Spin columns

For solid phase permethylation, partially purified plant extract (7 mg) was dried in a high vacuum concentrator and re-suspended in high purity DMSO (30 µl) with H₂O (1.2 µl) and methyl iodide (20 µl). Mixed sample was applied to spin columns (Harvard Apparatus, Holliston, MA) packed with sodium hydroxide beads (3 cm height) and rinsed with DMSO; the mixture was allowed to react at room temperature for 25 minutes. The column was then centrifuged at 1.6 K rpm for two minutes and the solution collected. A second aliquot of methyl iodide (20 µl) was added to the solution and the reaction mixture reapplied to the column, incubated for 15 minutes and re-centrifuged for two minutes. The column was rinsed with ACN (50 µl) centrifuged two minutes at 1.6K rpm and combined column elutions dried in vacuo.

PNGase F was obtained from New England Biolabs (Ipswich, MA). The mouse anti-human haptoglobin antibody was purchased from Abcam (Cambridge, MA). Borane-ammonia complex, sodium hydroxide beads, and iodomethane were acquired from Sigma-Aldrich (St. Louis, MO). Dimethyl sulfoxide (DMSO) was bought from Mallinckrodt Chemicals (Phillipsburg, NJ). HPLC grade methanol and acetonitrile were purchased from Fisher Scientific (Fair Lawn, NJ). HPLC water was obtained from Avantor (Central Valley, PA). Spin columns were purchased from Harvard Apparatus (Holliston, MA).
Serum samples were provided by the University Hospital, Ann Arbor, Michigan according to IRB approval (10 cases of hepatocellular carcinoma (HCC) and 10 cases of liver cirrhosis.) The clinical information associated with the samples used in this study are summarized in Table 1 and Table S1.