Pssm 8 automated peptide synthesizer

The C-ICD and C-ICDmut peptides were synthesized by Fmoc chemistry using the PSSM-8 automated peptide synthesizer (Shimadzu, Kyoto, Japan) and purified by reverse-phase high-performance liquid chromatography on a C18 column with a linear gradient from 0 to 90 % CH₃CN in 0.1 % trifluoroacetic acid for 60 min at a flow rate of 1 ml/min. The purified peptides were digested with trypsin and separated using the Smart System (GE Healthcare, Little Chalfont, UK) on a RP300 column with a linear gradient from 0 to 90 % CH₃CN in 0.1 % trifluoroacetic acid for 50 min at a flow rate of 1 ml/min, and the fractions were collected. The individual fractions were analyzed by matrix-assisted laser desorption/ionization time-of-flight collision-induced dissociation tandem mass spectroscopy using the Axima Performance system (Shimadzu), and their amino acid sequences were confirmed by the 492HT protein sequencer (Applied Biosystems, Foster City, CA, USA). FITC-I (Dojindo) was used to label the peptides according to the manufacturer’s instructions. Briefly, the peptide and FITC-I were mixed at a weight ratio of 10:1 in phosphate-buffered saline (PBS) containing 2.5 mM carbonate and were reacted at 4 °C for 4 h. The FITC-labeled peptide was washed with PBS by five centrifugation cycles using an Amicon Ultra-10 K membrane filter (Millipore, Billerica, MA, USA).

ELISA method was used to examine the levels of plasma anti-heart antibody (AHA) including antibodies (Abs) against the adenine nucleotide translocator (ANT), β1 adrenergic receptor (β1-AR), myosin heavy chain (MHC) and L-type calcium channel (CC) according to the procedures described in previous studies13 (link)14 (link)15 (link)16 (link)17 (link). Briefly, the peptides derived from different human cardiac proteins including ANT (PIERVKLLLQ-YDEIKKFV), β1-AR (HWWRAESDEARRCYNDPKCCDFVTN RC), MHC (EIERKLAEKD-VDKLQLKV-AKSRDIGAKGLNE) and CC (VNENTRMYIPEENHQ) were synthesized by a PSSM-8 automated peptide synthesizer (Shimadzu, Japan). The purities of the synthetic peptides confirmed by high liquid chromatography were up to 96%. A horseradish peroxidase-labeled rat antihuman IgG (Gibco, USA) was used to detect AHA, and the absorbance (A) of the dye is measured at a wavelength of 450 nm by using a Beckman DU-600 Spectrophotometer. All of the samples were measured in triplicate.