced in the liver and existed at low levels in serum, increased their fucosylation on N-glycans in patients with PC, using glycoprotein microarrays with multi-lectin detection after removing haptoglobin and other major serum proteins.12 However, it was confirmed in previous study that fucosylated haptoglobin was also produced from certain kinds of PC cells.4 Therefore, it is important to determine if the increase in fucosylated haptoglobin results directly from PC or is secondary to cancer-induced inflammation of the pancreas. Previously, we found that the appearance rates of fucosylated haptoglobin were higher in patients with PC than those in other types of cancer and normal volunteers (NV).4 In contrast, it was reported that fucosylated haptoglobin increased in patients with other types of cancer, such as ovary cancer,13 hepatocellular carcinoma14 and lung cancer15 that had progressed to an advanced stage. To gain insight into the mechanism and location of fucosylation up-regulation, more detailed analyses of haptoglobin N-glycans are required.

Human haptoglobin is comprised of 406 amino acid residues,16, 17 which consists of a signal peptide (Met1 to Ala18), one α chain (Val19 to Gln160) and one β chain (Ile162 to Asn406). The haptoglobin β chain contains four potential N-glycosylation sites (see supplemental Fig. 1): Asn184 (site 1), Asn207 (site 2), Asn211 (site 3) and Asn241 (site 4).18, 19 In the present study, PA-labeled haptoglobin N-glycans from patients with chronic pancreatitis (CP) and PC were analyzed using normal-phase HPLC (NP-HPLC) to measure the amount of fucosylated N-glycans and to identify the type of fucosylated N-glycans (i.e., the linkage position of Fuc). Furthermore, precise analyses of site-specific N-glycan structures of haptoglobin were carried out using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS, see glossary) to identify fucosylated N-glycans unique to PC.

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Figure 1. Purification of haptoglobin using an anti-haptoglobin affinity column and AAL blot analysis. (a and b), all gels were stained with CBB after SDS-PAGE analysis (10% polyacrylamide). (a) SDS-PAGE analysis of serum (S), pass through fraction (P), and eluted fraction (E) from sera of patients with pancreatic cancer (PC1). (b) purified haptogoboin from sera of patients with pancreatic cancer (PC), chronic pancreatitis (CP), and sera of normal volunteers (NV). The volume of NV applied was 3-fold greater than the sample volumes of CP and PC. (c) equal amounts of purified haptoglobin from PC, CP, and NV were electrophoresed, followed by CBB staining and AAL blot analysis (2 samples each).

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Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Glossary
  7. References
  8. Supporting Information

Lysylendopeptidase was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Sequencing-grade modified trypsin was purchased from Promega (Madison, WI). Polyclonal rabbit anti-human haptoglobin antibody was obtained from DakoCytomation (Glostrup, Denmark). Endoprotease Glu-C and Peptide-N4-(acetyl-β-D-glucosaminyl) asparagine amidase (PNGase F; E.C. 3.5.1.52, recombinant) were purchased from Roche Molecular Biochemicals (Tokyo, Japan). Aleuria aurantia lectin (AAL) conjugated with biotin was purchased from Honen Corp. (Tokyo, Japan). Horseradish peroxidase-conjugated streptavidin was obtained from Pierce, (Rockford, IL). Alpha 1-3/4 fucosidase and PA-labeled glycan standards for HPLC analysis were obtained from Takara Bio Inc (Shiga, Japan). Beta 1-4 galactosidase derived from S. pneumoniae was obtained from PROenzyme Co. (San Leandro, CA). Other reagents were either of the highest quality or were LC/MS grade, commercially available.

Serum samples

Serum samples from patients with PC (PC 1–5; n = 5, male 2, female 3, mean age 65 years) and CP (CP 1–5; n = 5, male 3, female 2, mean age 66 years) were obtained from Osaka University-related Hospitals. Serum samples from NV (NV 1-8; n = 8, male 5, female 3, mean age 46 years) were obtained in our laboratory with the participants' informed consent. NV 1-5 and NV 6–8 sera samples were respectively obtained from NV 26–38 and 63–75 years of age. All serum samples were stored at −80°C until used. The present project was approved by the ethics committees of the participating hospitals and Osaka University.

For a clear understanding, all procedures we performed in the present study are described in the workflow (Supplemental Fig. 2).

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Figure 2. NP-HPLC analysis of PA-labeled N-glycans. (a) chromatogram of standard PA-labeled N-glycans (STD, 10 pmol each) and N-glycans released from tryptic peptides of purified haptoglobin (NV5, CP3, and PC4). The peak numbers and the structures of standard PA-labeled N-glycans are shown in (b). More detailed structures thereof are shown in supplemental Figure 3a. (c) the candidate structures of N-glycan from peak 5*. (d) 100% stacked bar graph indicates the percentage of types of detected PA-labeled N-glycans derived from purified haptoglobins (NV, CP, and PC; 3 samples each). Fuc, fucose; Gal, galactose; Man, mannose; GlcNAc, N-acetylglucosamine; NeuAc, N-acetylneuramic acid; PA, 2-aminopyridine.

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Purification and SDS-PAGE analysis of haptoglobin from human sera

The sera of patients with PC (100 μL), CP (100 μL) and of NV (300 μL) were filtered using a 0.45-μm filter (Minisart RC 15, Sartorius, Goettingen, Germany) and then diluted with buffer A (50 mM sodium phosphate buffer (pH 7.4), 0.5 M NaCl, 0.02% NaN3) to a final volume of 7 mL. The diluted serum samples were circulated 5 times on an anti-haptoglobin affinity column, which was coupled with 300 μL of anti-human haptoglobin, at room temperature, according to the standard protocols of HiTrap NHS-activated HP (1 mL) (GE Healthcare, Uppsala, Sweden), using a peristaltic pump. After washing the column with 15 mL of buffer A, the haptoglobin bound to the column was eluted with 5 mL of elution buffer (100 mM Glycine, 0.5 M NaCl, pH 3.0). The eluate was immediately neutralized with 100 μL of 2 M Tris-HCl (pH 8.0). The neutralized eluate containing haptoglobin was passed through a PD-10 column (GE Healthcare) equilibrated with water to remove glycine, NaCl and Tris-HCl. The aqueous solution containing haptoglobin was evaporated to dryness. The residue was dissolved in 100 μL of water (sample solution A), and a portion (5 μL) was used for SDS-PAGE analysis to determine the haptoglobin yield. The solution (7 mL) containing proteins, which were not adsorbed to the anti-human haptoglobin column, were mixed with the washed column solution (15 mL), and the mixture was evaporated to dryness. The residue, which contained the pass-through fraction, was dissolved in 1 mL of water. The 10-fold diluted serum (5 μL), the pass-through fraction (5 μL) and the eluted fraction (sample solution A, 5 μL) were all subjected to SDS-PAGE (10% polyacrylamide) under reduced conditions and then stained with CBB.

LC-ESI MS identification of proteins purified using an anti-human haptoglobin column

The eluted solutions from the anti-human haptoglobin column (sample solution A, 80 μL) were evaporated to dryness, and 500 μL of a reducing solution containing 250 mM Tris-HCl (pH 8.5), 6 M guanidine hydrochloride, 2 mM EDTA and dithiothreitol (10 mg) were added to the residue. The mixture was incubated at 50°C for 1 hr to reduce Cys residues. After adding iodoacetamide (20 mg) to the mixture, the reaction was allowed to proceed for 30 min at room temperature in the dark. The reaction mixture was passed through a Nap-5 column (GE Healthcare) equilibrated with 0.05 N HCl to remove salts from the reducing solution and excess iodoacetamide. The eluate containing S-carbamidomethylated haptoglobin (1 mL, in 0.05 N HCl) was immediately neutralized with 100 μL of 1 M Tris-HCl (pH 9.0). The neutralized solution was mixed with 20 μL of 50 mM Tris-HCl (pH 8.5) containing an enzyme mixture of lysylendopeptidase (2 μg) and trypsin (2 μg) and incubated for 16 hr at 37°C.20 After boiling, the solution was evaporated to dryness. The residue was dissolved in 100 μL of water (sample solution B). The tryptic peptide mixture (sample solution B, 10 μL) was separated using an ODS column (Develosil 300ODS-HG-5, 150 × 1.0 mm i.d., Nomura Chemical, Aichi, Japan) under the following gradient conditions. The mobile phases were: (A) 0.1% TFA and (B) 0.1% TFA/80% acetonitrile. The gradient elution was performed at 10–60% B for 80 min with a flow rate of 50 μL/min using an Agilent 1100 series HPLC system (Agilent Technologies, Santa Clara, CA). The eluate was continuously introduced into an electrospray ionization (ESI) source (Esquire HCT, Bruker Daltonics GmbsH, Bremen, Germany). The proteins were identified using the NCBInr database with the MASCOT (Matrix Science, Boston, MA) database-searching algorithm.

Lectin blot analysis

Purified haptoglobin was subjected to SDS-PAGE (10% polyacrylamide) under reduced conditions. The gels were visualized by CBB staining, and the bands on the gels were quantified using an Image Reader LAS-3000 mini/Multi Gauge ver 2.2 (Fuji Photo Film, Tokyo, Japan). On the basis of the quantification results, equivalent amounts of purified haptoglobin were applied in duplicate to two SDS-PAGE gels. The proteins on one gel were stained with CBB, while those on the other gel were transferred to a PVDF membrane under semi-dry conditions by means of a Trans-blot (Bio-Rad, Hercules, CA). All AAL blot procedures have previously been described in detail.4 Briefly, after blocking by incubation with 2% BSA, the membrane was incubated with AAL conjugated with biotin followed by incubation with horseradish peroxidase-conjugated streptavidin. The chemiluminescence method with the ECL-Plus kit (GE Healthcare) was employed to detect the peroxidase activity. The samples were visualized using a LAS-3000 mini.

NP-HPLC analysis of PA-labeled N-glycans

The aqueous solution containing tryptic peptides treated with iodoacetamide (sample solution B, 10 μL) was mixed with 2 M acetic acid (100 μL), and the mixture was incubated at 80°C for 2 hr to remove sialic acids. After evaporating to dryness, the residue was digested with PNGase F (0.1 U in 50 μL of 50 mM NH4HCO3) to release the N-glycans. The released N-glycans were applied to a GlycoTAG apparatus (Takara Bio Inc) for derivatization with PA.21 Furthermore, a portion of PA-labeled N-glycans was digested with α1-3/4 fucosidase22 followed by digestion with β1-4 galactosidase. The PA-labeled N-glycans were subjected to NP-HPLC analysis. Eight types of commercially available PA-labeled N-glycans, shown in Figure 2b, were analyzed (10 pmol each) as standards. HPLC was performed with a Waters Alliance System equipped with a Waters 2475 fluorescence detector. Separation was done at 50°C using a polymer-based Asahi Shodex NH2P-50 4E column (Showa Denko, Tokyo, Japan; 4 × 250 mm) with a linear gradient formed by 2% acetic acid in acetonitrile (solvent A) and 5% acetic acid in water containing 3% triethylamine (solvent B). The column was initially equilibrated and eluted with 30% solvent B for 2 min, at which point the concentration of solvent B was increased to 50% over 60 min. The flow rate was 1.0 mL/min throughout the analysis. Detection was performed by fluorometry at λex = 320 nm and λem = 400 nm.

LC-ESI MS analysis of haptoglobin sialo-glycopeptides at sites 1 and 4

Analyses of haptoglobin sialo-glycopeptides containing N-glycans at binding sites 1 and 4 simultaneously were performed with identification of haptoglobin using LC-ESI MS (see the section “LC-ESI MS identification of proteins purified using an anti-human haptoglobin column”).

Preparation of asialo-glycopeptide and digestion with endoprotease Glu-C

Enrichment of glycopeptides from tryptic digests was carried out using affinity separation by partitioning with Sepharose CL4B, according to the method described by Wada et al.23 Briefly, water (150 μL) was added to the tryptic digests (sample solution B, 50 μL), and the solution was mixed with 1 mL of an organic solvent of 1-butanol/ethanol (4:1, v/v). The mixture was added to a 1.5-mL polypropylene tube containing 100 μL packed volume of Sepharose CL4B equilibrated with 1-butanol/ethanol/H2O (4:1:1, v/v). After gentle shaking for 30 min, the gel was washed three times with the same organic solvent (1 mL). The gel was then incubated with an aqueous solvent, ethanol/H2O (1:1, v/v), for 10 min, and the liquid-phase was evaporated to dryness. The residue was dissolved in 2 M acetic acid (100 μL) to remove sialic acids. After incubation for 2 hr at 80°C, the solution was evaporated to dryness. The residue was dissolved in 40 μL of water, and a portion (10 μL) was mixed with endoprotease Glu-C (1 μg in 30 μL of 50 mM NH4HCO3). The mixture was incubated for 16 hr at 37°C, and then boiled (sample solution C).

LC-ESI MS analysis of haptoglobin asialo-glycopeptides at sites 2 and 3

The solution containing asialo-glycopeptides digested with trypsin and endoprotease Glu-C (sample solution C, 10 μL) was subjected to LC-ESI MS analysis. The asialo-glycopeptides were separated using an ODS column (Develosil 300ODS-HG-5, 150 × 1.0 mm i.d., Nomura Chemical, Aichi, Japan) under the following gradient conditions. The mobile phases were: (A) 0.08% formic acid and (B) 0.15% formic acid/80% acetonitrile. The column was eluted with solvent A for 5 min, at which point the concentration of solvent B was increased to 50% over 75 min at a flow rate of 50 μL/min. The eluate was continuously introduced into an ESI source (Esquire HCT).

Relative abundance of N-glycan structures at each haptoglobin site

The relative abundance of each glycoform at sites 1–4 was calculated based on the signal intensities of the corresponding glycopeptides obtained by LC-ESI MS analysis.24 Briefly, total signal intensities of glycopeptides detected as sialylated-N-glycopeptides were set to 100% for each site of each sample, and the average ratio for each sialo-glycopeptide glycoform was calculated for young NV (under 40), old NV (over 60), CP and PC samples. When the ratio was expressed for asialoforms, the average ratios of sialo-glycopeptides thus obtained were converted to those of asialo-glycopeptides based on the predicted structures of asialo-glycoforms, while the value obtained by direct calculation was used for the desialylated samples (sites 2 and 3).

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Glossary
  7. References
  8. Supporting Information

Purification of human haptoglobin from sera and AAL blot analysis

Haptoglobin was purified from the sera of five patients with PC, CP and the sera of eight NV. A representative result of purified haptoglobin from a patient with PC (sample PC1) is shown in Figure 1a. A 40-kDa band, a haptoglobin β-chain, was detected as the major band without contaminant bands in the eluted fraction, demonstrating that haptoglobin was obtained with high-purity. A portion of the eluted fraction was digested with a combination of trypsin and lysylendopeptidase,19 and the digests were analyzed by LC-ESI MS to confirm that the 40-kDa protein was haptoglobin. The peptides detected by ESI MS were confirmed to be derived from haptoglobin with high probability scores by peptide mass fingerprinting against the NCBInr database (data not shown).

To evaluate the levels of haptoglobin in the serum of patients with PC and CP compared to those of NV, the samples of purified haptoglobin were electrophoresed on 10% polyacrylamide gels, followed by staining with CBB (Fig. 1b). Haptoglobin slightly increased in all 5 patients with CP; furthermore, the 3 patients with PC also had elevated haptoglobin levels. This result may be attributed to the fact that haptoglobin is an acute-phase protein.

To determine the variation in fucosylated glycans, AAL blot analysis of purified haptoglobin was performed. AAL interacts with α1-2 Fuc, α1-3/4 Fuc and α1-6 Fuc on glycans.25, 26 Equal amounts of purified haptoglobin were subjected to AAL blot analysis. As shown in Figure 1c, haptoglobin from patients with PC exhibited strong AAL binding. This result indicated that haptoglobin N-glycan structures changed in patients with PC-especially in terms of the number of Fuc residues.

NP-HPLC analysis of PA-labeled N-glycans derived from haptoglobin

Sialic acids were removed from N-glycan on tryptic peptides using acidic treatment. The asialo N-glycans were released with PNGaseF and were derivatized with PA. PA-labeled N-glycans were analyzed by NP-HPLC using an amino column. Eight commercially available PA-labeled N-glycans, shown in Figure 2b, were used as standards (Fig. 2a). More detailed structures of the standard N-glycans are shown in supplemental Figure 3a. The chromatograms for NV5, CP3 and PC4 are shown in Figure 2a. Tri-antennary N-glycan containing core Fuc (i.e., α1-6 Fuc, peak 4) was not detected in any of the samples. Bi-antennary N-glycan (peak 1*) was the most abundant peak in all samples. Peaks 5* and 8* increased in the PC4 sample compared to the NV5 and CP3 samples. Although the composition of N-glycan in peak 5* was predicted to be tri-antennary N-glycan containing a Lewis X-type Fuc (i.e., α1-3 Fuc), there were at least 4 possible structures (Lewis X, Lewis A, H1 and H2 in Fig. 2c) that were eluted at the same time. These 4 types of N-glycans have the same molecular weight and consist of the same numbers of the same kinds of monosaccharides, but the linkage position and linkage type of the Gal residues and a Fuc residue are different. To determine the precise structure of the N-glycan in peak 5*, sequential exoglycosidase digestion with α1-3/4 fucosidase and β1-4 galactosidase was performed. A Fuc residue and all Gal residues were removed from the N-glycan in peak 5*, indicating that the N-glycan in peak 5* could be predicted to be a tri-antennary containing a Lewis X type Fuc, which has an α1-3 Fuc and three β1-4 Gals. The detailed procedure and the resultant chromatograms are shown in supplemental Figure 4.

The amount (mol) of N-glycans detected in this experiment was calculated based on the peak areas of standard N-glycan samples. In each sample, the amount (mol) of each N-glycan was summed to 100%, and the results are represented in stacked bar graphs to compare the contribution of each N-glycan to the total for each sample (Fig. 2d). The average values are shown in supplemental Figure 3b. The percentage of each N-glycan in the NV samples was similar to previous values reported by Ferens-Sieczkowska and Olczak7 Although there was not much difference between NV and CP, the composition of N-glycans in PC changed markedly. Tri- and tetra-antennary N-glycans containing a Lewis X-type Fuc (peaks 5* and 8*) significantly increased in PC.

LC-ESI MS analysis of haptoglobin sialo-glycopeptides at sites 1 and 4

To identify the site responsible for the increase in N-glycans containing a Lewis X-type Fuc, site-specific analysis of haptoglobin N-glycans was performed in patients with PC, CP and in NV. The tryptic peptide mixture after reduction and alkylation was separated by reverse-phase HPLC followed by ESI MS analysis. Digestion of glycopeptides with a combination of trypsin and lysylendopeptidase should, in theory, yield (Supplemental Fig. 1): Met179-Lsy202, including one N-glycan binding site (site 1: Asn184); Asn203-Lsy215, including two N-glycan binding sites (site 2 and site 3: Asn207 and Asn211); and, Val236-Lsy251, including one N-glycan binding site (site 4: Asn241). The results of these analyses are presented in Figure 3a. The representative data shown in Figure 3a is the result of the analysis of serum from a normal volunteer (NV 5). Many ions derived from peptides were detected in the base peak chromatogram (BPC, see glossary) shown in Figure 3a. The ions detected in the extracted ion chromatogram (EIC, see glossary) of MSMS at m/z 657.3, which is the mass number of NeuAc-Gal-GlcNAc, indicated the presence of glycopeptides. Three robust peaks were detected at 40, 61 and 68 min. EIC of the MS scans at the mass number for each site containing a disialo-biantennary N-glycan was used to find the elution time for the glycopeptide of each site, because the majority of the N-glycans on haptoglobin were disialo-biantennary N-glycans. The ion at m/z 1221.8–1222.8 represents [M+4H]4+ of the glycopeptide containing site 1 with a disialo-biantennary N-glycan. The ion at m/z 1467.8–1468.8 represents [M+4H]4+ of the glycopeptide containing sites 2–3 with two disialo-biantennary N-glycans. The ion at m/z 1333.9–1334.9 represents [M+3H]3+ of the glycopeptide containing site 4 with a disialo-biantennary N-glycan. In the three EIC of MS, single peaks were observed at 68 (site 1), 40 (site 2–3) and 61 min (site 4). CP and PC samples were analyzed in the same manner as the NV samples. Averaged MS spectra during 67–72 min of EIC of MS at m/z 1221.8–1222.8 (site 1) were performed. The results for NV5, CP4 and PC5 are shown in Figure 3b. The abbreviations for N-glycan structures in the glycopeptide are summarized in Figure 4. For example, the peptide containing the tri-antennary N-glycan with two NeuAc residues and one Fuc residue is represented as 3-N2-F1. The first numeral indicates the branch number (tri-antennary in this case), N2 indicates the presence of two NeuAc residues, and F1 indicates the presence of one Fuc residue. “0” means the absence of both Fuc and NeuAc. Eight glycopeptides (2-N1, 2-N1-F1, 2-N2, 2-N2-F1, 3-N2, 3-N2-F1, 3-N3 and 3-N3-F1) were detected as quadruply charged ions-proton adducts- at site 1 in NV5. Although triply charged ions were also observed in the averaged MS spectra (data not shown), quadruply charged ions were selected as target ions for relative quantification of the highest ionic intensity. In Figure 3b for site 1, 2-N2-F1 in CP4 increased in comparison to NV5. In the case of PC5, allN-glycans containing Fuc increased.

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Figure 3. LC-ESI MS analysis of sialo-glycopeptides for sites 1 and 4 on haptoglobin. (a) BPC (MS+MSMS), EIC at m/z 657.3 (MSMS), EIC at m/z 1221.8–1222.8 (MS), EIC at m/z 1467.8–1468.8 (MS), and EIC at m/z 1333.9–1334.9 (MS) obtained from the analysis of NV5. Scan range for MS: m/z 400–3,000, for MSMS: m/z 200–3,000. EIC at m/z 657.3 suggested the presence of sialo-glycopeptides. EIC at m/z 1221.8–1222.8, EIC at m/z 1467.8–1468.8 and EIC at m/z 1333.9–1334.9 were indicative of: glycopeptides having 2-N2 at site 1; glycopeptide containing both site 2 and 3 with two 2-N2; and glycopeptides having 2-N2 at site 4, respectively. B, averaged MS spectra during 67–72 min of EIC at m/z 1221.8–1222.8 of MS (site 1) derived from NV5, CP4 and PC 5. Peak abbreviations and structures are summarized in Figure 4.

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Figure 4. Abbreviations, structures and calculated mass numbers for sialo- or asialo-glycopeptides detected in our study. Sialo-glycopeptides which include site 1 or 4 were calculated as quadruply and triply charged ions, respectively. Asialo-glycopeptides which include site 2 or 3 were calculated as doubly charged ions.

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Concerning site 4, the spectra during 59–64 min of EIC of MS at m/z 1333.9–1334.9 from all samples were also averaged (averaged MS spectra not shown). The relative abundances of sialo-N-glycan peptides in sites 1 and 4 in all samples were calculated as described in Material and methods section and the results are shown in Figure 6. Concerning sites 2 and 3, sialo-glycopeptide (Asn203-Lsy215), which includes sites 2 and 3, was strongly detected by ESI MS (spectrum not shown). To find each distribution pattern of the N-glycans in site 2 and site 3, sequential digestion with endoprotease Glu-C was carried out, and glycopeptide (Asn203-Glu210, including site 2) and glycopeptide (Asn211-Lsy215, including site 3) were separated. But, unfortunately, the separated glycopeptides were barely detectable. This phenomenon may be due to overlapping of the nonglycosylated peptides.

LC-ESI MS analysis of haptoglobin asialo-glycopeptides at sites 2 and 3

To detect each glycopeptide which include sites 2 and site 3, glycopeptides from tryptic digests were enriched using affinity separation by partitioning with Sepharose CL4B, according to the method of Wada et al.23 Furthermore, sialic acids were removed from the enriched glycopeptides using acidic treatment to reduce the complicated heterogeneity of the sialic acids. The samples including asialo-glycopeptides were digested with endoprotease Glu-C. In theory, digestion of asialo-glycopeptides with endoprotease Glu-C should yield (Supplemental Fig. 1): Met179-Glu194, including one N-glycan binding site (site 1: Asn184); Asn203-Glu210, including one N-glycan binding site (site 2: Asn207); Asn211-Lsy215, including one N-glycan binding site (site 3: Asn211); and Val236-Lsy251, including one N-glycan binding site (site 4: Asn241). The sample was separated by reverse-phase HPLC and the eluate was analyzed using ESI MS. The representative data shown in Figure 5a are the result of analysis of the serum from a normal volunteer (NV 5). Several peaks derived from glycopeptides and other peptides were detected by BPC in Figure 5a. Sepharose CL4B treatment reduced the peptides that interfered with ionization of the glycopeptides (compare with BPC in Fig. 3a). The ions detected in EIC of MSMS at m/z 366.3, which is the mass number of Gal-GlcNAc, indicated the presence of glycopeptides. EIC of MS scans at the mass number corresponding to the peptide of each site containing an asialo-biantennary N-glycan was selected to find the elution time of the glycopeptide at each site. The ion at m/z 1298.4–1299.4 represents [M+2H]2+ of the glycopeptide containing site 2 with an asialo-biantennary N-glycan. The ion at m/z 1063.8–1064.8 represents [M+2H]2+ of the glycopeptide containing site 3 with an asialo-biantennary N-glycan. In the two EIC of MS, single peaks were observed at 52 (site 2) and 5 min (site 3). CP and PC samples were analyzed in the same manner as the NV samples. MS spectra during 3–7 min of EIC of MS at m/z 1063.8–1064.8 (site 3) were averaged; the results of NV5, CP4 and PC5 are shown in Figure 5b. The abbreviations for N-glycan structures in the glycopeptide are summarized in Figure 4. Six types of glycopeptides (2-0, 2-F1, 3-0, 3-F1, 4-0 and 4-F1) were detected as doubly charged ions-proton adducts-at site 3 in NV5. There was not much difference between CP4 and NV5. In contrast, N-glycans containing a Fuc (2-F1, 3-F1 and 4-F1) dramatically increased in PC5. Moreover, tetra-antennary N-glycans with two Fuc (4-F2) were observed only in PC samples. The results obtained from all samples are summarized in Figure 6.

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Figure 5. LC-ESI MS analysis of asialo-glycopeptides for sites 2 and 3 on haptoglobin. (a) BPC (MS+MSMS), EIC at m/z 366.3 (MSMS), EIC at m/z 1298.4–1299.4 (MS) and EIC at m/z 1063.8–1064.8 (MS), obtained from the analysis of NV5. Scan range for MS: m/z 400–3,000, for MSMS: m/z 200–3000. EIC at m/z 366.3 suggested the presence of asialo-glycopeptides. EIC at m/z 1298.4–1299.4 and EIC at m/z 1063.8–1064.8 were indicative of glycopeptides having 2-0 at site 2 and site 3, respectively. (b) averaged MS spectra during 3–8 min of EIC at m/z 1063.8–1064.8 of MS (site 3), derived from NV5, CP4 and PC 5. Peak abbreviations and structures are summarized in Figure 4.

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Figure 6. Relative abundance (%) of glycopeptide glycoforms, including the N-glycan binding site of haptoglobin from sera of patients with pancreatic cancer, chronic pancreatitis and sera of old normal volunteers [over 60, NV(>60)] and young normal volunteers (under 40 NV [<40)]. The relative abundance was calculated based on the signal intensities of corresponding glycopeptides obtained from the results presented in Figures 3 and 5. The error bar represents the standard deviation (S.D.) calculated from each type of sample. The abbreviations in this figure are summarized in Figure 4. To compare 4 groups [NV (<40), NV (>60), CP and PC] nonrepeated measured ANOVA (two-tail) was used, and then each groups of two was compared by Bonferroni correction (two-tail). The annotations with asterisks in this figure indicate p < 0.05, and those without an asterisk indicate p < 0.01.

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Concerning site 4, observation of asialo-glycopeptides at this site was attempted. All types of N-glycan structures detected in the analysis of sialo-glycopeptides were observed at site 4, with the exception of the peptide having an N-glycan (2-F1) (data not shown). The triply charged ion for 2-F1 (m/z 1188.6) was hidden by a doubly charged ion derived from another unknown peptide. Therefore, the data for sialo-glycopeptides at sites 1 and 4, and for asialo-glycopeptides at sites 2 and 3 were used for comparison of relative abundance.

Identification of Lewis X structure and development of a novel tumor marker for pancreatic cancer

Average relative abundances (%) of N-glycans were calculated for patients with PC (n = 5), CP (n = 5), and for young NV (n = 5) and old normal volunteer (n = 3), and they were graphed with error bars representing standard deviations (Fig. 6). Site 3 was a unique N-glycan binding site because of the branching patterns and fucosylation. Concerning young NV samples, the relative abundances of 2-0 at sites 1–4 were 68.0%, 72.8%, 31.2% and 75.3%; the relative abundances of 3-0 at sites 1–4 were 26.8%, 22.5%, 42.8% and 17.6%. Tri-antennary N-glycans (3-0) were the major N-glycan for site 3 only, although it has been reported that the major haptoglobin N-glycan is bi-antennary N-glycan (75%).7 Highly branched N-glycans (3-0 and 4-0) were abundant at site 3; therefore, fucosylated N-glycans (3-F1 and 4-F1) could increase at site 3.27 The distribution of N-glycan structure types of at sites 1–4 were similar among PC, CP and NV. We also analyzed the N-glycan structures of 3 cases of old NV who were more than 60-year-old in order to match age of CP and PC samples. There was no significant difference between old NV (over 60-year-old) and young NV (under 40-year-old) except 4F-1 on site 4, although fucosylated N-glycans from old NV exhibited an upward trend in comparison with young NV. The relative abundance of fucosylated N-glycans from old NV was very similar to that from CP, because the patients with CP determined in our study were also old persons (55- to 81-year-old). In contrast, significant increases in fucosylation, such as 3-F1 on site 1, 2-F1 on site 2, 3-F1 and 4-F1 on site 3, were observed only in PC samples, compared to the others type of samples (young NV, old NV or CP). These 3-F1 on site 1 and site 3 significantly increased with a concomitant decrease in 3-0. On the basis of the results of analyses of PA-labeled N-glycans with α1-3/4 fucosidase and β1-4 galactosidase, the increased 3-F1 had N-glycans with Lewis X-type Fuc. This change was most likely due to the formation of Lewis X-type Fuc as a result of fucosyltransferase activity.

Moreover, it should be noted that tetra-antennary N-glycan with two Fuc (4-F2) was observed at site 3 only in PC samples (3.2%). MSMS analysis for the glycopeptide (4-F2) was performed and the result is shown in Figure 7. The fragment ion at m/z 659.8 represents [M+H]1+ of Gal-GlcNAc with two Fuc residues. The structure is suggested to be the Lewis Y-type (Fucα1-2Galβ1-4GlcNAcα1-3Fuc) according to the report by Kim et al.28

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Figure 7. MSMS spectrum of ion at m/z 1575.6 represented [M+2H]+2 of the glycopeptide which contains site 3 with 4-F2. The data present MSMS analysis of asialo-glycopeptides derived from haptoglobin in pancreatic cancer (PC5) after digestion with trypsin and endoprotease Glu-C.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Glossary
  7. References
  8. Supporting Information

Ectopic production of haptoglobin has received attention as a novel tumor marker for several cancers.13–15 However, most haptoglobin is produced in the liver. Glycan analysis is a promising technique to identify the organ that produces haptoglobin, because each type of fucosyltransferase is specifically expressed in each organ. Our previous study demonstrated that fucosylated haptoglobin increased in the sera of patients with PC compared to other cancer patients.4 To be clinically useful, it is important to clarify whether fucosylated haptoglobin is increased directly by the PC or indirectly because of inflammation of the pancreas. Interestingly, the haptoglobin N-glycan structures at site 3 were unique compared to the structures at the other sites. While small amounts of fucosylated glycans could be detected at site 3, derived from NV and patients with CP, the 4-F2 structure was uniquely observed only in patients with PC.

In general, it has not been clarified how glycosyltransferase recognizes a specific N-glycan site in proteins. Several glycosyltransferases involved in branch formation, as well as fucosyltransferases, may recognize site 3 via unique amino acid sequences around this site or by the conformational structure of haptoglobin. The reason for increased fucosylation at site 3 in individuals with PC may be induction of glycosyltransferases in PC cells themselves or in the liver secondary to PC. Glycan changes in haptoglobin have been reported in hepatocellular carcinoma.14, 29 These changes could be caused by up-/down-regulation of glycosyltransferases in the liver and in hepatocellular carcinomas. In the case of PC, the diseased pancreas may induce a factor capable of producing fucosylated haptoglobin.4 This hypothesis is based on the observation of fucosylated haptoglobin in the sera of patients with tumor-forming pancreatitis (data not shown). In addition, obstruction of the pancreatic duct may induce this factor, which should be identified in future studies.

To make a practical application of 4-F2 at site 3 as a tumor marker for PC, a simpler method with high throughput would be required. The procedures we used in our study might not be suitable for clinical application directly. Although ELISA systems to detect Lewis Y and/or X structures on haptoglobin are more useful, the sensitivity might not be high enough.30 Site-specific analysis of N-glycans on transferrin has been incorporated into screening for congenital disorders of glycosylation (CDG) by MALDI-TOF MS in Japan.31 Tumor marker 4-F2 at site 3 on haptoglobin could easily be detected using the same method that is used when screening for CDG. If site-specific glycan analyses of glycopeptides were possible, using crude serum samples, it would be a powerful tool as a novel glyco-marker. This analytical system is undergoing in our laboratory in next our study.

Glossary

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Glossary
  7. References
  8. Supporting Information

Glycomics, an analogous term to genomics and proteomics, is the comprehensive study of glycomes, including genetic, physiologic, and other aspects. The term glycomics was formed to follow the naming convention established by genomics (which deals with genes) and proteomics (which deals with proteins). The identity of the entirety of carbohydrates in an organism is thus collectively referred to as the glycome. LC-ESI MS, a mass spectrometric ionization method based on the ionization of molecules in solution introduced from LC in an electric field at atmospheric pressure. BPC, Base Peak Chromatogram: a chromatogram in which the y-axis for each point on the chromatogram represents the intensity of the most abundant ion in the mass spectrum. EIC, Extracted Ion Chromatogram: graphical representation of the abundance of one (individual) or more (summed) selected ion(s) versus retention time.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Glossary
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Glossary
  7. References
  8. Supporting Information

This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0020-7136/suppmat .

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