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. 2015 Dec 8;3(4):454-466.
doi: 10.3390/proteomes3040454.

Bead Based Proteome Enrichment Enhances Features of the Protein Elution Plate (PEP) for Functional Proteomic Profiling

Xing Wang  1 , Michael Davies  2 , Swapan Roy  3 , Matthew Kuruc  4
Affiliations

Bead Based Proteome Enrichment Enhances Features of the Protein Elution Plate (PEP) for Functional Proteomic Profiling

Xing Wang et al. Proteomes. .

Abstract

A novel functional proteomics technology called PEP(Protein Elution Plate) was developed to separate complex proteomes from natural sources and analyze protein functions systematically. The technology takes advantage of the powerful resolution of two-dimensional gel electrophoresis (2-D Gels). The modification of electrophoretic conditions in combination with a high-resolution protein elution plate supports the recovery of functionally active proteins. As 2DE(2-Dimensional Electrophoresis) resolution can be limited by protein load, we investigated the use of bead based enrichment technologies, called AlbuVoid™ and KinaSorb™ to determine their effect on the proteomic features which can be generated from the PEP platform. Using a variety of substrates and enzyme activity assays, we report on the benefits of combining bead based enrichment to improve the signal report and the features generated for Hexokinase, Protein Kinase, Protease, and Alkaline Phosphatase activities. As a result, the PEP technology allows systematic analysis of large enzyme families and can build a comprehensive picture of protein function from a complex proteome, providing biological insights that could otherwise not be observed if only protein abundances were analyzed.

Keywords: 2-D gel; PEP; biomarkers; cancer; functional proteomics; protein enrichment.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Simplified workflow of the combined enrichment and the PEP technology.
Figure 2
Figure 2
Hexokinase activity of normal and lung cancer patient serum, with and without AlbuVoid™ enrichment.
Figure 3
Figure 3
Hexokinase activity at 180 min (a) and 300 min (b) after KinaSorb™ treatment of human serum. The readings at 340 nm were from each of the 384-well microplate with a portion of the proteins recovered from the PEP plate.
Figure 4
Figure 4
Hexokinase activity at 180 min (a) and 300 min (b) after KinaSorb™ treatment of beef liver protein. The readings at 340 nm were from each of the 384-well microplate with a portion of the proteins recovered from the PEP plate.
Figure 5
Figure 5
Protein kinase from beef liver after KinaSorb™ enrichment.
Figure 6
Figure 6
Alkaline phosphatase after AlbuVoid™ treatment of human serum at 60 min (a) and 120 min (b). The readings at 405 nm were from each of the 384-well microplate with a portion of the proteins recovered from the PEP plate.
Figure 7
Figure 7
Protease activity overnight after AlbuVoid™ treatment of human serum.
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