Plasma proteomics

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Plasma proteomics is conducted in the project. Because plasma can be obtained by less invasive examination, it is an ideal resource for biomarkers for early diagnosis. Critical points of plasma proteomics is sample preparation. Abundant plasma proteins such as albumin and transferrin always interfere the observation of less abundant proteins, and the protocols to deplete such abundant proteins should be optimized according to the purpose of studies. A novel protocol based on the combination of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis (2D-DIGE) was established in the project, and used to study the plasma samples from the cancer patients.

In the protocol, highly abundant proteins are firstly depleted by immuno-affinity column. Many types of immuno-affinity column for plasma proteomics are commercially available. The important proteome data may be lost by extensive depletion, because certain proteins to be depleted by commercially available column are aberrantly regulated in cancer patients. In addition, as the proteins to be depleted by commercial immuno-affinity column function as carrier proteins, certain biomarker candidate proteins can be unexpectedly depleted with such carrier proteins. The purpose of experiments and the nature of target proteins may determine the type of immuno-affinity column. The proteins not to be depleted out are further separated by anion-exchange column. The reproducible fractionation is achieved by step-wise fractionation. The fractionated proteins are labeled with high sensitive fluorescent dyes and separated by a large format 2D gel apparatus.

The 2D gel images of plasma proteins are analyzed by image analysis software. Progenesis SameSpots may be the best solution for image analysis of plasma proteins, because the protein spots with irregular shape can be well manipulated by users.