*4.4. Preparation of Troponin I-Peptide-AntiTn I Antibody Immune Complex-Containing Solution*

The human cardiac Troponin I epitope peptide (ENREVGDWRKNIDAL; peptides&elephants, Hennigsdorf, Germany) was obtained as lyophilized powder. The peptide was dissolved in 200 mM ammonium acetate buffer, pH 7, to obtain a peptide concentration of 1.31 µg/µL. The antiTroponin I antibody [MF4] (product code ab38210; Abcam, Cambridge, UK) was obtained dissolved in PBS buffer, pH 7.4. Buffer was exchanged to 200 mM ammonium acetate buffer, pH 7, by loading 21 µL (40 µg) of the antibody stock solution into a centrifugal filter (Microcon with a cutoff of 50 K; Merck Millipore Ltd., Tullagreen, Carrigtwohill, Co. Cork, Ireland). Then 200 mM ammonium acetate buffer, pH 7, were added to reach a volume of 500 µL. The solution was centrifuged at 13,000 rpm for 10 min. The filtrate was discarded. To the retentate on the filter (ca. 30 µL) 470 µL of 200 mM ammonium acetate buffer, pH 7, were added to reach a total volume of 500 µL. The solution was centrifuged again. This centrifugation/re-filling procedure was repeated eight times. After the last spinning, the filter unit was inverted into a new vial and was centrifuged at 4500 rpm for 5 min to collect the retentate (52 µL). Protein concentration was determined to be 0.33 µg/µL using the QubitTM 2.0 Fluorometer (Carlsbad, CA, USA) assay. To obtain the immune complex with molar ratio of 2.2:1 of peptide to antibody the antiTroponin I antibody solution (0.225 µM) was diluted 1:2 with 200 mM ammonium acetate and 4 µL were mixed with 1.37 µL of the Troponin I peptide solution 1 which previously had been diluted 1:100 with 200 mM ammonium acetate. The immune complex-containing mixture was incubated at room temperature for at least 1 h. For each measurement, 3 µL of antibody-peptide complex-containing solution were loaded into nanoESI capillaries using a microloader pipette tip (Eppendorf, Hamburg, Germany).
