Quantitative LC-MS Analysis of CC3-Fc

CC3-Fc concentrations in plasma were analysed by quantitative LC-MC method described by Steven et al. [50 (link)]. Briefly, 50 μl Protein A-Sepharose was added to wells of a Millipore filter plate and conditioned with PBS pH 7.4. Plasma samples were diluted 1 : 4 with PBS and added to the filter plate, incubated at room temperature and agitated at 700 rpm for 1 h. Wells were washed four times with 200 μl PBS using a vacuum manifold. Bound Fc protein was eluted twice with 25 μl of 100 nM glycine pH 2.0 into a low-bind deep-well block and neutralised by adding 7.5 μl 2M Tris pH 8.0. Eluted protein samples were then trypsin-digested by addition of 20 μl of proteomics-grade trypsin (made up at 20 μg/mL in 10 mM CaCl₂, 50 mM Tris-HCl, pH 8.0) followed by incubation at 37°C for 18 h prior to LC-MS/MS analysis. Samples were analysed using LC-MS/MS by monitoring tryptic signature peptides resulting from CC3-Fc present in the samples.