Analysis Platforms

ActivX has developed three unique platforms for analyzing ABP treated samples (Page 2, Figure 1). These platforms can function independently or in concert depending on the level of detail and accuracy required in the results as well as the required throughput.

Xhibit ®

Whole Protein Based Activity Analysis

Xhibit is an automated, high-throughput gel electrophoresis platform capable of sensitive activity-based probe analysis. Xhibit uses 1-dimensional (1D) SDS-PAGE to separate activity-based probe labeled proteins according to their size. Xhibit is operated in a standard 96-well format and is compatible with all fluorescent ABPs. In addition, live cells in a 96-well format can be analyzed with ABPs following treatment with inhibitors, growth factors etc. Proprietary software is used for lane detection on the high density, 96-well Xhibit gels.

An example Xhibit gel image is shown in Figure 2. In this example, proteomic samples were treated with multiple concentrations of inhibitors for IC50 determination. Following automated lane finding and data extraction, each lane/sample is converted to an intensity trace as shown in Figure 3.

Xceed™ and Xsite ®

Active Site Peptide Profiling

Two stand-alone analysis technologies, Xceed and Xsite, utilize the active site peptide-profiling concept. These technologies differ fundamentally from Xhibit in that Activity Based Probe (ABP) labeled samples are digested with a protease (Trypsin) to yield probe-labeled peptide fragments prior to analysis. Several advantages are gained by peptide based analysis including higher resolution, non-size based separations, and the ability to precisely define probe labeling sites on target proteins.

ActivX has perfected methods for generating extremely reproducible protease digests in a broad range of proteomic samples such as raw plasma, and protease rich tissues. Protease digested ABP-labeled samples can be subjected to two platforms for analysis, capillary electrophoresis based Xceed, or mass spectrometry based Xsite.

Xceed The capillary electrophoresis based Xceed technology is ideally suited for high-throughput separations using minimal amounts of sample. The resolution and sensitivity achieved by Xceed are unparalleled by other high-throughput proteomics methods, with detection limits in the high attomolar range (10 -18 moles) and resolution of up to 300 species per run. Quantitation is also achieved with high precision and enables the characterization of IC 50 values for small molecule protein interactions, as well as measurement of activity levels across diverse proteomes. All sample preparation steps for the Xceed method have been automated in the 96-well format, and a highly customized 96-channel free-zone capillary electrophoresis instrument is used for high-throughput separations. Xceed is compatible with all fluorescent ABPs.

An example Xceed run is shown in Figure 3. In this example, the mouse submaxillary proteome was analyzed in the presence/absence of a protease inhibitor. The submaxillary gland is rich with proteases in the kallikrein family which are highly related, all having molecular masses of approximately 25 kDa. Thus these enzymes cannot be separated by the SDS-PAGE.

Xsite is a high content, quantitative mass spectrometry (MS) based platform for ABP analysis. XsiteTM is ideally suited for the analysis of probes that have highly complex labeling profiles (such as the Kinase/ATPase probes) that can’t be fully resolved using either the Xhibit or Xceed platforms. Additionally, XsiteTM is used to identify ABP targets discovered using the Xhibit and Xceed platforms. The Xsitesystem is the highest resolution, highest sensitivity technology in use at ActivX, but the throughput is substantially lower than Xhibit and Xceed. The identity and site of probe labeling can be determined for more than 1000 labeled proteins in a single Xsiteanalysis. Additionally, ActivX has developed proprietary software for automated, quantitative comparison of MS data. The quantitative accuracy of the XsiteTM platform is suitable for identifying targets of small molecules, IC50 determinations, and characterizing changes in protein expression/activity following cell or live animal treatments.

Examples of XsiteTM data can be found on the Kinase/ATPase probe page.