Cell debris was eliminated by centrifugation of the suspension (14,000 x for 45 minutes at 15 C)

Cell debris was eliminated by centrifugation of the suspension (14,000 x for 45 minutes at 15 C). they were excised from gels, trypsin-digested, and analyzed by nanoLC-electrospray tandem MS. Forty unique proteins were identified and these include proteins that are associated with cardiac hypertrophy (mimecan, myozenin), cardiac remodeling (periostin), cardiomyopathy (desmin, desmoplakin), cell survival (laminin), structural integrity (filamin), chaperone proteins (crystalline, HSP70), mitochondrial enzymes and channel proteins. Ingenuity Pathway Analysis showed multiple pathways were involved including those that regulate energy metabolism, redox, fibrosis, cardiac hypertrophy and degeneration. Conclusions and clinical relevance Autoantibodies are present in patients with POTS. These autoantibodies cross-react with a wide range of cardiac proteins and may induce alterations in cardiac function. Autoimmune pathogenetic mechanisms should be further explored in these patients. fibromyalgia,Imitrex, Inderal,for 1 hour at 4 C) were resuspend in Protein Extraction Reagent Type 4 and sonicated on ice. Cell debris was eliminated by centrifugation of the suspension (14,000 x for 45 minutes at 15 C). The supernatant was then reduced by freshly prepared tributylphosphine for 1 hour at room temperature. Membrane proteins were alkylated with iodoacetamide (IAA) for 1.5 h at room temperature with a concentration of 1 1.9 mg/ml. 2.3 2DE Immunoblot analysis First-dimension IEF was carried out using the Protean IEF Cell (BioRad). For each sample, 100g of the protein was diluted in 2DE rehydration buffer [7M urea, 2M thiourea, 4% CHAPS, 30mM dithiothreitol (DTT), 0.1% 3C10 Pharmalyte (GE) and 0.25% pH 3-10NL (non-linear) IPG buffer (GE)] and overlaid with 11 cm 3-10NL IPG strip. The strips were passively rehydrated overnight for 11 h, then focused for a total of 25KVhr. IPG strips were stored at ?80C prior to separation by SDS-PAGE. IPG strips were equilibrated in SDS equilibration buffer (6 M urea, 2% SDS, 50 mM Tris pH 8.8, 30% glycerol) for 10 min with 1% DTT, then 15 min in fresh buffer with 2% IAA, to maximize alkylation of any free cysteines. 10.5-14% Criterion gradient gels (133 87 1.0 mm) were used for the second dimension separation. After electrophoresis, gels for spot excision and MS analysis ADU-S100 were fixed and silver-stained. Gels for immunoblot analysis were transfered to PVDF (HyBond, GE) using CAPS buffer with 10% methanol. Some transferred blots were also stained with Sypro-Ruby fluorescent blot stain (Invitrogen) for identifying spot location by comparing with immunoblot results. Serum IgG ADU-S100 was used for immunoblotting at ADU-S100 0.2 mg/ml. IgGs were diluted with blocking buffer (PBS made up of 0.05% Tween 20, 10% milk). Immunoblot analysis was performed as we have previously described [9]. Antibody binding was detected with a horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Invitrogen) and chemiluminescence using SuperSignal West Pico Chemiluminescence Substrate (Pierce Biotechnology). Specific antibodies against desmin (1:1000, Cell Signaling Technology, Inc), and ADU-S100 periostin (1:1000, Sigma, St. Louis) were used for 2DE immunoblot analysis to identify their spot locations in 2DE gels. 2.4 Protein identification and data analysis Immunoreactive protein spots were selected by comparing immunoblot with a silver-stained 2-DE gel. Each spot was given a standard spot number and by its position on a grid of (x by y). The spots were selected visually and by analysis of overlaid images of the immunoblot and the fluorescent stained protein spots around the membrane using PDQuest 7.4.0. image analysis software (Bio-Rad). Protein spots specifically reacting with patient IgGs were selected and processed for protein identification by in-gel trypsin digestion and nano-LC-ESI-tandem mass spectrometry using anLTQ Orbitrap ( ThermoElectron, Bremen, Germany) coupled to a nano-LC-2D HPLC system (Eksigent, Dublin, CA). The mass spectrometer experiment was set to perform an Oribtrap full scan from 375 to 1600 m/z, ( 60,000 resolutionat 400 m/z), followed by linear ion trap MS/MS scans on the top five [M+2H]2+ or [M+3H]3+ ions. All MS/MS spectra were analyzed by using Mascot (version 2.2.04; Matrix Science, London, UK;), Sequest (ThermoFinnigan, San Jose, CA; version 27, rev. 12), and X! Tandem (www.thegpm.org; version 2006.09.15.3) Each analysis was set up to search the most current Swiss-Prot database, assuming semitrypsin or full trypsin digestion with a fragment ion mass tolerance of 0.80 CD117 Da and a parent ion tolerance.