13c6 lysine

QconCAT DNA constructs were synthesized de novo by Genscript and expressed in Escherichia coli auxotrophic strain BL21(l)DE3-LysA ArgA in minimal medium supplemented with 13C₆ arginine and 13C₆ lysine at 0.1 mg/mL (Sigma Aldrich). The cells were grown to mid-log phase (absorbance at 600 nm [A₆₀₀] = 0.6–0.8), at which point expression was induced by adding 1 mM isopropyl-D-1-thiogalactopyranoside. After 4 hours of growth at 37°C, the cells were harvested by centrifugation and processed as previously described with minor modifications.^{13 (link)} Briefly, the cells were lysed with the Bug Buster Protein Extraction Reagent (EMD Millipore). Inclusion bodies were suspended in 20 mM phosphate buffer, 6 M guanidinium chloride, 0.5 M NaCl, and 20 mM imidazole, pH 7.4. QconCAT proteins were desalted by affinity chromatography using a nickel-based resin. The purified QconCAT was desalted by three rounds of dialysis against 100 volumes of 10 mM ammonium bicarbonate, pH 8.5.

In agreement with Pratt et al. [26 (link)], QConCAT constructs were designed to quantify plasma and PLTs specific proteins. The selection of target peptides was based on the result of previous non-targeted experiments of plasma and publicly accessible databases, including PeptideAtlas (www.peptideatlas.org) SRM Atlas (www.srmatlas.org) and Global Proteome Database (http://gpmdb.thegpm.org/). The QConCAT DNA construct was synthesized de novo and cloned into pET21a by Genscript (Piscataway, NJ ). E. coli strain BL21(l)DE3 was transformed with the vector and cultured in minimal medium supplemented either with unlabeled or ¹³C₆ arginine and ¹³C₆ lysine at 0.1 mg/ml (Sigma Aldrich). The cells were grown to mid log phase (A600 0.6–0.8), at which point expression was induced by adding 1 mM isopropyl-D-1-thiogalactopyranoside. The cells were lysed with the BugBuster Protein Extraction Reagent (EMD Millipore). Inclusion bodies were suspended in 20 mM phosphate buffer, 6 M guanidinium chloride, 0.5 M NaCl, 20 m M imidazole, pH 7.4. QConCAT proteins were purified by affinity chromatography using a nickel-based resin. The purified QConCAT was desalted by three rounds of dialysis against 100 volumes of 10 mM ammonium bicarbonate, pH 8.5.