Proteominer

The use of hexapeptide libraries (ProteoMiner^®, Biorad Laboratories Inc., Hercules, CA, USA) can significantly increase the detection of medium- and low-abundance proteins by the partial removal of high abundance proteins [14 (link)]. ProteoMiner® treatment was therefore applied to the entire cohort of serum samples. All serum samples were processed in 5 days according to manufacturer’s instructions. After removal of unbound fraction with PBS buffer (3 × 5 min of incubation), proteins were eluted with 3 × 100 µl of 25 mM Tris, 7 M urea, 2 M thiourea, 4 % CHAPS. A quality control (serum provided from a healthy volunteer) was run throughout the 5 days of ProteoMiner® process to control the reproducibility across the time. It is presented in Additional file 1: Appendix Figure 1.

The secretory proteins present in low abundance in comparison with the BSA or serum proteins. Enrichment of secretory proteins was carried out using ProteoMiner™ Bio-Rad (Hercules, CA, USA) protein enrichment technology based on binding of proteins to a library of combinatorial peptide ligands that act as unique binders for proteins [52 (link),53 (link)].
The supernatant was dialyzed against PBS (containing 150 mM NaCl, pH 7.4) to facilitate optimum binding condition to ProteoMiner™. Slurry from ProteoMiner™ Large-Capacity column (100 μL settled beads) was washed two times with 1 mL of PBS and added to 100 mL of the supernatant and allowed to bind overnight (>8 h) under shaking at 4 °C. After binding, the beads were allowed to settle and we removed the clear volume of supernatant, repacked in the ProteoMiner™ column, and carried out 2 × 100 µL washes with PBS. The elution 2 × 20 µL was made using Elution Reagent (8 M urea, 2% CHAPS) supplied by the manufacturer. The eluted secretory protein was subjected to quantification, electrophoretic characterization, and trypsin digestion to obtain peptides and further high-resolution LC-MS/MS based proteomics.

Enrichment of low-abundance plasma proteins using the CPLL column (ProteoMiner; #163-3006, Bio-Rad Laboratories, Inc., CA, USA) was performed according to the manufacturer's instructions. Briefly, the CPLL column was prepared by adding 200 μl wash buffer (BioRad) and rotating the column several times over a 5 min period. The wash buffer was removed by centrifugation at 1,000 × g for 1 min. This step was repeated once. Thereafter, 200 μl of plasma was added to the column followed by incubation for 2 h at room temperature with gentle mixing. The unbound proteins were then removed by 1000 x g centrifugation for 1 min, and the column was washed twice using 200 μL wash buffer (BioRad) and additionally washed by 200 μL deionized water to remove unbound proteins and salt contamination. The bound proteins were eluted by adding 20 μl of elution reagent (BioRad) and then incubation for 15 min with intermittent gentle mixing. The eluted proteins were collected by centrifugation at 1,000 × g for 30-60 sec. This elution step was repeated twice. The eluate was kept at -80°C until further analysis.