Proteomelab pps system

Haptoglobin was purified from a 20-μL aliquot of serum for each patient by using an HPLC-based antibody-immobilized column developed in-house as previously reported [38 (link)]. Briefly, the mouse anti-human haptoglobin antibody was covalently immobilized on the UltraLink hydrazide resin (Thermo Scientific, Rockford, IL) and then packed into a PEEK column (4.6 mm × 50 mm). The immunoaffinity purification of haptoglobin was performed on a Beckman Coulter ProteomeLab PPS system (Fullerton, CA) based on the HPLC platform developed previously [38 (link)]. The bound haptoglobin fraction was eluted with stripping buffer (0.1 M Glycine, pH 2.5) and then immediately neutralized with 0.1 M Tris-HCl (pH 8.0). Subsequently, the enriched haptoglobin was desalted using a YM-3 centrifugal filter device (Millipore, Billerica, MA) by buffer exchange with water for three times and then dried down in a SpeedVac concentrator (Thermo). Before glycan release, the purity of the eluted haptoglobin was confirmed by 1D SDS-PAGE followed by silver staining using ProteoSilver™ Plus Silver Stain Kit (Sigma).

Haptoglobin was purified from up to 20 μL patient serum using an anti-haptoglobin antibody immobilized HPLC column developed in-house as reported previously¹⁴ . The purification was performed on a Beckman Coulter Proteome^™ Lab PPS system (Fullerton, CA) with a flow rate of 0.5 mL/min for 40 min. A custom-made antibody conjugated resin column (4.6 mm × 50 mm) was used for serum haptoglobin enrichment. Briefly, the serum sample was diluted with 200 μL of dilution buffer (10 mM TBS, 150 mM NaCl, pH 7.4) and then loaded onto the HPLC column. The bound fraction was eluted with stripping buffer (0.1 M Glycine, pH 2.5) and neutralized with the neutralization buffer (0.1 M Tris-HCl, pH 8.0). The eluted haptoglobin fraction (~ 3.5 mL) was desalted using a 4 mL YM-3 centrifugal device (Millipore, Billerica, MA) by buffer exchange three times with HPLC water and then dried down in a SpeedVac concentrator. One-tenth of the eluted haptoglobin was checked by SDS-PAGE followed by silver staining using ProteoSilver^™ Plus Silver Stain Kit (Sigma) to confirm the purity of haptoglobin.