A System Based on Principle of Two-Dimensional Liquid Chromatography of Proteins as an Alternative to Two-Dimensional Electrophoresis

Abstrakt

New analytical systems alternative to two-dimensional polyacrylamide gel electrophoresis (2D-E) of proteins in very complex biological samples have been developed recently. One of them, two-dimensional liquid protein fractionation system PF 2D, has been used in our study and compared with previously used 2DE. PF 2D is based on 2D HPLC technique, where proteins are separated according their isoelectric point and hydrophobicity in the 1st and 2nd dimension, respectively. Fractions are collected in 96-well microplates; proteins remain in liquid phase during the entire separation process. Omitting solid phase is beneficial to subsequent protein identification by mass spectrometry. Separation data are processed using ProteoVue software and the resulting 2D protein maps are compared using DeltaVue software package generating a differential display view of up-regulated and down-regulated proteins. PF 2D approach have been used for interlysate comparison between cultured T-lymphoblastic leukemia CEM cells grown in the presence or absence of new synthetic cyclin-dependent kinase inhibitor, bohemine. The results indicate that both proteomics technologies applied to the same sample provide complementary data. The use of PF2D contributes to a more complete understanding of the biological system studied.