Motivated by the desire to improve upon the ICAT reagent technology, Andrew Emili and his colleagues at the University of Toronto have devised a tagging technique for performing de novo peptide sequencing and quantitative protein profiling experiments that they claim is cheaper and less complicated than its more established cousin.
In a paper published in the February issue of Nature Biotechnology, Emili and his colleague Gerard Cagney, also at the University of Toronto, described how their tagging reagent, which they termed MCAT (mass-coded abundance tagging), could be used for peptide sequencing and quantitative protein profiling using liquid chromatography-electrospray tandem mass spectrometry.
Although the MCAT reagent, more commonly known as O-methylisourea, has previously been shown to ameliorate peptide sequencing using MALDI mass spectrometry with post-source decay, Emili said that using the MCAT reagent with LC/MS allows researchers to analyze much more complex mixtures of peptides, and could lead to the wider adoption of techniques for identifying and quantifying the amounts of proteins on a genome-wide scale.
Compared to ICAT, the most well-known tagging technique for protein quantification used by Celera and Oxford GlycoSciences, the MCAT reagent is significantly cheaper because it does not rely on a stable isotope-containing tag, Emili said. In addition, the MCAT reagent may also present fewer sample handling difficulties because of its greater solubility in water, added John Yates, a protein mass spectrometrist at the Scripps Research Institute who is familiar with Emili’s research.
“ICAT was prohibitively expensive even for my lab, which is a proteomcs lab so I have a relatively big budget,” said Emili, who along with Cagney has filed a patent application on the MCAT method. “We could do maybe 12 experiments a year, but I don’t know what sort of biology we’re going to learn in 12 experiments.”
In addition to potentially making tagging techniques cheaper, the MCAT technique might also allow researchers to more easily study proteins isolated from species whose genomes have not yet been sequenced, Emili said. This is because MCAT reagents can also be used to perform de novo peptide sequencing experiments, which become necessary when a particular protein’s amino acid sequence is not yet cataloged in a database.
The MCAT technique is not the only way to perform de novo peptide sequencing, however, and the advantage of integrating both database-assisted and de novo peptide sequencing will fall mostly to those less experienced in protein mass spectrometry, said Don Hunt, a professor of analytical chemistry at the University of Virginia. “For those not skilled in the art, this is a helpful tool,” he said.
Hunt called the development of the MCAT reagent “a nice contribution,” but said that the technique “doesn’t dramatically push the envelope” because it fails — like ICAT — to address the need to identify low-abundance proteins. “ICAT has the same problem of trying to derivatize low-level materials and getting good spectra on [them],” he said, although he noted that Ruedi Aebersold, the inventor of the ICAT reagent technology, has recently made some improvements in this area.
Where the MCAT tag significantly differs from the ICAT reagent, in addition to cost, is in its ability to reduce the complexity of a mixture of peptides. Because the ICAT reagent technique contains an avidin purification step — and the reagent only binds to cysteine residues — the number of peptides entering the mass spectrometer is reduced by a factor of 10. In contrast, the MCAT reagent method does not include a purification step, which could make identifying peptides more confusing, Yates said.
But Emili countered that using the MCAT technique does not preclude researchers from combining it with other methods for simplifying protein mixtures. Furthermore, members of his research group are currently attempting to modify the chemistry of the reagent to select out proteins with specific functional groups or post-translational modifications, he said.
Emili’s group is currently one of many attempting to make improvements to the concept of an ICAT reagent-type technique. But Emili insists his goal is to take the technique out of the mass spectrometry community, and make it more widely available to researchers in the molecular biology.
“I don’t want to say we’ve supplanted any of these [other] strategies,” said Emili. “There’s no one solution to the proteomics problem, but I’m satisfied that we’ve made it more accessible, so that more people can participate in proteomics exploration.”