When Applied Biosystems introduced a commercial version of the ICAT reagent technology last March, proteomics researchers had high hopes that at long last a method for performing high-throughput differential protein analysis had emerged to provide an alternative to the drudgery of 2D gel electrophoresis.
Nine months later, however, only two companies engaged in industrial-scale proteomics Celera Genomics and Oxford GlycoSciences [OGS] have fully adopted the technology. Most academic and industry researchers are reluctant to integrate the technique into their platforms because of the cost and challenge of optimizing the experiments.
An ICAT starter kit containing the necessary column cartridges, buffers, and reagents for 10 assays costs $2,595. In addition, users point to the lack of software for analyzing the data obtained from their mass spectrometers and the difficulty optimizing the purification steps as primary reasons for the markets reticence to adopt the technology.
Given enough time and some more development, this will be better than 2D gels, said Igor German, who recently began trying to use the technique in his research as a postdoc at the University of Florida core facility for protein chemistry. But if I had a wish list I would put down that somebody should simplify and miniaturize [the amount of sample and reagents required]. Software to analyze the data would also be on his list, German said.
Other commercial users agree that the technology is at an early stage and requires a certain degree of lead time before it can churn out consistently reliable data. On the other hand, they say that adopting any new technology will require a significant investment in resources to optimize and validate the technique, and that the ICAT reagents are too promising to pass by.
In short, the ICAT reagents allow researchers to label peptides taken from two samples for example normal and disease tissue samples with different stable isotope tags. The samples can then be simultaneously analyzed with mass spectrometry to determine which proteins are differentially expressed.
Its a very useful reagent, said Scott Patterson, vice president for proteomics at Celera. Its a lot better than anything else out there.
[In addition to Celera and OGS, GeneProt is also considering whether to integrate the ICAT reagent technology into its arsenal of proteomics capabilities, but GeneProt chief scientific officer Keith Rose declined to comment on the technique, saying only that we are currently evaluating the technology in collaboration with ABI.]
Vendors Recognize Shortcomings
Vendors themselves realize that improvements to the ICAT reagent technology are required, and are currently taking steps to at least partially rectify the gap between the promise and reality of the technique. Next month, ABI will release a software package called Pro ICAT for use with ICAT and its QSTAR pulsar hybrid LC/MS/MS mass spectrometers. Thermo Finnigan is also working on software to allow ICAT analysis using its ion trap mass spectrometers, according to David Goodlett, a proteomics researcher at the Institute for Systems Biology.
As for the front-end chromatography steps required to purify and isolate the ICAT labeled peptides, current versions of ABIs Vision multidimensional chromatography workstation allow researchers to automate the strong cation exchange and affinity purification steps, said Tony Hunt, senior marketing director for proteomics consumables at ABIs proteomics facility in Framingham, Mass.
ABI also has plans to expand the capabilities of the ICAT reagents, including developing different chemistries to pull down proteins with post-translational modifications and to analyze more than two samples simultaneously. We see product-line extensions in functional groups to look at different peptides or different types of proteins, and different strategies for isotopic labeling of the linker, [which] will get us potentially into areas of things like multiplexing, said David Hicks, director of proteomics applications marketing at ABI.
Aebersolds Group Moves Ahead
Further down the road, Ruedi Aebersold, who led the development of the ICAT reagent technique while at the University of Washington, and other scientists at ISB are working to expand the applications of the technology, and make it more amenable to users. Aebersolds group recently received a grant from the National Cancer Institute to develop an annotated peptide database for cataloging reverse phase chromatography data from experiments using the ICAT reagents. The database would help researchers identify ICAT-labeled peptides based on where they elute on the reverse phase column, avoiding unnecessary peptide sequencing with tandem mass spectrometry.
Its just one small step, said Goodlett at a conference earlier this month sponsored by NIH, but the hope is that we can go to a table to look up [a peptide], and we wont need MS/MS to identify it.
ISB scientists are also experimenting with multiple ICAT reagents, Goodlett said in an interview, so that instead of comparing two samples, one would compare three to four biological states with three to four different ICAT reagents. Aebersolds lab is also making progress developing a phosphate-specific ICAT reagent, Goodlett said, and expects to transfer the technology into the ISB proteomics facility some time in 2002. Commercializing these unpublished advances will fall to ABI.
Whether these advances make it into the mainstream depends a lot on cost, but Hicks and Hunt at ABI claim the cost is already dropping, particularly if users buy the ICAT reagents in bulk. [A 200 assay kit without column cartridges or buffers costs $19,995.] Furthermore, the cost of the reagents would presumably fall as the volume of reagent kits sold rises in response to mass spectrometry vendors releasing software customized for ICAT reagents, Hicks said.
Goodlett compares the initial versions of the ICAT reagents to cDNA arrays when they first became available several years ago: Just as with cDNA arrays, people wanted to use the technology before it was analytically ready and before there was good software to support it and analyze the data. With ICAT I think we have the analytical control part pretty far along and the software support area is coming along. It is expensive but the cost will come down as the product life cycle advances.