Haptoglobin (Hp) was purified from 20 μL of patient serum using an antibody-immobilized HPLC column which has a recovery of 40–50% of Hp from serum samples as described previously.37 (link) The immunopurification was performed on a Beckman Coulter HPLC system (Fullerton, CA) with a PEEK column (4.6 mm × 50 mm) packed in-house with the UltraLink hydrazide resin (Thermo Scientific, Rockford, IL) conjugated with an antihuman Hp antibody (Abcam, Cambridge, MA). The eluted haptoglobin fraction was desalted using a 4 mL YM-3 centrifugal device by buffer exchange 3 times with deionized water and then dried down in a SpeedVac concentrator (Thermo). The HPLC peak area of the eluted Hp fraction was measured and compared among patients (Supplemental Table S1), where SDS-PAGE gel analysis of the Hp eluent showed the purity of haptoglobin fraction (Supplemental Figure S1). One-tenth of the Hp eluent was loaded onto the gel, with 0.3 μg of a haptoglobin standard protein (Abcam) as a reference. On the basis of the gel result, the amount of haptoglobin used for the subsequent trypsin digestion was estimated to be around 3 μg.