Fig. 2.

Flowchart of the AH proteome analysis. Pooled AH samples were further prepared via two processes (with or without depletion). A portion of the sample was separated with an ALB/IgG depletion column, and the fractions (flow-through and eluent) were subjected to SDS-PAGE and in-gel digestion (8 bands), followed by multistep fractionation methods (with a strong-anion exchanger and C18 reversed-phase fractionation) at the peptide level. The other portion was subjected directly to in-solution digestion or PLRP-S column chromatography (8 fractions) at the protein level. In-solution-digested samples were further subjected to the fractionation method of high-pH reverse fractionation (15 fractions) at the peptide level, and each PLRP-S fraction was individually digested with trypsin in solution. A total of 50 LC-MS/MS runs were completed, and the total LC-MS/MS running time was more than 100 h