February 1, 2014 (Vol. 34, No. 3)

Xing Wang, Ph.D.
Michael Davies
David Wang Vanderbilt University

PEP Technology Can Also Be Used to Speed Protein Purification

Over the past few decades, much progress has been made in the effort to understand the biological processes involved in many diseases. Genomics, proteomics, metabolomics, and other systematic approaches have provided an understanding of many disease mechanisms. However, one area of systematic biological analysis that is still relatively underserved is the rapid elucidation of the biological functions of a proteome.

Compared with the systematic analysis of gene expression or gene mutations from a genome, protein functions can currently only be analyzed individually or on a few proteins from a proteome. Array Bridge’s PEP technology is intended to fill in this knowledge gap, providing a tool to systematically analyze the function of proteins and create an overall picture of the functional proteome.

Compared with the relatively simple physiochemical properties of DNA and mRNA, the complexity of proteins is far greater. Different proteins have different sizes, shapes, and charges. In addition, the function of a protein is directly related to its 3D structure. However, some of the factors involved in defining a protein’s 3D structure are weak chemical bonds, allowing proteins to relatively easily lose shape under harsh conditions, making it challenging to develop a suitable technology for systematic analysis of protein functions.

Two-dimensional gel electrophoresis (2D PAGE) is an effective method for separating thousands of proteins within a proteome; however, the traditional 2D PAGE method results in denaturation of proteins, making protein function analysis impossible.

In PEP technology, the powerful separation capability of 2D gel was retained but factors that cause denaturation of protein function were modified. Furthermore, an effective protein-recovery method was developed to collect the separated proteins from 2D gel into microplates to facilitate systematic protein function analysis and mass spectrometry identification. A diagram of PEP technology is provided in Figure 1.

Basically, proteins are first separated by isoelectric focusing (IEF), followed by a modified 2D gel process. In these steps, no reducing agent is used so that the disulfide bonds are maintained, and free cysteines are not blocked by iodoacetamide. Furthermore, a refolding process is implemented after the IEF step to allow the diffusion of 8 M urea and the refolding of the proteins after IEF. During protein elution from the 2D gel to the PEP plate, a recovery solution was developed to reduce protein diffusion from the plate and also help to protect protein function. The large-format PEP plate has 1,536 wells to fit with large-format 2D gels for complex protein separation and recovery, and also provide a high-resolution so that each well contains one or just a few proteins.

Following PEP transfer and collection, the proteins from each well can be analyzed for protein function and size determination by standard SDS-PAGE. This information will help to determine the identity of the protein based on its size and function if more than one protein is identified by mass spectrometry in a single well.

During the development of PEP technology, several important questions were addressed: 1.) Would the proteins be separated and transferred effectively? 2.) After the transfer to the PEP plate, could the proteins be recovered effectively? 3.) After transfer of the proteins to the microplate, could enzyme assay/protein function be analyzed and MS be used for protein identification?

Figure 1. With PEP technology, proteins are separated by isoelectric focusing and then subjected to a modified 2D gel process. The large-format PEP plate has 1,536 wells. Following PEP transfer and collection, the proteins from each well can be analyzed by standard SDS-PAGE.

Application Areas

With the ability to effectively recover and analyze enzyme activities systematically, PEP technology can be used in many areas of biological research and drug discovery. For example, PEP technology can be used for the systematic analysis of enzymes involved in metabolism. Figure 2 showed that NADH-dependent oxidase (or dehydrogenases if using NAD as cofactor) could be analyzed from a proteome of interest systematically; many fractions with putative enzyme activity were detected from a proteome, thus providing a snapshot of NADH oxidase activities.

Another area of application is for activity-based biomarker discovery; NADH oxidase activity from human serum or other enzymes such as proteases that exist in the human serum in large numbers can also be analyzed to look for disease signatures or treatment biomarkers. A third potential application is evaluating drug safety.

Figure 2. PEP technology can be used for the systematic analysis of metabolic enzymes. In this example, NADH-dependent oxidase is analyzed from a proteome of interest.

Developing a protein kinase inhibitor that only targets the kinase of interest remains challenging. PEP technology can be used as an effective screening tool to test the specificity of the kinase inhibitor to find out if the protein kinase inhibitor also inhibits other protein kinases besides the intended target. By expanding the application further, PEP technology can be used to answer more general questions, i.e., besides hitting the protein kinase, is this inhibitor hitting other classes of enzymes or functional protein classes, especially those using ATP as a cofactor?

Finally, PEP technology can be used as a simple protein purification tool. For example, if the protein mixture is already fractionated, the fraction can be applied to 1D gels, and the protein of interest can be separated and recovered by small-format PEP plate (Figure 3).

If, however, the protein of interest is in a protein mixture of hundreds to thousands of proteins, a large-format PEP plate can be used, as can be seen in Figure 1; multiple proteins with NADH oxidase activity can be purified directly from a complex proteome. Because no reducing agent is used, some proteins with heterologous subunits and linked by disulfide bonds will appear as two or more protein bands in the standard SDS-PAGE in the presence of reducing agent.

In many cases, however, the protein of interest can still be positively identified by mass spectrometry and bioinformatics analysis because it is unlikely that more than one protein from among the two or a few protein bands from one well in the PEP plate will share the same enzyme activity and molecular size.

PEP technology was developed to fill a knowledge gap in proteomics research. By analyzing enzyme activity or protein function systematically, an overall function-based picture can be constructed. This technology can also be used in drug development for target identification, drug safety evaluation, and biomarker discoveries. With the integration of this activity-based knowledge to other knowledge bases from genomics, proteomics, and metabolomics, it will advance our understanding of complex disease mechanisms thus targeting diseases more rationally.

Figure 3. If the protein mixture is already fractionated, the fraction can be applied to 1D gels, and the protein of interest can be separated and recovered by small-format PEP.

Xing Wang, Ph.D. (xing.wang@arraybridge.com), is president and Michael Davies is a senior scientist at Array Bridge. David Wang is a student at Vanderbilt University who did summer research at Array Bridge.