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.
Highlights
The field of proteomics endeavors to systematically identify, construct and contrast proteomes based on protein quantities or abundances of individual gene products
Isoelectric Focusing (IEF) unit capable of running IEF at different lengths is from Bio-Rad (PROTEAN IEF Cell, Hercules, CA, USA)
The protein concentration was determined by BCA before 2-D gel electrophoresis
Summary
The field of proteomics endeavors to systematically identify, construct and contrast proteomes based on protein quantities or abundances of individual gene products. The development of analytical technologies in genomics and proteomics have made it possible to systematically study gene regulation, protein expression and post-translational modifications on a large scale. This has advanced our understanding of complex biological systems tremendously. Chemically synthesized probes were used to study enzyme families such as serine hydrolases, cysteine proteases, and tyrosine phosphatases These approaches included the use of a complex naturally derived proteome, and the proteins with high affinity towards the probes were isolated and further identified with LC-MS. These enzyme activities can be measured systematically from hundreds to thousands of fractions depending on the complexity of the proteome and degree of resolution necessary for the profile and analysis
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