April 16, 2004 | Despite all the attention being given to the development of protein biochips (see "Fishing Chips," Sept. 2003 Bio·IT World, page 60), proteomics research labs remain dominated by 2-D gel electrophoresis (2DGE). 2DGE has historically been hampered by low throughput and poor reproducibility, and seldom detects low-abundance proteins — frequently the most important disease-related proteins in a human proteome, estimated to comprise hundreds of thousands of individual proteins. However, the predominance of 2DGE technology continues to spur refinements and the development of increasingly high-throughput modalities.
The digital ProteomeChip is Protein Forest's miniaturized chip for the separation of proteins in complex mixtures by both charge and size. Separation by isoelectric point in the first dimension is effectively digitized, avoiding the smearing that often occurs in a linear gradient, whereas the second dimension uses standard electrophoresis. "The greater accuracy of the separation by isoelectric point is the crux of the method, giving rise to greater reproducibility," says CEO Russell Garlick.
Separation through the microchip takes merely minutes, resulting in significantly increased productivity. The sensitivity of the chips also allows the identification of proteins of low abundance and/or unique migration characteristics. Proteins can be quantified, allowing proteomewide expression profiling in one chip format, while a second format is designed for subsequent analysis by mass spectrometry (MS) for identification of unknown proteins.
The digital ProteomeChip package includes analytical software to decode the grayscale image of the silver-stained chips to create a contour map and a 3-D image (see picture). The software allows users to develop internal databases that are compatible with external databases, facilitating the identification of proteins and data exchange.
No More Messy Cleanup
Another innovative solution is Proteome Systems' Chemical Inkjet Printer (ChIP). ChIP uses piezoelectric printing technology to dispense minute amounts of reagents onto selected protein spots electroblotted from 2-D gels onto polyvinylidene fluoride (PVDF) membranes. The device allows researchers to skip the cumbersome excision, digestion, extraction, cleanup, and target-spotting steps. After processing, the membrane can be placed directly into the mass spectrometer for peptide analysis. The system can easily lay down a 4x4 reagent grid on a 2mm protein spot, enabling the simultaneous analysis of an individual sample with various enzymes.
SEEING THE FOREST: Image from Protein Forest's Proteome-Chip of proteins separated and visualized from human serum, showing pI addresses of separated proteins and their abundance (by silver staining) in the pH range 5 to 6.
Protein separation is not limited to solid-phase tools. Beckman Coulter provides a fluidic system called ProteomeLab for 2-D protein separation and analysis by high-performance liquid chromatography (HPLC). Chromatofocusing is followed by nonporous reverse-phase chromatography for high-resolution separation of proteins from complex mixtures. Liquid fractions can be stored or analyzed by electrospray ionization — MS or MALDI plate spotters.
Similarly, Eksigent Technologies' NanoLC-2D Proteomics System combines automated 2-D HPLC with precise nanoscale gradients and simple operation, facilitating multidimensional peptide analysis. The NanoLC-2D System uses Eksigent's direct pumping nanoscale microfluidics, providing enhancements in detection sensitivity and reproducibility for LC/MS proteomics studies. It also offers added convenience by automatically running 2-D peptide separations, such as the MudPIT method used for analysis of membrane proteins.
Robert M. Frederickson is a biotech writer based in Seattle. E-mail: firstname.lastname@example.org.