Kari Gurtner (Presenter)
Mayo Clinic
Bio: I have spent the last 10 years in patient care at Mayo Clinic in Rochester, MN in a laboratory role. Currently, I serve as a development technologist for the Protein Immunology laboratory. I have a great interest in mass spectrometry and looking towards its platform for alternative testing in the protein immunology field. I enjoy balancing my interests with work and raising two children with my husband. We enjoy being outdoors, being involved in team sports such as soccer and hockey, and traveling.
Authorship: Kari M. Gurtner¹, Patrick M. Vanderboom², Christine M. Charlesworth², Surendra Dasari³, David R. Barnidge¹, Melissa R. Snyder¹, and David L. Murray¹
¹Department of Labratory Medicine and Pathology, ²Medical Genome Facility – Technology Development Team, ³Department of Biomedical Stats and Informatics, Mayo Clinic, Rochester, MN
| Short Abstract Celiac Disease (CD) is characterized by an intolerance to dietary gluten. Patients with CD often have high levels of IgA autoantibody against tissue transglutaminase (tTG). To characterize tTG reactive IgA, the autoantibodies were isolated and then analyzed by TOF-LC/MS and LC-MS/MS, using top-down and bottom-up proteomic approaches. This provided information from the variable regions of both IgA light and heavy chains to characterize tTG reactive antibodies. Comparison of positive and negative sample groups demonstrated significant light chain profile differences, indicating that celiac patients exhibit an oligoclonal response to tTG. |
Long Abstract
Introduction: Celiac Disease (CD) is a multisystem inflammatory disease characterized by intolerance to dietary gluten. CD has a strong link to a patient’s immune system with a well-documented concordance with the patient’s HLA type. Current practice for celiac disease screening includes serological Enzyme Linked Immunosorbant-Assay (ELISA) detection of immunoglobulins (IgA) directed towards human tissue transglutaminase (tTG), an enzyme involved with processing dietary gluten. The ELISA method is limited to simply quantifying the amount of tTG reactive IgA present and is unable to provide additional information on the number IgA clones or the similarity of the IgA structure between patients. This study aimed to develop an isolation method capable of obtaining a sufficient quantity of IgA directed towards human tTG characterized by top-down and bottom-up proteomic approaches.
Methods: Residual sera was chosen for comparative proteomic analysis of tTG reactive and non-reactive IgA. Samples were selected based on the value of anti-tTG response using our ELISA testing method. Both non-detectable and very high level response sera were compared. Bottom-up: To confirm antibody isolation, purified samples were first run by SDS-PAGE. Using a vendor supplied ELISA kit; antibodies from a single high IgA titered patient were stripped from wells of the tTG-IgA plate and pooled. The pooled eluent was dried, reconstituted, and then fractionated by SDS-PAGE. Isolated IgA light and heavy chain bands were cut from the gel and analyzed using high resolution LC-MS/MS (Thermo Q-Exactive Orbitrap and Orbitrap Elite) and PEAKS proteomic software. Top-down: Antibody isolation was performed using a tTG pull-down purification method. Patient serum was incubated with biotinylated tTG, followed by the addition of streptavidin coated magnetic beads, used to capture and purify the tTG/anti-tTG antibody complex. The purified tTG reactive antibodies were then eluted and reduced prior to intact light chain analysis, using an Agilent 6224 TOF-LC/MS and its supporting software.
Results: To characterize tTG reactive IgA, analysis was performed using both a top-down and bottom-up proteomic approach. Using PEAKS software for the bottom-up approach, peptide data generated by nano LC-MS/MS, provided information from the variable regions of both IgA light and heavy chains to characterize tTG reactive antibodies. By utilizing the biotin/streptavidin interaction to purify tTG reactive IgA directly from patient serum, a top-down proteomic approach was possible. The results of a comparison between six patients and six controls, demonstrated significant light chain profile differences between the two groups. Multiple clones were detected above the polyclonal background in the patient group, while the control group light chain clones were indistinguishable from background levels. The results of this study have led to the indication that celiac patients have an extensive variable oligoclonal immune response to tTG, while further demonstrating the utility of this method for tTG isolation.
Conclusion: Proteomic analysis using nano LC-MS/MS and TOF-LC/MS technology has allowed the characterization of antibody reactive tTG in patients with Celiac Disease. This study has provided convincing data to continue pursuing the unique characteristics of tTG reactive IgA. With the combination of information obtained from both top-down and bottom-up proteomic analysis, we hope to provide a more complete picture of autoantibody production and variable region gene selection in patients with Celiac Disease.
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