Multiplexed proteomics

The use of new fluorescence technologies in the application of proteomics to drug discovery is discussed by Molecular Probes' Peter Roberts

Proteomics has rapidly become an important tool in drug discovery, triggering increased interest in its applications and associated technologies. However, research in the field poses many technical challenges, not least in meeting increasing demands for high throughput and information rich techniques.One of the core methods in proteomics is 2-D gel electrophoresis (2-DGE), which separates proteins on a gel in one direction on the basis of molecular weight and then in a second direction depending on the charge of a protein. The resulting 2-dimensional image reveals to the researcher how many proteins are present and a comparison of gels will show altered protein expression levels identifying potential targets for future research.

sensitivity issues

The demands of proteomics research have exposed the limitations of these conventional protein detection methods and have driven the development of fluorescence-based techniques with enhanced detection sensitivity, high throughput capabilities and quantitative accuracy. Through these new technologies phosphorylated and glycosylated proteins can now be identified in 2-DGE. These post-translational modifications of proteins yield clues as to their functionality and hence their potential importance in drug discovery.

2-DGE is the principal protein separation technology capable of separating thousands of proteins on a single gel and presenting the proteome in such a way as to make it relatively easy to analyse changes in protein expression.

Requiring little specialised equipment and being low in cost, 2-DGE is likely to remain a key proteomics tool with new fluorescence technologies adding to the information yielded from a single gel.

Traditionally, it has relied on protein detection by silver staining or Coomassie blue. These were the leading non-radioactive visualisation methods for detecting low, nanogram levels of protein. However, these staining methods fail to meet today's requirements in proteomics research and it has been necessary to develop staining with increased detection sensitivity, quantitative accuracy and compatibility with modern higher throughput protein identification and characterisation procedures.

Silver staining is a complex procedure requiring multiple carefully timed steps. This leads to variability in results, makes automation difficult, and limits the ability to quantify small changes in signal intensity. In addition, quantification is restricted by the limited dynamic range of response for silver stains.

SYPRO Ruby protein stain (Molecular Probes) was developed to address these issues, providing background free staining without the need for destaining and giving a linear dynamic range over three orders of magnitude - unmatched by any other single non-radioactive protein imaging methodology.

While total protein stains show the expression levels of proteins, they do not provide information on their functionality. It is important to yield further information from 2-D gels, such as which proteins are phosphorylated and have altered phosphorylation, and which are glycosylated and which are altered.

Phosphorylation is a common and important post-translational modification involved in the regulation of proteins in almost every biological pathway. Glycosylation is another common modification with importance in cell adhesion, cell communication, immune recognition, inflammation and protein folding. Changes in glycosylation are also associated with many types of cancer.

Fluorescent dyes Pro-Q Emerald and Pro-Q Diamond were developed to show glycosylated and phosphorylated proteins respectively.

multiplexed proteomics

With the development of fluorescent dyes using different excitation and emission spectra to highlight specific functional attributes of proteins in addition to total protein expression levels, it is now possible to determine expression and functional attributes in a single 2-DGE experiment. Initially a gel is fluorescent stained to detect a functional attribute such as post-translational modification or drug binding capabilities. Then SYPRO Ruby is used on the same gels to show total protein. Differential display analysis is used to compare gels and changes in expression levels.

With the introduction of multiplexed proteomics, traditional 2-DGE has been combined with non-overlapping fluorescent dyes to give a complete picture of altered protein expression. This has applications in highlighting potential drug targets for future research and is achieved in an efficient, high throughput and reproducible manner.

With the potential development of further fluorescent dye technologies, it would be expected that future analysis of proteins in a single 2-DGE would be capable of showing an even greater variety of functional attributes, therefore increasing research potential.