Deciphering Membrane-Associated Molecular Processes in Target Tissue of Autoimmune Uveitis by Label-Free Quantitative Mass Spectrometry

Abstract

Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immuneprivileged target tissue retina. The molecular events enabling T cells to invade and attack the tissue have remained elusive. Changes in membrane protein expression patterns between diseased and healthy stages are especially interesting because initiating events of disease will most likely occur at membranes. Since disease progression is accompanied with a break-down of the blood-retinal barrier, serum-derived proteins mask the potential target tissue-related changes. To overcome this limitation, we used membrane-enriched fractions derived from retinas of the only available spontaneous animal model for the disease equine recurrent uveitis, and compared expression levels by a label-free LC-MSMS-based strategy to healthy control samples. We could readily identify a total of 893 equine proteins with 57% attributed to the Gene Ontology project term "membrane." Of these, 179 proteins were found differentially expressed in equine recurrent uveitis tissue. Pathway enrichment analyses indicated an increase in proteins related to antigen processing and presentation, TNF receptor signaling, integrin cell surface interactions and focal adhesions. Additionally, loss of retina-specific proteins reflecting decrease of vision was observed as well as an increase in Mü ller glial cell-specific proteins indicating glial reactivity. Selected protein candidates (caveolin 1, integrin alpha 1 and focal adhesion kinase) were validated by immunohistochemistry and tissue staining pattern pointed to a significant increase of these proteins at the level of the outer limiting membrane which is part of the outer blood-retinal barrier. Taken together, the membrane enrichment in combination with LC-MSMS-based label-free quantification greatly increased the sensitivity of the comparative tissue profiling and resulted in detection of novel molecular pathways related to equine recurrent uveitis. Molecular & Cellular Proteomics 9: 2292-2305, 2010.

Peptide serum markers in islet autoantibody-positive children

“…The RAW files (Thermo Fisher Scientific) were analysed using the Progenesis LC-MS software (version 4.0; Nonlinear Dynamics, Waters, Eschborn, Germany), as previously described [27,29].…”

A Combined Omics Approach to Generate the Surface Atlas of Human Naive CD4+ T Cells during Early T-Cell Receptor Activation

“…Therefore an omics approach was performed, combining quantitative proteomic and transcriptomic methods. For the identification of surface proteins of CD4 ϩ /CD45RA ϩ naive T cells by massspectrometry the recently described PAL (periodate oxidation and aniline-catalyzed oxime ligation) approach (18) was applied and adapted for the use with primary T cells and combined with quantitative LC-MS/MS (19). This method allows an efficient labeling of cell surface sialic acid-containing glycans on living cells.…”

“…Database Search, Label-Free Relative Quantification, Data Processing, and Identification of Cell Surface Proteins-The RAW files (Thermo Fisher Scientific) were further analyzed using the Progenesis LC-MS software (version 4.0, Nonlinear Dynamics, Newcastle, UK), as described previously (19), with the following changes: For peptide identification, using the search engine Mascot (Matrix Science, Release number 2.4), assuming tryptic digestion, one missed cleavage was allowed, a fragment ion mass tolerance of 0.6 Da and a parent ion tolerance of 10 ppm. Carbamidomethylation was set as fixed modification, methionine oxidation, and asparagine or glutamine deamidation were allowed as variable modifications.…”

Loss of Caveolin-1 Impairs Retinal Function Due to Disturbance of Subretinal Microenvironment

“…Global deletion of Cav-1 results in loss of morphologically identifiable caveolae and a variety of abnormalities, including microvessel hyperpermeability (19), alterations in lipid metabolism (20,21), and insulin resistance (22). Recently, dysregulation of or abnormalities in Cav-1 have been linked to diabetic retinopathy (23) and inflamed uveitic retinas (24). In addition, a frameshift mutation in Cav-1 is associated with a rare congenital lipodystrophy with severe retinitis pigmentosa-like ocular abnormalities (25).…”