Application of 2D-HPLC system for plasma protein separation

Abstract

Summary : The ProteomeLab¥PF 2D protein fractionation system is a rapid, semi-automated, 2 D-HPLC instrument that uses two different methods to separate plasma serum proteins: ion-exchange chro-matography using a wide range of pH in the first dimension and non-porous reverse-phase chromatography inthe second dimension. Because this methodology has only very recently been introduced in proteomic labora-tories, little is known about the characteristics of PF 2D fractionation of human serum proteins. To evaluate thesystem’s application in a clinical laboratory setting, the characteristics of the ion-exchange chromatography-ba-sed separation were analyzed. Following fractionation of human serum proteins on a linear gradient of pH (ran-ging from 8.5 to 4.0), each fraction was collected in a cool module of the instrument. Different fractions obta-ined from the first dimension were then pooled together and loaded on classic 2D gel electrophoresis instru-mentation. The different spots obtained were then checked against the Swiss-Prot Database. A total of 36 hu-man serum proteins were identified in different PF 2D-generated fractions. Some important features of the se-paration system were observed. Different eluted fractions contained different proteins, thus demonstrating thereliability of the fractionation system. The proteins were also fractionated according to the theoretical isoelec-tric point (p