Selected Reaction Monitoring to Differentiate and Relatively Quantitate Isomers of Sulfated and Unsulfated Core 1 O-Glycans from Salivary MUC7 Protein in Rheumatoid Arthritis
2013; Elsevier BV; Volume: 12; Issue: 4 Linguagem: Inglês
10.1074/mcp.m113.028878
ISSN1535-9484
AutoresSarah A. Flowers, Liaqat Ali, Catherine Lane, Magnus Olin, Niclas G. Karlsson,
Tópico(s)Galectins and Cancer Biology
ResumoRheumatoid arthritis is a common and debilitating systemic inflammatory condition affecting up to 1% of the world's population. This study aimed to investigate the immunological significance of O-glycans in chronic arthritis at a local and systemic level. O-Glycans released from synovial glycoproteins during acute and chronic arthritic conditions were compared and immune-reactive glycans identified. The sulfated core 1 O-glycan (Galβ1–3GalNAcol) was immune reactive, showing a different isomeric profile in the two conditions. From acute reactive arthritis, three isomers could be sequenced, but in patients with chronic rheumatoid arthritis, only a single 3-Gal sulfate-linked isomer could be identified. The systemic significance of this glycan epitope was investigated using the salivary mucin MUC7 in patients with rheumatoid arthritis and normal controls. To analyze this low abundance glycan, a selected reaction monitoring (SRM) method was developed to differentiate and relatively quantitate the core 1 O-glycan and the sulfated core 1 O-glycan Gal- and GalNAc-linked isomers. The acquisition of highly sensitive full scan linear ion trap MS/MS spectra in addition to quantitative SRM data allowed the 3- and 6-linked Gal isomers to be differentiated. The method was used to relatively quantitate the core 1 glycans from MUC7 to identify any systemic changes in this carbohydrate epitope. A statistically significant increase in sulfation was identified in salivary MUC7 from rheumatoid arthritis patients. This suggests a potential role for this epitope in chronic inflammation. This study was able to develop an SRM approach to specifically identify and relatively quantitate sulfated core 1 isomers and the unsulfated structure. The expansion of this method may afford an avenue for the high throughput investigation of O-glycans. Rheumatoid arthritis is a common and debilitating systemic inflammatory condition affecting up to 1% of the world's population. This study aimed to investigate the immunological significance of O-glycans in chronic arthritis at a local and systemic level. O-Glycans released from synovial glycoproteins during acute and chronic arthritic conditions were compared and immune-reactive glycans identified. The sulfated core 1 O-glycan (Galβ1–3GalNAcol) was immune reactive, showing a different isomeric profile in the two conditions. From acute reactive arthritis, three isomers could be sequenced, but in patients with chronic rheumatoid arthritis, only a single 3-Gal sulfate-linked isomer could be identified. The systemic significance of this glycan epitope was investigated using the salivary mucin MUC7 in patients with rheumatoid arthritis and normal controls. To analyze this low abundance glycan, a selected reaction monitoring (SRM) method was developed to differentiate and relatively quantitate the core 1 O-glycan and the sulfated core 1 O-glycan Gal- and GalNAc-linked isomers. The acquisition of highly sensitive full scan linear ion trap MS/MS spectra in addition to quantitative SRM data allowed the 3- and 6-linked Gal isomers to be differentiated. The method was used to relatively quantitate the core 1 glycans from MUC7 to identify any systemic changes in this carbohydrate epitope. A statistically significant increase in sulfation was identified in salivary MUC7 from rheumatoid arthritis patients. This suggests a potential role for this epitope in chronic inflammation. This study was able to develop an SRM approach to specifically identify and relatively quantitate sulfated core 1 isomers and the unsulfated structure. The expansion of this method may afford an avenue for the high throughput investigation of O-glycans. Glycosylation is a prevalent and significant post-translational modification, exemplified by the existence of a number of congenital and genetic diseases; 64 gene defects have been identified that affect glycan biosynthesis (1Hennet T. Diseases of glycosylation beyond classical congenital disorders of glycosylation.Biochim. Biophys. Acta. 2012; 1820: 1306-1317Crossref PubMed Scopus (107) Google Scholar). This can result in quite devastating diseases, such as leukocyte-adhesion deficiency type II, which is caused by a loss of sialyl Lewis x production leading to mental retardation, or Wiskott-Aldrich syndrome, which results from problems with O-linked glycans in lymphocytes, leading to immunodeficiency (2Durand G. Seta N. Protein glycosylation and diseases: blood and urinary oligosaccharides as markers for diagnosis and therapeutic monitoring.Clin. Chem. 2000; 46: 795-805Crossref PubMed Scopus (211) Google Scholar). 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Role of sulfated O-glycans expressed by high endothelial venule-like vessels in pathogenesis of chronic inflammatory gastrointestinal diseases.Biol. Pharm. Bull. 2009; 32: 774-779Crossref PubMed Scopus (14) Google Scholar), and sulfated mucins have also been identified in patients with Helicobacter pylori infection (14Cohen M.C. Rúa E.C. Balcarce N. Drut R. Sulfomucins in Helicobacter pylori-associated chronic gastritis in children: is this incipient intestinal metaplasia?.J. Pediatr. Gastroenterol. Nutr. 2000; 31: 63-67Crossref PubMed Scopus (14) Google Scholar) and in severe ventilator-associated pneumonia (15Dennesen P. Veerman E. van Nieuw Amerongen A. Jacobs J. Kessels A. van der Keybus P. Ramsay G. van der Ven A. High levels of sulfated mucins in bronchoalveolar lavage fluid of ICU patients with ventilator-associated pneumonia.Intensive Care Med. 2003; 29: 715-719Crossref PubMed Scopus (15) Google Scholar). In this study, O-glycosylation changes were addressed in immunological arthritic patients. Arthritis is a large group of joint conditions, with the most common being rheumatoid arthritis (RA) 1The abbreviations used are:RArheumatoid arthritisEPIenhanced product ionSRMselected reaction monitoringPGCporous graphitized carbonPGMporcine gastric mucinReAreactive arthritisSDS-AgPAGESDS-agarosePAGESFsynovial fluidCEcollision energyDPdeclustering potentialCXPcollision cell exit potentialGalNAcolN-Acetylgalactosaminitol. 1The abbreviations used are:RArheumatoid arthritisEPIenhanced product ionSRMselected reaction monitoringPGCporous graphitized carbonPGMporcine gastric mucinReAreactive arthritisSDS-AgPAGESDS-agarosePAGESFsynovial fluidCEcollision energyDPdeclustering potentialCXPcollision cell exit potentialGalNAcolN-Acetylgalactosaminitol. and osteoarthritis. There are many other forms of arthritis, including the more acute forms such as allergic arthritis and reactive arthritis (ReA). These latter two are generally agreed to be caused by molecular mimicry, resulting in cross-reactive antibodies from gastrointestinal or genitourinary tract bacterial infections, as in ReA, or allergens (16Morris D. Inman R.D. Reactive arthritis: developments and challenges in diagnosis and treatment.Curr. Rheumatol. Rep. 2012; 14: 390-394Crossref PubMed Scopus (33) Google Scholar, 17Carter J.D. Treating reactive arthritis: insights for the clinician.Ther. Adv. Musculoskelet. Dis. 2010; 2: 45-54Crossref PubMed Scopus (12) Google Scholar). ReA onset is acute from 1 to 6 weeks post-infection, and usually lasts weeks to months, except for those 30–50% of patients that go on to form a chronic inflammatory response (17Carter J.D. Treating reactive arthritis: insights for the clinician.Ther. Adv. Musculoskelet. Dis. 2010; 2: 45-54Crossref PubMed Scopus (12) Google Scholar). RA is, however, a systemic chronic autoimmune inflammatory disease, affecting 0.5–1% of the world's population. It leads to an altered immune response and hyperproliferation of the synovial lining, causing debilitating joint pain and swelling (18Firestein G.S. Evolving concepts of rheumatoid arthritis.Nature. 2003; 423: 356-361Crossref PubMed Scopus (2785) Google Scholar, 19Gay S. Gay R.E. Koopman W.J. Molecular and cellular mechanisms of joint destruction in rheumatoid arthritis: two cellular mechanisms explain joint destruction?.Ann. Rheum. 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Human synovial lubricin expresses sialyl Lewis x determinant and has L-selectin ligand activity.J. Biol. Chem. 2012; 287: 35922-35933Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The level of sulfation correlated with the quantity of rheumatoid factor, indicating a role in chronic state inflammation. However, sulfation has also been indicated to participate in acute phase inflammation, including neutrophil migration (20Jin C. Ekwall A.K. Bylund J. Björkman L. Estrella R.P. Whitelock J.M. Eisler T. Bokarewa M. Karlsson N.G. Human synovial lubricin expresses sialyl Lewis x determinant and has L-selectin ligand activity.J. Biol. Chem. 2012; 287: 35922-35933Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 21Subramanian H. Grailer J.J. Ohlrich K.C. Rymaszewski A.L. Loppnow J.J. Kodera M. Conway R.M. Steeber D.A. Signaling through L-selectin mediates enhanced chemotaxis of lymphocyte subsets to secondary lymphoid tissue chemokine.J. Immunol. 2012; 188: 3223-3236Crossref PubMed Scopus (32) Google Scholar). Hence, in acute immunological joint conditions like ReA, there may be a different role for sulfation than has been suggested in chronic inflammatory joint disease. The low abundance of these potentially immunologically relevant sulfated oligosaccharides makes them challenging to study. Mass spectrometry has developed into the most accepted method for analysis of O-glycans (22Jensen P.H. Kolarich D. Packer N.H. Mucin-type O-glycosylation–putting the pieces together.FEBS J. 2010; 277: 81-94Crossref PubMed Scopus (184) Google Scholar, 23North S.J. Hitchen P.G. Haslam S.M. Dell A. Mass spectrometry in the analysis of N-linked and O-linked glycans.Curr. Opin. Struct. Biol. 2009; 19: 498-506Crossref PubMed Scopus (176) Google Scholar, 24Schulz B.L. Packer N.H. Karlsson N.G. Small-scale analysis of O-linked oligosaccharides from glycoproteins and mucins separated by gel electrophoresis.Anal. Chem. 2002; 74: 6088-6097Crossref PubMed Scopus (169) Google Scholar). The large scale analysis of O-glycans is inhibited by the laborious nature and the expertise required for this analysis, as well as the lack of standards to expedite the annotation process. Furthermore, the need to analyze low abundance glycans requires more sensitive and specific methods and relative quantitation to allow comparison of samples. Selected reaction monitoring (SRM), also known as multiple reaction monitoring, is just such a sensitive and specific quantitative mass spectrometric technique (25Boja E.S. Rodriguez H. Mass spectrometry-based targeted quantitative proteomics: achieving sensitive and reproducible detection of proteins.Proteomics. 2012; 12: 1093-1110Crossref PubMed Scopus (128) Google Scholar, 26Picotti P. Aebersold R. Selected reaction monitoring-based proteomics: workflows, potential, pitfalls, and future directions.Nat. Methods. 2012; 9: 555-566Crossref PubMed Scopus (991) Google Scholar). This method uses a triple quadrupole or triple quadrupole/linear ion trap hybrid mass spectrometer (27Le Blanc J.C. Hager J.W. Ilisiu A.M. Hunter C. Zhong F. Chu I. Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (Q TRAP) used for high sensitivity proteomics applications.Proteomics. 2003; 3: 859-869Crossref PubMed Scopus (127) Google Scholar, 28Hager J.W. Yves Le Blanc J.C. Product ion scanning using a Q-q-Q linear ion trap (Q TRAP) mass spectrometer.Rapid Commun. Mass Spectrom. 2003; 17: 1056-1064Crossref PubMed Scopus (132) Google Scholar), where the two mass analyzers are used for filtering. The first quadrupole, Q1, selects the parent mass of interest, which is then fragmented in Q2, and fragment ions analyzed in Q3 for specific ions. Specific transitions are used, i.e. pairs of parent and fragment masses that identify an ion of interest, making it highly specific for the target and sensitive due to the reduction in noise. The hybrid mass spectrometer gives the advantage of scanning MS/MS capabilities; this means when an SRM signal satisfies certain threshold criteria, a full scan linear ion trap MS/MS spectrum (enhanced product ion (EPI) spectrum) is triggered of the parent compound. This is referred to as the MIDAS (multiple reaction monitoring initiated detection and sequencing) methodology (29Anderson L. Hunter C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.Mol. Cell. Proteomics. 2006; 5: 573-588Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar). SRM methodologies are used extensively with small molecules for large scale screening purposes, for example the testing for up to 240 pesticides in food (30Zhang K. Wong J.W. Yang P. Hayward D.G. 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It has also become prevalent in the proteomics field on a large scale (26Picotti P. Aebersold R. Selected reaction monitoring-based proteomics: workflows, potential, pitfalls, and future directions.Nat. Methods. 2012; 9: 555-566Crossref PubMed Scopus (991) Google Scholar). SRM methodologies have been used successfully with glycans, although all have focused on N-glycans. Most have used glycopeptides, with methods developed for the quantitation of N-glycosylated peptides that have had their glycosylation sites removed (33Kim Y.J. Zaidi-Ainouch Z. Gallien S. Domon B. Mass spectrometry-based detection and quantification of plasma glycoproteins using selective reaction monitoring.Nat. Protoc. 2012; 7: 859-871Crossref PubMed Scopus (30) Google Scholar). Using oxonium ions, which are diagnostic or characteristic ions of glycoproteins, has been effective in identifying and quantifying glycopeptides. Less emphasis has been placed on the identification of specific transitions, instead focusing on compositional information of the identified glycans (34Song E. Pyreddy S. Mechref Y. Quantification of glycopeptides by multiple reaction monitoring liquid chromatography/tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2012; 26: 1941-1954Crossref PubMed Scopus (64) Google Scholar). This approach allows for the general identification of glycopeptides rather than identification of specific ones. A similar method has also been used to analyze variation in N-glycan site occupancy in glycopeptides using a standard indicator fragment ion for all glycans (35Gil G.C. Velander W.H. Van Cott K.E. N-Glycosylation microheterogeneity and site occupancy of an Asn-X-Cys sequon in plasma-derived and recombinant protein C.Proteomics. 2009; 9: 2555-2567Crossref PubMed Scopus (30) Google Scholar). Other methods have focused on specific sugar epitopes. Twenty five SRM transitions for 2-aminopyridine-labeled sialic acid, containing glycopeptides from enriched mouse serum, were successfully created using a fragment ion containing an innermost GlcNAc fragment and quantitatively used to compare normal and diabetic mice (36Kurogochi M. Matsushista T. Amano M. Furukawa J. Shinohara Y. Aoshima M. Nishimura S. Sialic acid-focused quantitative mouse serum glycoproteomics by multiple reaction monitoring assay.Mol. Cell. Proteomics. 2010; 9: 2354-2368Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). A method directed at identification of core-fucosylated proteins has also been developed. Simplified core-fucosylated peptides, containing the fucosyl-GlcNAc glycan, were enriched and used for SRM analysis. This proved to be an effective method, even if unable to differentiate glycoforms (37Zhao Y. Jia W. Wang J. Ying W. Zhang Y. Qian X. Fragmentation and site-specific quantification of core-fucosylated glycoprotein by multiple reaction monitoring-mass spectrometry.Anal. Chem. 2011; 83: 8802-8809Crossref PubMed Scopus (46) Google Scholar). Lectins, however, have been used to enrich for various glycosylations associated with disease, before SRM analysis of peptides of interest to analyze glycoforms (38Ahn Y.H. Lee J.Y. Lee J.Y. Kim Y.S. Ko J.H. Yoo J.S. Quantitative analysis of an aberrant glycoform of TIMP1 from colon cancer serum by L-PHA-enrichment and SISCAPA with SRM mass spectrometry.J. Proteome Res. 2009; 8: 4216-4224Crossref PubMed Scopus (79) Google Scholar). Much less work has focused on released glycans; compositional analysis of N-glycans has been investigated using a general approach. Compositions of interest were set as the Q1 transition (parent mass) and general N-glycan masses for Q3 to identify all N-glycans of that mass in a sample (39Zhang H. Wang Z. Stupak J. Ghribi O. Geiger J.D. Liu Q.Y. Li J. Targeted glycomics by selected reaction monitoring for highly sensitive glycan compositional analysis.Proteomics. 2012; 12: 2510-2522Crossref PubMed Scopus (15) Google Scholar). An SRM approach has yet to be developed for O-glycans. The highly specific nature of an O-glycan SRM approach has the potential to not only allow relative quantitation but also to drastically improve the time-consuming annotation process. Here, the investigation of sulfated core 1 O-glycans in acute ReA SF compared with that of chronic RA patients was undertaken. To further investigate the possibility of a systemic sulfation effect and as SF sampling is an invasive technique, sulfation changes in mucin rich- saliva were analyzed. This led to the development of an SRM approach to identify and relatively quantitate the isomers of the sulfated and unsulfated core 1 O-glycans in RA and control salivary MUC7. SF samples from arthritis patients were collected during therapeutic joint aspiration at the Rheumatology Clinic, Sahlgrenska University Hospital (Gothenburg, Sweden). All patients used in this study gave informed consent, and the procedure was approved by the Ethics Committee of the University of Gothenburg. All RA patients fulfilled the American College of Rheumatology 1987 revised criteria for RA (40Arnett F.C. Edworthy S.M. Bloch D.A. McShane D.J. Fries J.F. Cooper N.S. Healey L.A. Kaplan S.R. Liang M.H. Luthra H.S. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis.Arthritis Rheum. 1988; 31: 315-324Crossref PubMed Scopus (18607) Google Scholar). The samples were clarified by centrifugation at 10,000 × g for 10 min and stored at −80°C before use. The acidic proteins were purified as described previously (41Estrella R.P. Whitelock J.M. Packer N.H. Karlsson N.G. The glycosylation of human synovial lubricin: implications for its role in inflammation.Biochem. J. 2010; 429: 359-367Crossref PubMed Scopus (70) Google Scholar). In brief, the SF sample was diluted with wash buffer (250 mm NaCl, 20 mm Tris-HCl, 10 mm EDTA, pH 7.5) before applying to a 1-ml DEAE-fast flow Hi-Trap column (GE Healthcare). Enriched acidic glycoproteins were eluted with wash buffer containing 1 m NaCl. This fraction was precipitated with 80% ethanol for 16 h at −20°C. The precipitate was collected by centrifugation at 12,100 × g for 20 min, air-dried, and then resuspended in PBS. Protein concentration was determined by the BCA protein assay kit (Thermo Scientific, San Jose, CA) using BSA as standard. The major Alcian blue-staining glycoprotein in the fraction was isolated by SDS-PAGE after reduction by adding 10 mm DTT and heating at 95°C for 20 min and alkylation (25 mm iodoacetamide for 1 h at room temperature) before separation on a 3–8% Tris acetate NuPAGE gel (Invitrogen). Gels were then transferred to PVDF membrane using a semi-dry blotter as described previously (42Hayes C.A. Nemes S. Issa S. Jin C. Karlsson N.G. Glycomic work-flow for analysis of mucin O-linked oligosaccharides.Methods Mol. Biol. 2012; 842: 141-163Crossref PubMed Scopus (8) Google Scholar). Membranes were stained for 20 min using Alcian blue solution (0.125% Alcian blue in methanol with acetic acid) and destained in methanol. Whole saliva samples were collected from control and RA patients a minimum of 2 h after eating. Saliva was centrifuged at 5000 × g for 3 min, and saliva was removed from any remaining pellet. MUC7 was isolated from whole saliva. The samples were reduced (50 mm DTT, 70°C for 2 h) and alkylated (125 mm iodoacetamide, 30 min at room temperature in the dark) before separation using composite agarose and polyacrylamide (SDS-AgPAGE) gels that were prepared as described previously (42Hayes C.A. Nemes S. Issa S. Jin C. Karlsson N.G. Glycomic work-flow for analysis of mucin O-linked oligosaccharides.Methods Mol. Biol. 2012; 842: 141-163Crossref PubMed Scopus (8) Google Scholar). Each saliva sample was loaded into 4 wells to be sure to account for low abundance of MUC7 in some individuals. Gels were transferred to PVDF and stained with Alcian blue as described for the SF samples, and the lower MUC7 band was excised. O-Glycans were released from MUC7 on Alcian blue-stained PVDF membranes by reductive β-elimination. All bands from a single sample were prepared on the same day with the same reagents. Bands were cut from the membrane, and each band was further cut into small squares. Membrane pieces were wet with methanol before the addition of 20 μl of freshly made reductive elimination solution (50 mm sodium hydroxide, 0.5 m sodium borohydride) and incubated at 50°C for 16 h. Samples were neutralized with 1 μl of glacial acetic acid before clean up. Cation exchange columns (AG50WX8 resin, Bio-Rad) were prepared in Ziptips (Millipore, Billerica, MA) using 25 μl of resin suspension (1:1 resin/methanol). Columns were prepared before neutralized samples were washed through, and residual sugar was eluted with water. Samples were dried using a speedvac (45°C) and washed five times with 1% glacial acetic acid in methanol until all borate salts were removed. Porous graphitized carbon (PGC) columns (5-μm particles) were used with an inner diameter of 250 μm and a length of 10 cm packed in-house. Mobile phases consisted of 10 mm ammonium bicarbonate for solvent A and 10 mm ammonium bicarbonate with 80% acetonitrile for solvent B. The gradient, after 5 min of 100% solvent A, increased solvent B to 45% in 41 min and stayed at 100% solvent B for 8 min and then equilibrated at 100% A for 25 min. Columns were attached to an Agilent 1100 series HPLC with a flow rate after passive splitting of 7–10 μl/min. An LTQ linear ion trap (Thermo Fisher Scientific) in negative ion mode was used for MS and MS/MS analysis. A top three data-dependent method was used with normalized collision energy of −35 eV, isolation width of 1.0 m/z, and an activation Q value of 0.250 and time of 0.250 ms. Mobile phases and PGC columns were used as above with a constant flow of 10 μl/min using an ekspertTM microLC 200 HPLC system (Eksigent, AB Sciex, Framingham, MA). Gradient was as follows: 100% A for 5 min, then a gradient up to 23% B in 21 min, and to 95% B in 5 min. A 20-min wash at 95% was used to keep the column sensitivity high and prevent carry-over, and a 25-min equilibration with 100% A completed the gradient. SRM analyses were created and carried out on a QTRAP® 5500 ESI-triple quadrupole linear ion trap hybrid mass spectrometer (AB Sciex, Framingham, MA) in negative mode. A ReA lubricin sample was used for creation of the sulfated core 1 O-glycan transitions, and O-glycans released from porcine gastric mucin (Sigma-Aldrich) were used for the core 1 structure. Optimization of collision energy (CE), declustering potential (DP), and collision cell exit potential (CXP) was performed for each transition tested. The final method for SRM included the following transitions and specifications: 384.1/101.1 (CE −29 eV, DP −25, and CXP −15), 464/241.1 (CE −41 eV, DP −15, and CXP −20), and 464/302.1 (CE −40 eV, DP −15, and CXP −20). A dwell time of 35 ms was used for all. EPI spectra were acquired for the 384 and 464 ions, providing full scan MS/MS data from which sequence analysis could be performed. MultiQuantTM software version 2.1.1 (AB Sciex, Framingham, MA) was used for semi-quantitation. GraphPad Prism version 5 (GraphPad Software, La Jolla, CA) was used for unpaired two-tailed t test statistical analysis. Glycans have been shown to be essential to a range of immune functions. Here, glycans from the acidic glycoprotein fraction in SF were investigated under either acute or chronic inflammatory conditions to iden
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