Over three years after Applied Biosystems licensed ICAT technology from inventor Ruedi Aebersold, the company announced this week, at the Association of Biomolecular Resource Facilities conference in Portland, Ore., that it will soon release a “next generation” of ICAT reagents. These reagents will be peptide-based, amine-reactive reagents that can be multiplexed for comparison of up to four samples, the company said.
“The first ICAT reagent [was a] protein-based, cysteine-specific tagging technology where all the quantitation [was] done in MS,” Tony Hunt, director of systems integration, proteomics, at ABI told ProteoMonitor. “The challenge is, how do you expand the family of chemistries so you get increased proteome coverage, and don’t lose PTM information.”
ABI says it has met this challenge with its new reagents, which are currently in “latter stages of development” and are set to be released later this year. Coinciding with the reagent release, ABI will also release accompanying software that is being developed specifically for analyzing output from experiments using the new reagents. “One of the lessons learned from the ICAT days is that to enable the chemistry, you really have to have software,” Hunt said.
A key criticism of ICAT in the past has been its dependence upon cysteine labeling, which means that only peptides that contain cysteine residues can be labeled (see, for example, PM 11-21-03). This limits coverage of the proteome and leads to the likelihood of missing detection of post-translational modifications, since they would only show up in the mass spectrum if the peptides that contained the modification also contained a cysteine residue that could be labeled. In addition, only two samples at a time can be compared. Since the new residues will be amine-reactive, they will have the ability to react with all peptides. The tags — each of which will consist of a reporter moiety, a balancing moiety, and an amine-reactive group — will all be mass-balanced to make them isobaric. Four tags will be available, ranging in mass from 114 to 117. The tags would be added to samples only after tryptic digestion, and thus mixing of samples would not occur until the peptide stage. All quantitation and identification would take place in MS/MS mode.
In addition to providing more extensive peptide coverage, Hunt said that another benefit of the new tags will be added flexibility from the 4-plexing capabilities of the tags. “You can do normal versus disease versus drug [treated], you can do time course studies, and you also have the capability to do duplicates and triplicates” of the experiment, Hunt said. He said that with researchers looking to fulfill more rigorous statistical standards, the ability to do a duplicate run of disease versus normal, for example, in the same mass spec run would be very beneficial. Plans for further extension of multiplexing capabilities are likely to come later, Hunt said, but for now “it’s important to commercialize the 4-plex.”
The arrival of new ICAT reagents does not, however, mean that the old reagents no longer have a place, however. The old reagents provide reduction of sample complexity due to their specificity for cysteine, Hunt said. They also allow for labeling and mixing of the samples while they are still at the protein stage, and quantitation in MS. The new reagents do not allow for mixing of samples until the proteins have already been digested. Some researchers may prefer the control at earlier stages of the process that the old reagents allow for, Hunt acknowledged.
As for when exactly ABI will release the new reagents on the market, Hunt would not say. “We are working with key labs to work through the experiments and really show proof of concept,” he said. “We anticipate as the year proceeds you will hear more.”
— KAM & JSM