**2.1 Materials and methods**

#### **2.1.1 Enrichments of lubricin from synovial fluid**

Synovial fluid samples from RA patients were collected during therapeutic joint aspiration at the Rheumatology Clinic, Sahlgrenska University Hospital (Gothenburg, Sweden). All patients gave informed consent and the procedure was approved by the Ethics Committee

Glycoproteomics of Lubricin-Implication

2010) and bovine fetuin (Sigma-Aldrich).

**2.1.4 Proteomic characterization of lubricin** 

manually.

electrophoresis.

identification.

loading buffer with boiling.

**2.1.3 Glycomic analysis of lubricin** *O***-glycan structures** 

of Important Biological Glyco- and Peptide-Epitopes in Synovial Fluid 135

Denmark). Mouse anti-3'-sulfo-Lea was kindly provided by Dr. Antoon J Ligtenberg (Department of Oral Biochemistry, University of Amsterdam, The Netherlands). The other anti-carbohydrate antibodies tested in this study were mouse anti- sialyl Lewis x (sLex , CD15s, or mAb CSLEX1, BD Biosciences, Franklin Lakes, NJ, USA), MECA-79 (CD62L, BD Biosciences), mouse anti-chondroitin sulfate (mAb CS56, Sigma-Aldrich), mouse anti-sLea (mAb CA19-9, Abcam, Cambridge, MA, USA), and mouse anti-Leb (mAb 2-25LE, Abcam). Biotinylated lectins were also used in this study including ConA (concanavalin A), MAA-I (*Maackia amurensis* lectin I), WGA (succinylated wheat germ agglutinin), and AAL (*Aleuria aurantia* lectin), all from Vector (Vector Laboratories, Burlingame, CA, USA). Biotinylated PNA (*Arachis hypogaea* lectin) and HAA (*Helix aspersa* agglutinin) were from Sigma-Aldrich. Secondary antibodies used were HRP conjugated rabbit anti-mouse IgG, HRP conjugated rabbit anti-rat IgG+IgM (Jackson ImmunoResearch, Suffolk, UK). For biotin labeled lectin, HRP conjugated streptavidin (Vector Laboratories) was used. The immunoassay was validated and optimized with human salivary mucin as described previously (Issa et al.,

*O*-linked oligosaccharides were released by reductive β-elimination (Schulz et al., 2002). In brief, membrane strips were incubated with 50 µL of 1.0 M NaBH4 in 100 mM NaOH for 16 hours at 50oC. Reactions were quenched with 1 µL of glacial acetic acid. Samples were then desalted and dried for capillary graphitized carbon LC-MS and LC-MS2 in negative ion mode using an LTQ Ion Trap (Thermo Scientific). Oligosaccharides were identified from their MS2 spectra using the UniCarb-DB (2011 version) (Hayes et al., 2011) and validated

For deglycosylation, the reduced and alkylated samples (20 µg) were incubated with 5 mU of sialidase A (Prozyme Inc., Oxford, UK) to remove sialic acids at 37oC for 16 hours. An aliquot of sample was also treated with 2.5 mU *O*-glycanase (endo-α-*N*acetylgalactosaminidase, Prozyme Inc.), which cleaves core 1 type *O*-linked glycan on glycoproteins and glycopeptides, at 37oC for 16 hours. The reaction was stopped by heating at 95oC for 10 minutes in SDS-loading buffer, and enzymes were removed by

Coomassie blue-stained protein bands in Tris/acetate NuPAGE gels were excised and digested with trypsin as described (Kuster et al., 1997). The resultant peptides were subjected to nano-LC-MS2 using LTQ-Orbitrap XL mass spectrometer (Thermo Scientific). Peptide MS/MS spectra were searched against UniProt and NCBI human protein databases using GPM (Zhang et al., 2011) and Mascot software (v.2.2.04, Matrix Science Inc., MA, USA). Only peptides with a mass deviation lower than 10 ppm were accepted and two peptide sequences with manual inspection were used for positive protein

Enriched synovial lubricin sample was also treated with *O*-sialoglycoprotein endopeptidase from *Pasteurella haemolytica* (Cedarlane Laboratories, Ontario, Canada)*.* 5 µg of samples were incubated with endopeptidase in PBS (pH 7.4) at 37oC; and small aliquots were taken out at 0, 3, 6, and 16 hours. The reaction was stopped by adding SDS-

of Sahlgrenska University Hospital. All patients fulfilled the American College of Rheumatology 1987 revised criteria for RA (Arnett et al., 1988). The samples were clarified by centrifugation at 10,000 g for 10 minutes and stored at -80oC before use. The acidic proteins were purified as previously described (Estrella et al., 2010). In brief, synovial fluid sample was diluted with washing buffer (250 mM NaCl, 20 mM Tris-HCl, 10 mM EDTA, pH 7.5) before applying to 1 mL DEAE FF Hi-Trap column (GE Healthcare, Uppsala, Sweden). Enriched glycoproteins were eluted with 1 M NaCl in washing buffer. Lubricin containing fractions were precipitated with 80% ethanol for 16 hours at -20oC. The precipitate was collected by centrifugation at 12,100 g for 20 minutes and re-suspended in phosphate buffered saline (PBS) at pH 7.4 after air-dry. Protein concentration was determined by BCA protein assay kit (Thermo Scientific, San Jose, CA, USA) using bovine serum albumin (BSA) as standard.

For sandwich ELISA, 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated with rabbit anti-lubricin polyclonal antibody (Thermo Scientific) in 0.1 M carbonate buffer, pH 9.5, at a concentration of 2 ng/mL and 4oC overnight. The plates were then blocked with 1% BSA in TBS-T buffer (Tris-buffered saline with 0.01% Tween 20) at 37oC for 1 hour. Fractions were diluted with 1% BSA in TBS-T buffer, added to each well, and incubated at 37oC for 1 hour. After washing with TBS-T buffer, diluted anti-lubricin mouse monoclonal antibody (Pfizer Research, Cambridge, MA, USA) was added to each well and incubated at 37oC for 1 hour. After extensive wash, horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin antibody (DakoCytomation, Glostrup, Denmark) was added. Color was developed by using tetramethyl benzidine (TMB) buffer (Sigma-Aldrich, St. Louis, MO, USA) as substrate for 10 minutes at room temperature; and reaction was stopped by adding 1 M H2SO4. The optical density was measured at 450 nm wavelength.

#### **2.1.2 Western blot and lectin immunoblot**

Samples were reduced with 10 mM dithiothreitol (Sigma-Aldrich) and denatured by heating at 95oC for 20 minutes, and then alkylated with 25 mM iodoacetamide (Sigma-Aldrich) for 1 hour at room temperature in the dark. As for non-reduced samples, protein samples were mixed with SDS loading buffer and heated at 95oC for 20 minutes. The samples were then applied to a 3-8% Tris/acetate NuPAGE gel (Invitrogen AB, Stockholm, Sweden) or agarosepolyacrylamide gel (AgPAGE) which was made as described previously (Schulz et al., 2002). The samples were blotted onto PVDF membrane (Immobilon P, Millipore, Billerica, MA, USA) using a semi-dry blotter (Bio-Rad, Hercules, CA, USA).

PVDF membranes were blocked for 1-2 hour at room temperature in TBS-T buffer containing 1% BSA at room temperature on a shaker, and then incubated with primary antibodies or biotinylated lectins at the appropriate concentration diluted in TBS-T buffer with 1% BSA for 1 hour at room temperature on a shaker. After washing the blots three times with TBS-T, blots were incubated with secondary antibodies or streptavidin labeled with HRP for 1 hour at room temperature. After wash, bound antibodies and lectins were detected by using SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific).

Anti-carbohydrate antibodies used in study including anti-T antigen (mAb 3C9), anti-Tn antigen (mAb 5F4 and 1E3), and anti-sialyl Tn (mAb TKH2 and 3F1), which were kindly provided by Prof. Henrick Clausen and Prof. Ola Blixt (University of Copenhagen,

of Sahlgrenska University Hospital. All patients fulfilled the American College of Rheumatology 1987 revised criteria for RA (Arnett et al., 1988). The samples were clarified by centrifugation at 10,000 g for 10 minutes and stored at -80oC before use. The acidic proteins were purified as previously described (Estrella et al., 2010). In brief, synovial fluid sample was diluted with washing buffer (250 mM NaCl, 20 mM Tris-HCl, 10 mM EDTA, pH 7.5) before applying to 1 mL DEAE FF Hi-Trap column (GE Healthcare, Uppsala, Sweden). Enriched glycoproteins were eluted with 1 M NaCl in washing buffer. Lubricin containing fractions were precipitated with 80% ethanol for 16 hours at -20oC. The precipitate was collected by centrifugation at 12,100 g for 20 minutes and re-suspended in phosphate buffered saline (PBS) at pH 7.4 after air-dry. Protein concentration was determined by BCA protein assay kit (Thermo Scientific, San Jose, CA, USA) using bovine serum albumin (BSA)

For sandwich ELISA, 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated with rabbit anti-lubricin polyclonal antibody (Thermo Scientific) in 0.1 M carbonate buffer, pH 9.5, at a concentration of 2 ng/mL and 4oC overnight. The plates were then blocked with 1% BSA in TBS-T buffer (Tris-buffered saline with 0.01% Tween 20) at 37oC for 1 hour. Fractions were diluted with 1% BSA in TBS-T buffer, added to each well, and incubated at 37oC for 1 hour. After washing with TBS-T buffer, diluted anti-lubricin mouse monoclonal antibody (Pfizer Research, Cambridge, MA, USA) was added to each well and incubated at 37oC for 1 hour. After extensive wash, horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin antibody (DakoCytomation, Glostrup, Denmark) was added. Color was developed by using tetramethyl benzidine (TMB) buffer (Sigma-Aldrich, St. Louis, MO, USA) as substrate for 10 minutes at room temperature; and reaction was stopped by adding 1 M H2SO4. The optical density was measured at 450 nm

Samples were reduced with 10 mM dithiothreitol (Sigma-Aldrich) and denatured by heating at 95oC for 20 minutes, and then alkylated with 25 mM iodoacetamide (Sigma-Aldrich) for 1 hour at room temperature in the dark. As for non-reduced samples, protein samples were mixed with SDS loading buffer and heated at 95oC for 20 minutes. The samples were then applied to a 3-8% Tris/acetate NuPAGE gel (Invitrogen AB, Stockholm, Sweden) or agarosepolyacrylamide gel (AgPAGE) which was made as described previously (Schulz et al., 2002). The samples were blotted onto PVDF membrane (Immobilon P, Millipore, Billerica, MA,

PVDF membranes were blocked for 1-2 hour at room temperature in TBS-T buffer containing 1% BSA at room temperature on a shaker, and then incubated with primary antibodies or biotinylated lectins at the appropriate concentration diluted in TBS-T buffer with 1% BSA for 1 hour at room temperature on a shaker. After washing the blots three times with TBS-T, blots were incubated with secondary antibodies or streptavidin labeled with HRP for 1 hour at room temperature. After wash, bound antibodies and lectins were detected by using SuperSignal West Femto maximum sensitivity substrate (Thermo

Anti-carbohydrate antibodies used in study including anti-T antigen (mAb 3C9), anti-Tn antigen (mAb 5F4 and 1E3), and anti-sialyl Tn (mAb TKH2 and 3F1), which were kindly provided by Prof. Henrick Clausen and Prof. Ola Blixt (University of Copenhagen,

as standard.

wavelength.

Scientific).

**2.1.2 Western blot and lectin immunoblot** 

USA) using a semi-dry blotter (Bio-Rad, Hercules, CA, USA).

Denmark). Mouse anti-3'-sulfo-Lea was kindly provided by Dr. Antoon J Ligtenberg (Department of Oral Biochemistry, University of Amsterdam, The Netherlands). The other anti-carbohydrate antibodies tested in this study were mouse anti- sialyl Lewis x (sLex , CD15s, or mAb CSLEX1, BD Biosciences, Franklin Lakes, NJ, USA), MECA-79 (CD62L, BD Biosciences), mouse anti-chondroitin sulfate (mAb CS56, Sigma-Aldrich), mouse anti-sLea (mAb CA19-9, Abcam, Cambridge, MA, USA), and mouse anti-Leb (mAb 2-25LE, Abcam). Biotinylated lectins were also used in this study including ConA (concanavalin A), MAA-I (*Maackia amurensis* lectin I), WGA (succinylated wheat germ agglutinin), and AAL (*Aleuria aurantia* lectin), all from Vector (Vector Laboratories, Burlingame, CA, USA). Biotinylated PNA (*Arachis hypogaea* lectin) and HAA (*Helix aspersa* agglutinin) were from Sigma-Aldrich. Secondary antibodies used were HRP conjugated rabbit anti-mouse IgG, HRP conjugated rabbit anti-rat IgG+IgM (Jackson ImmunoResearch, Suffolk, UK). For biotin labeled lectin, HRP conjugated streptavidin (Vector Laboratories) was used. The immunoassay was validated and optimized with human salivary mucin as described previously (Issa et al., 2010) and bovine fetuin (Sigma-Aldrich).

### **2.1.3 Glycomic analysis of lubricin** *O***-glycan structures**

*O*-linked oligosaccharides were released by reductive β-elimination (Schulz et al., 2002). In brief, membrane strips were incubated with 50 µL of 1.0 M NaBH4 in 100 mM NaOH for 16 hours at 50oC. Reactions were quenched with 1 µL of glacial acetic acid. Samples were then desalted and dried for capillary graphitized carbon LC-MS and LC-MS2 in negative ion mode using an LTQ Ion Trap (Thermo Scientific). Oligosaccharides were identified from their MS2 spectra using the UniCarb-DB (2011 version) (Hayes et al., 2011) and validated manually.

For deglycosylation, the reduced and alkylated samples (20 µg) were incubated with 5 mU of sialidase A (Prozyme Inc., Oxford, UK) to remove sialic acids at 37oC for 16 hours. An aliquot of sample was also treated with 2.5 mU *O*-glycanase (endo-α-*N*acetylgalactosaminidase, Prozyme Inc.), which cleaves core 1 type *O*-linked glycan on glycoproteins and glycopeptides, at 37oC for 16 hours. The reaction was stopped by heating at 95oC for 10 minutes in SDS-loading buffer, and enzymes were removed by electrophoresis.

## **2.1.4 Proteomic characterization of lubricin**

Coomassie blue-stained protein bands in Tris/acetate NuPAGE gels were excised and digested with trypsin as described (Kuster et al., 1997). The resultant peptides were subjected to nano-LC-MS2 using LTQ-Orbitrap XL mass spectrometer (Thermo Scientific). Peptide MS/MS spectra were searched against UniProt and NCBI human protein databases using GPM (Zhang et al., 2011) and Mascot software (v.2.2.04, Matrix Science Inc., MA, USA). Only peptides with a mass deviation lower than 10 ppm were accepted and two peptide sequences with manual inspection were used for positive protein identification.

Enriched synovial lubricin sample was also treated with *O*-sialoglycoprotein endopeptidase from *Pasteurella haemolytica* (Cedarlane Laboratories, Ontario, Canada)*.* 5 µg of samples were incubated with endopeptidase in PBS (pH 7.4) at 37oC; and small aliquots were taken out at 0, 3, 6, and 16 hours. The reaction was stopped by adding SDSloading buffer with boiling.

Glycoproteomics of Lubricin-Implication

recognized both peripheral and core fucosylated glycans.

of Important Biological Glyco- and Peptide-Epitopes in Synovial Fluid 137

hybrid type *N*-glycans, suggesting *N*-glycans were absent on lubricin or in very low amounts. The same negative results were obtained with *Aleuria aurantia* lectin (AAL), which

Fig. 2. Glyco-epitope on lubricin analyzed by immunoblot. Reduced and alkylated enriched lubricin sample (6 µg/lane) was separated by 3-8% Tris/acetate NuPAGE and blotted to PVDF membrane. Strips were incubated with various lectins or anti-carbohydrate antibodies after blocking with 1% BSA in TBS-T buffer. After incubating with HRP

conjugated corresponding secondary antibodies and streptavidin, bands were developed by SuperSignal West Femto maximum sensitivity substrate. CB, Coomassie blue stained gel; PNA, peanut agglutinin; WGA, wheat germ agglutinin; AAL, *Aleuria aurantia* lectin; HAA,

When synovial lubricin was investigated by anti-carbohydrate antibodies (Fig. 2), lubricin was suggested to have T antigen and sialyl Lewis x (sLex, structure in Fig. 3C). Western blot showed (data not presented) that lubricin was negative for anti-carbohydrate antibodies specific to chondroitin sulfate (mAb CS56), sLea [NeuAcα2,3Galβ1,3(Fucα1,4)GlcNAcβ1-], (mAb CA19-9), 3'-sulfo-Lea [NeuAcα2,3Gal(3S)β1,3(Fucα1,4)GlcNAc-], Leb [Fucα1,2Galβ1,3(Fucα1,4)GlcNAcβ1- ], (mAb 2-25LE], MECA-79 epitopes, Tn antigen (mAb 5F4 and 1E3), and sialyl Tn antigen [NeuAcα2,6GalNAc-*O*-Ser/Thr], (mAb TKH2 and 3F1). Results obtained from anticarbohydrate antibodies agree with results from the lectin immunoblot except for lectin HAA. Together with the lectin immunoblot, these results demonstrated synovial lubricin had sialylated glycans, core 1 *O*-glycan and peripheral sLex epitope. In order to reveal the identity of the sLex containing *O*-glycans and identify other glycan epitopes not recognized by the

Though immunoassay with lectins and anti-carbohydrate antibodies is convenient to detect glyco-epitopes, inner structural information is commonly scant. Furthermore, some glyco-

*Helix aspersa* agglutinin; Anti-sLex, sialyl Lewis x-specific antibody; T, T antigen,

Galβ1,3GalNAc-*O*-Ser/Thr; Tn, Tn antigen, GalNAc-*O*-Ser/Thr.

antibodies and lectin used, additional experiments were carried out.

**2.2.3 Glyco-epitope on synovial lubricin verified by LC-MS** 
