ProGRP isoform 1 (AA 1-125 + 8) was cloned from human cDNA (OriGene Technologies), expressed in Escherichia coli (Promega) using pGEX-6P-3 constructs (GE Healthcare) and purified as described elsewhere.16 (link) The concentration of the ProGRP stock solution was determined by absorbance at 280 nm (A280). Working solutions were prepared by dilution with 50 mM ammonium bicarbonate solution (ABC solution) and stored at 4 °C.
Initially, complex samples were prepared by adding Lys-C or trypsin digested ProGRP to trypsin digested human serum from healthy subjects. The digested standards were added to the digested serum immediately before performing the extraction. In later experiments, spiked serum samples were prepared by adding intact ProGRP immediately before digestion with trypsin beads.
Initially, complex samples were prepared by adding Lys-C or trypsin digested ProGRP to trypsin digested human serum from healthy subjects. The digested standards were added to the digested serum immediately before performing the extraction. In later experiments, spiked serum samples were prepared by adding intact ProGRP immediately before digestion with trypsin beads.