P182-M Reproducibility of the Protein Forest digital Proteome Chip as a Protein Isoelectric Fractionation Tool.

Protein fractionation is often necessary in mass spectrometry (MS) proteomic studies to help detect low-abundance proteins. Protein Forest has developed the digital ProteomeChip (dPC) technology, which is a rapid, reproducible, and easy to use protein MS pre-fractionation tool. The dPC consists of discrete pH gel features with pH intervals of 0.1 or smaller, which, when placed in an electric field for 30 min, will each trap and collect proteins simultaneously from a complex mixture according to the proteins’ respective isoelectric points (pI). The resultant protein fractions can then be tryptically digested for subsequent MS identification analysis, transferred to traditional SDS-PAGE, or blotted to a membrane for antibody staining. The individual pH features allow researchers to reproducibly extract protein fractions from a complex mixture for detailed analysis, such as in biomarker discovery and clinical assay applications. We evaluated the isoelectric fractionation reproducibility of the dPC technology using I125-labeled human growth hormone (hGH) as radioactive tracers spiked into a model complex protein mixture (E. coli lysate). We collected focused I125-hGH predominantly in the pH 5.2 to 5.4 fractions, and the average radioactive counts recovered were very reproducible, with a coefficient of variance of 16%. We also demonstrated the utility of a dPC in fractionating E. coli lysates when coupled to subsequent LC/ESI-MS, SDS-PAGE, and immunoblotting analyses. The dPC is a novel fractionation technology that provides improved throughput and reproducibility compared to existing protein fractionation techniques. Our technology can be easily coupled to most existing downstream analysis techniques, allowing great research flexibility. Finally, the robustness of the chip format, along with the digital nature, also makes the dPC amenable to customization and automation.