Figure 1: (a) Schematic work flow. PBMCs were obtained from heart failure subjects (HF, ) and normal healthy (NH, ) subjects. Each sample was divided into two fractions, and S-NO cysteines were reduced with ascorbate (Asc+) in one fraction and stabilized with neocuproine in 2nd fraction (Asc). All fractions were labeled with BODIPY FL -(2-aminoethyl) maleimide (binding to reduced cysteine) and resolved by 2-dimensional gel electrophoresis. Gel images were normalized against a reference gel. Ratiometric calculation of differential protein abundance from BODIPY-fluorescence units in Asc+ aliquots (normal versus experimental) was calculated for all the protein spots (protein abundance = Asc+ HF/Asc+ NH). The S-NO modification levels were quantified by calculation of the ratio of fluorescence units from Asc aliquots (S-NO = Asc HF/Asc NH). The ratio of ratios (RoR), that is, S-NO/protein abundance = [Asc HF/Asc NH]/[Asc+ HF/Asc+ NH], was calculated to obtain the change in S-NO levels normalized for protein abundance. The fold changes in abundance and S-NO-modification of the protein spots in all gels were log transformed and subjected to statistical analysis as described in Materials and Methods. Protein spots that changed in abundance or S-NO modification by |≥1.5-fold| at were submitted to mass spectrometry analysis for protein identification. The protein datasets were analyzed by ingenuity pathway analysis and MARS modeling, and selected proteins were confirmed for differential abundance and S-NO modification levels by multiple assays. (b) Two-dimensional gel images of protein spots in PBMCs of heart failure (HF) subjects and normal healthy controls. BD-labeled PBMC lysates were separated in the 1st-dimension by isoelectric focusing on 11 cm nonlinear pH 3–11 immobilized pH gradient strips and in the 2nd-dimension by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on an 8–16% gradient gel. Gel images were obtained at 100 µm resolution using the Typhoon Trio Variable Mode Imager (GE Healthcare) to quantify BD-labeled proteins (). Shown are representative gel images of Asc+ ((A) and (B)) and Asc ((C) and (D)) PBMCs from NH ((A) and (C)) controls and HF ((B) and (D)) subjects and approximate size (vertical) and pI (horizontal) ranges.