Rapid imaging of proteins
After the enormous impact genomics has had on understanding biological systems through genome sequencing, proteomics promises even greater rewards.
CCD-based system may speed 2-D gel electrophoresis for proteomic analysis.
After the enormous impact genomics has had on understanding biological systems through genome sequencing, proteomics promises even greater rewards. In fact, clues to the solution of many biological problems in health and disease may be found through proteomics by revealing the identity of proteins at any given time in a population of cells and, ultimately, in single cells.
Advanced proteomic mapping and analysis requires separation and isolation of individual proteins from a complex mixture using 2-D gel electrophoresis (2-DGE). The “gel” in this case is a crosslinked polymer whose composition and porosity is chosen based on the specific weight and size of the target to be analyzed. The sample is applied to this gel. In first-dimension separation it is put through isoelectric focusing that separates proteins according to their isoelectric points. The second-dimension process separates proteins according to their molecular weights.
Imaging issues with all 2-DGE processes make reliable analysis challenging, including the fact that protein spots tend to overlap and show poor definition, and gels can be cluttered with “noise.” Other issues during the initial electrophoresis process can cause odd migration patterns. Therefore, it‘s not just a matter of creating the protein gels, taking an image of them, and then starting analysis. Much depends on the ability of the imaging system to produce high-quality scans or digital images and the ability of software to make adjustments to the resulting images.
To map protein spots for true proteomic analysis, 2-D gels must be imaged into a format that the analyst can manipulate and work with. Typically, this is done using high-resolution laser scanning with fluorescent protein labels and then using one of the available software packages.
Reaping the benefits
Originally CCD camera imaging was not seen as competitive as laser scanning because of image resolution. However, advances in CCD technology are ending that disparity. Syngene, a division of Synoptics (Cambridge, England), developed the Dyversity 6 2-D gel imaging system that features a 16-bit, 6-megapixel CCD-based camera setup with a variety of lighting options housed within its own light-tight, compact darkroom. The system can generate 2-D protein gel images up to ten times faster than a conventional laser-based scanner and comes with a software suite that adapts to laboratory analytical needs.
The imaging system is designed to capture 2-D gel images from fluorescent, visible stained gels and chemiluminescent blots. The 16-bit cooled CCD camera can detect 65,536 gray levels, which allows it to resolve the high density and large dynamic range of proteins found on 2-D gels. It handles the capture of a range of gel sizes in a single shot, eliminating defects resulting from image stitching.
The Dyversity 6 uses a 1.4-in.-format sensor with 6.3 million pixels in a 3072 × 2048 format, with a pixel size of 9 × 9 μm. This can be greatly extended by on-chip binning for high sensitivity up to 72 × 72 μm. The CCD-based system can produce 2-D gel images in 10 s compared to the 8 min of a typical high-end laser scanner.
In 2006, the Imperial College London‘s Proteomics Laboratory invested in the Dyversity system. Judit Nagy, director, says research programs include proteomic characterization of stem cells and mapping the insulin-resistance pathway. She needed to set up her new facility with equipment to perform critical 2-D gel electrophoresis and subsequent gel analysis of protein samples. Previously, three separate scanning systems were used to perform separate types of protein imaging and analysis—one that could image fluorescent gels, another that could detect chemiluminescence, and a third to image stained gels in the visible range.
“I wanted to replace these three systems with something new and not too expensive,” Nagy says. “The CCD camera can handle chemiluminescence, and can image visible and fluorescent stains. At the moment I use four filters, but that is less than half the options in the filter wheel.”
To test the system, the researchers used one of the most challenging applications—imaging gels that were treated with three different Cyanine dyes. These required specific wavelengths for excitation and specific filters for emission. They found they could speed up the image-acquisition time considerably without compromising image quality.
CONARD HOLTON is editor in chief of Vision Systems Design; e-mail: firstname.lastname@example.org.