Carboxylink kit

N^α-(tert-butoxycarbonyl)-L-Lys (Boc-Lys), ε-aminocaproic acid (6-ACA), AA, FA, BSA, trifluoroacetic acid, 3,3′,5,5′-tetramethylbenzidine (TMB), human MPO, and H₂O₂ were purchased from Sigma-Aldrich (St. Louis, MO). Malondialdehyde bis(dimethyl) acetal, Dynabeads M-270 Amine, CarboxyLink Kit, the Imject EDC mcKLH Spin Kit, goat anti-rabbit IgG (H + L) antibody with HRP, goat anti-human IgG/IgM secondary antibody, and goat anti-mouse IgM secondary antibody were obtained from Thermo Scientific (Rockford, IL). Protein A column and autoLDL™ Cholesterol kit were purchased from GE Healthcare Life Sciences (Pittsburgh, PA) and Pointe Scientific (Lincoln Park, MI), respectively. EnVision + Single Reagents anti-mouse/rabbit-HRP were from Dako North America, Inc. (Carpentaria, CA). The anti-M2AA mouse monoclonal antibody (1F83)²⁴ and rabbit polyclonal antibody^{48 (link)} were provided by Drs. Koji Uchida (Nagoya University) and Todd A. Wyatt (University of Nebraska), respectively.

Synthetic peptide TS9 (1 mg) was conjugated to the agarose beads using the CarboxyLink Kit (Thermo Scientific) according to the manufacturer recommendations. LLC-MK₂ cells were then lysed in PBS 2% Nonidet P-40, centrifuge at 10.000g for 30 minutes and the supernatant incubated with the peptide-coupled resin overnight at 4°C. After extensive wash with PBS and NaCl 1M, the resin was eluted sequentially with SDS 1% (1^st elution) and 8 M urea (2^nd elution). The eluted proteins were analyzed by SDS-PAGE and the gel stained with Coomassie brilliant blue G250 before processing for mass spectrometry. Protein bands with calculated molecular masses of 49, 53, 59 and 66 kDa were excised from the gel, sliced into small pieces, treated with dithiothreitol DTT (10 mM), iodoacetamide (100 mM) and then digested with sequencing grade trypsin (5 μg/ml) (Promega, WI, USA). The resulting peptides were then analyzed by HPLC-ESI-MS at the Analytical Center facility of the Chemistry Institute, University of São Paulo (São Paulo, Brazil). The proteins present in each gel fragment were identified by peptide fingerprinting using the MASCOT software [52 (link)].