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).