Omer Deutsch1, Guy Krief1, Yrjö T Konttinen2, Batia Zaks1, David T Wong1, Doron J Aframian1, Aaron Palmon3. 1. Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University of Jerusalem, Israel, Department of Medicine, Biomedicum Helsinki, Helsinki University Central Hospital, ORTON Orthopaedic Hospital, Helsinki, COXA Hospital for Joint Replacement, Tampere, Finland, School of Dentistry and Dental Research Institute, University of California at Los Angeles, Los Angeles, CA, USA and Salivary Gland Clinic and Saliva Diagnostic Laboratory, Department of Oral Medicine, Hebrew University of Jerusalem, Hadassah Medical Center, Jerusalem, Israel. 2. Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University of Jerusalem, Israel, Department of Medicine, Biomedicum Helsinki, Helsinki University Central Hospital, ORTON Orthopaedic Hospital, Helsinki, COXA Hospital for Joint Replacement, Tampere, Finland, School of Dentistry and Dental Research Institute, University of California at Los Angeles, Los Angeles, CA, USA and Salivary Gland Clinic and Saliva Diagnostic Laboratory, Department of Oral Medicine, Hebrew University of Jerusalem, Hadassah Medical Center, Jerusalem, Israel Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University of Jerusalem, Israel, Department of Medicine, Biomedicum Helsinki, Helsinki University Central Hospital, ORTON Orthopaedic Hospital, Helsinki, COXA Hospital for Joint Replacement, Tampere, Finland, School of Dentistry and Dental Research Institute, University of California at Los Angeles, Los Angeles, CA, USA and Salivary Gland Clinic and Saliva Diagnostic Laboratory, Department of Oral Medicine, Hebrew University of Jerusalem, Hadassah Medical Center, Jerusalem, Israel Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University of Jerusalem, Israel, Department of Medicine, Biomedicum Helsinki, Helsinki University Central Hospital, ORTON Orthopaedic Hospital, Helsinki, COXA Hospital for Joint Replacement, Tampere, Finland, School of Dentistry and Dental Research Institute, University of California at Los Angeles, Los Angeles, CA, USA and Salivary Gland Clinic and Saliva Diagnostic Laboratory, Department of Oral Medicine, Hebrew University of Jerusalem, Hadassah Medical Center, Jerusalem, Israel. 3. Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University of Jerusalem, Israel, Department of Medicine, Biomedicum Helsinki, Helsinki University Central Hospital, ORTON Orthopaedic Hospital, Helsinki, COXA Hospital for Joint Replacement, Tampere, Finland, School of Dentistry and Dental Research Institute, University of California at Los Angeles, Los Angeles, CA, USA and Salivary Gland Clinic and Saliva Diagnostic Laboratory, Department of Oral Medicine, Hebrew University of Jerusalem, Hadassah Medical Center, Jerusalem, Israel apalmon@cc.huji.ac.il.
Abstract
OBJECTIVE: SS is an autoimmune exocrinopathy affecting ∼1 million patients in the USA that is diagnosed mostly in middle-aged women. Oral fluids (OFs) serving as the mirror of the body were suggested as an ideal non-invasive diagnostic tool. Previously we developed depletion techniques for OF high-abundance proteins to increase visualization of low-abundance proteins. Therefore the aim of this study was to examine the effect of depletion pretreatments on the identification potential of SS OF biomarker candidates. METHODS: Unstimulated OFs were collected from 18 female SS patients and 18 healthy age- and gender-matched controls. High-abundance proteins were depleted using affinity and immunodepletion methodologies followed by semi-quantitative two-dimensional gel electrophoresis and quantitative dimethylation liquid chromatography tandem mass spectrometry (LC-MS/MS). To initially validate the MS results, western blotting was performed. RESULTS: The use of depletion strategy before proteomics analysis increased identification ability by 3-fold. Overall, 79 biomarker candidates were identified. Proteins with the most pronounced fold changes were related to SS serum or tissue factors. In addition, bioinformatics analysis of proteins with a >3-fold increase in SS patients showed calcium-binding proteins, defence-response proteins, proteins involved in apoptotic regulation, stress-response proteins and cell motion-related proteins. Preliminary validation by western blotting of profilin and CA-I indicated similar expression profile trends to those identified by quantitative MS. CONCLUSION: The significance of OF novel depletion methodologies is clearly demonstrated for increased visibility of biomarker candidates as well as for unveiling possible mechanisms involved in this syndrome. This represents a major contribution to our ability to use OF as a future diagnostic fluid.
OBJECTIVE: SS is an autoimmune exocrinopathy affecting ∼1 million patients in the USA that is diagnosed mostly in middle-aged women. Oral fluids (OFs) serving as the mirror of the body were suggested as an ideal non-invasive diagnostic tool. Previously we developed depletion techniques for OF high-abundance proteins to increase visualization of low-abundance proteins. Therefore the aim of this study was to examine the effect of depletion pretreatments on the identification potential of SS OF biomarker candidates. METHODS: Unstimulated OFs were collected from 18 female SS patients and 18 healthy age- and gender-matched controls. High-abundance proteins were depleted using affinity and immunodepletion methodologies followed by semi-quantitative two-dimensional gel electrophoresis and quantitative dimethylation liquid chromatography tandem mass spectrometry (LC-MS/MS). To initially validate the MS results, western blotting was performed. RESULTS: The use of depletion strategy before proteomics analysis increased identification ability by 3-fold. Overall, 79 biomarker candidates were identified. Proteins with the most pronounced fold changes were related to SS serum or tissue factors. In addition, bioinformatics analysis of proteins with a >3-fold increase in SS patients showed calcium-binding proteins, defence-response proteins, proteins involved in apoptotic regulation, stress-response proteins and cell motion-related proteins. Preliminary validation by western blotting of profilin and CA-I indicated similar expression profile trends to those identified by quantitative MS. CONCLUSION: The significance of OF novel depletion methodologies is clearly demonstrated for increased visibility of biomarker candidates as well as for unveiling possible mechanisms involved in this syndrome. This represents a major contribution to our ability to use OF as a future diagnostic fluid.
Authors: Feng Li; Karolina Elżbieta Kaczor-Urbanowicz; Jie Sun; Blanca Majem; Hsien-Chun Lo; Yong Kim; Kikuye Koyano; Shannon Liu Rao; So Young Kang; Su Mi Kim; Kyoung-Mee Kim; Sung Kim; David Chia; David Elashoff; Tristan R Grogan; Xinshu Xiao; David T W Wong Journal: Clin Chem Date: 2018-04-23 Impact factor: 8.327