Workflow for proteomic analysis. Proteins in the plasma or urine of healthy controls and hepatitis E patients were labelled with Cy2, Cy3 or Cy5 dyes as described in Methods. The labelled samples were mixed and subjected to different fractionation strategies. In workflow A, the labelled samples were first subjected to Multi Lectin Affinity Chromatography (MLAC) to obtain fractions enriched in glycoproteins (G fraction) and non-glycoproteins (NG fraction). These fractions were then separately fractionated by mixed cation-anion exchange chromatography (CAX). The six fractions obtained by step salt elution were then analysed by SDS-PAGE. In workflow B, the labelled samples were subjected to MLAC and the enriched fractions were resolved by two-dimensional gel electrophoresis (2DGE). Workflows A and B were followed for plasma samples. Workflow C was followed for urine samples for which the labelled samples were directly fractionated by 2DGE. The differentially expressed spots were analysed with DeCyder 2D v6.5, the selected spots were cut out and the proteins identified by mass spectrometry.