1. IDENTIFICATION OF SALIVARY GLYCOPROTEINS INVOLVED IN IMMUNE PROCESS BY MudPIT TECHNOLOGY M. Gonzalez-Begne, DDS, PhD 1 *, B. Lu PhD 3, I. Sanz MD 2, and J.R. Yates ІІІ, PhD 3. ( 1 Center for Oral Biology and 2 Department of Allergy/Immunology and Rheumatology; University of Rochester, Medical Center, Rochester. NY. 3 The Scripps Research Institute, La Jolla, California). Saliva is secreted by the major and minor salivary glands and is vital for the maintenance of oral homeostasis. Physiological changes associated with different diseases are reflected by changes in the glycoproteome composition. Thus mapping the salivary glycoproteome will provide a better understanding of health and disease processes. Lectin chromatography and MudPIT (multidimensional protein identification technology) mass spectrometry were used to isolate glycopeptides and glycoproteins from submandibular/sublingual (SM/SL) ductal saliva. 903 proteins were catalogued in human SM/SL saliva. The Gene ontology analysis revealed that salivary glycoproteins are involved in different biological processes, such as cellular process (17.9%), response to stimulus (13.2%), biological regulation (12.7%), metabolic process (11.8%), developmental process (8.0%) and immune system process (7.5%) among others. Of significance is the presence of proteins which over expression is associated with rheumatoid arthritis (S100A8, S100A9, rheumatoid factor C6 light chain, MMP9), Sjögren’s syndrome (CRISP3, IGHG1, α-enolase) and Systemic lupus erythematosus (IGLV3-21, IGLV2-14, V1-19) suggesting the possible use of saliva as a diagnostic tool for immune diseases. The results showed that MudPIT technology on saliva was crucial for identifying glycoproteins with high mass accuracy and analytical sensitivity, two important parameters for biomarker validation. This research project was supported by 5 KL2RR024136-04. ABSTRACT Materials: Con-A, RCA-I and UEA-I covalently linked to agarose beads were obtained from EY-labs (San Mateo, CA). All other chemicals, supplies, and reagents were obtained from Sigma-Aldrich (St. Louis MO). Methods: Salivary collection: Submandibular/sublingual saliva collection was performed at the University of Rochester following standardized protocols (Burlage et al., 2005; Navazesh, 1993). A healthy control donor was asked to refrain from eating, drinking, smoking or performing any hygiene procedures for at least 1 hr prior collection. Using 0.4% citrate solution and a Block and Brotman collector (Block and Brotman, 1962) saliva was collected on ice pre-chilled tubes containing protease inhibitors, between 7:00 to 10:00 am. Collected samples were immediately centrifuged, dialyzed (3.5 kDa molecular mass cutoff) and lyophilized. Lectin chromatography procedure: 10 mg/ml of lyophilized submandibular/sublingual saliva was subjected to lectin fractionation for isolating the glycoproteins of interest. All chromatography procedures were carried out in a cold room at 4 o C following the manufacturer’s protocol. Elution of glycoproteins were performed using the method of gravity flow in combination with carbohydrate buffers made of 0.2 M Methyl α-D-Mannopyranoside for Con A, 0.1M Lactose for RCA-I and 0.05 M α-Fucose for UEA-I lectins respectively. Mass Spectrometry Analysis: Mass spectrometry analysis was performed on each eluted fractionations. 10 mg of digested protein was used for multidimensional protein identification (MudPIT) analysis. As peptides were eluted from the microcapillary column they were electrosprayed directly into LTQ Orbitrap instrument. Tandem mass spectra were searched with SEQUEST algorithm against the European Bioinformatics Institute (EBI) human International Protein Index (IPI) database version 3.01 to get the identified protein. Saliva is a complex fluid secreted by the major and minor salivary glands. The composition of saliva varies according to many factors including the gland type, collection time, stimulus used, etc. Mostly it is composed of water, and other components such as inorganic compounds (electrolytes), organic compounds (urea, uric acid, glucose, fatty acids) and a complex mixture of peptides, proteins and enzymes (Melvin et al., 2005; Denny et al., 2008; Yan et al., 2009). Because of its complex chemistry, saliva has a wide range of biological activities which maintain oral homeostasis (Denny et al., 2008; Yan et al., 2009; Gonzalez-Begne et al., 2009). In fact, salivary proteins can act alone or forming complexes with multifunctional activities in the oral cavity and have been used in the proteomics field as important biomarkers associated with oral and systemic diseases (Zhang et al., 2009). However, relatively few major salivary proteins have been explored. Identification of all secreted salivary proteins pose a significant challenge. Highly abundant proteins tend to mask the isolation of other salivary components. Therefore, conventional fractionation methods are required for the isolation and identification of medium and low- abundant salivary proteins. Taking into consideration that glycosylation is the primary cause of micro-heterogeneity in proteins, and that glycoproteins make up a major and significant part of the salivary proteome, lectin chromatography was used as a preferred method for the separation and purification of glyco-conjugates. The advantages of lectin affinity over other physico- chemical separation techniques are high specificity for a certain carbohydrate structure, mild separation conditions for preserving the biological activity of the molecule of interest, and the ability to concentrate the product from a dilute sample (Mechref and Novotny, 2002). It is well known that different lectins display a variety of binding specificities. In this study Con-A (Canavalia ensiformis), RCA-I (Ricinus Communis Agglutinin I) and UEA-I (Ulex europaeus) were chosen due to their capacity to bind a) branched α-mannosidic structures; high-mannose type, hybrid-type and biantennary complex type N-glycans, b) oligosaccharides ending in Beta-D- galactose and/or N-acetyl-alpha-D-galactosamine and c) Fuca1-2Gal-R carbohydrate structures respectively. Many glycoproteins have been purified using different affinity chromatography methods, however this is the first study carried out using a combination of lectin chromatography and multidimensional protein identification technology (MudPIT) mass spectrometry, offering an in depth high throughput identification of submandibular/sublingual salivary glycoproteins that will serve as a baseline for understanding glycoprotein function in healthy vs. disease status. RESULTS METHODS INTRODUCTION CONCLUSION OBJECTIVES 1) To identify and catalogue α-mannose or α-glucose glycoproteins in submandibular/sublingual ductal saliva using Con A lectin chromatography and MudPIT mass spectrometry. 2) To identify and catalogue b-linked galactose glycoproteins in submandibular/sublingual ductal saliva using RCA-I lectin chromatography and MudPIT mass spectrometry. 3) To identify and catalogue α-L-fucose glycoproteins in submandibular/sublingual ductal saliva using UEA-I lectin chromatography and MudPIT mass spectrometry. Con-A, RCA-I and UEA-I lectin chromatographies and MudPIT technology allowed the identification of high, medium and low-copy glycoproteins with efficient online peptide separation, high mass accuracy and analytical sensitivity. Future studies are needed to elucidate the biological role of these submandibular/sublingual salivary glycoproteins in health and disease processes. Strategy for isolating and identifying submandibular/sublingual glycoproteins. Gene Ontology annotation of submandibular/sublingual glycoproteins by biological process. Con A RCA-I UEA-I