Agilix has recently won a patent for a new isobaric protein-labeling technology that allows for the simultaneous quantitative analysis of multiple samples in one mixture, the company announced last week.
The technology, called i-PROT, enables quantitative comparison between proteins expressed in a number of different conditions, for example at six or seven different time points. According to Darin Latimer, vice president of Agilix and co-inventor of the new technology, i-PROT has been used to label up to 29 peptides at six different time points.
“This is ground breaking,” said Agilix’s CEO Martin Mattessich. “With the standard technology, you can do two, at most four samples at a time. With this technology, instead of processing things in a serial manner, which introduces large, inherent errors, you can do all samples at the same time and all samples will go through the same process variations.”
Gregg Morin, the head of proteomics at Genome Sciences Center, a division of the British Columbia Cancer Agency, is currently beta-testing i-PROT by using it to study time-dependent changes of proteins associated wtih prostate hormone stimulation. The new technology is ideal for the study because time-dependent studies require at least six labels in order to obtain data points for a sigmoidal time curve, said Morin.
“With time experiments in the past, you would have to go from time zero to a, time zero to b, time zero to c, and then extrapolate between the different experiments,” said Morin. “Here, [with i-PROT], you can look at any relationship among six or seven samples directly, and by having [all the samples] multiplexed into one experiment, you improve the accuracy of the experiment.”
Isotope Coded Affinity Tags, or ICAT, is currently one of the most popular techniques for quantitative assays. Using ICAT, researchers can determine which proteins are present in a sample and how much of each protein is expressed in one condition relative to another.
With i-PROT, researchers are not limited to comparing protein expression between two different conditions. They can compare between multiple different conditions as is needed for time-dependent or dose-dependent experiments.
“Researchers want to compare numerous sample states in one experiment resulting in high quality, quantitative data untarnished by ‘noise’; i-PROT labels achieve multiplexing up to previously impossible levels through an innovative isobaric design described in our patent,” said Mattessich.
Experiments using i-PROT are done in much the same way as those using ICAT, said Mattessich. First the samples are labeled using i-PROT isobaric labels. Next, the samples are separated to reduce complexity using high performance liquid chromatography or 2D gels. Finally, the samples are introduced into a mass spectrometer. Analysis of mass-spectrometry data can then tell the researcher both the identity of peptides and the relative quantitation of each peptide labeled under one condition relative to the other conditions.
“There’s nothing different about the labeling step or the sample preparation, or the reduction of mixture complexity,” said Mattessich. “You’re just doing what you’re doing, but instead of doing it serially in many different batches, you just throw it all together.”
Development of i-PROT began two to three years ago, and the product should be commercially available by the middle of next year, said Mattessich. The cost of the i-PROT kit will be competitive with other quantitation systems such as ICAT, he added.
The i-PROT technology is protected under US Patent No. 6,824,981, entitled “Ultra-sensitive detection systems using alterable peptide tags.” The patent has over 500 allowed claims, according to Agilix.
Morin said that the i-PROT labeling system fills a very important niche in the proteomics industry.
“The need for doing mass tags for measuring and labeling proteins is important to the industry, in particular where you’re trying to look at time-dependent or dose-dependent experiments,” he said. “Getting all time points in one sample and having all controls internal allows a high level of accuracy to experiments that was not before achievable.”
During beta tests of i-PROT, the main problems that came up were glitches in software, said Morin.
“Where the labels are involved, the experiments are working exactly as they should and giving results that are reproducible,” he said.
Ruedi Aebersold, the inventor of ICAT, said that the ability to analyze more than two samples concurrently is certainly an advance, but that he could not comment on the new i-PROT technology until he had seen some concrete data on it.
Mattessich said that he is not aware of any products on the market that would compete with i-PROT, since the only products on the market so far used for quantitative proteomics are ICAT, which compares two different conditions, and Applied Biosystem’s iTRAQ, which compares four different conditions.
“This is a very nice, very interesting product that has a lot of capabilities to it,” said Morin. “Once it’s out there, people will come up with a lot of imaginative ways to deploy [the product], and that’s going to be very exciting.”