Five years after the birth of ICAT, many labs are still struggling with how — or whether — to label their samples for mass spec-based quantitation experiments. Quantitation was the subject of several sessions at the American Society for Mass Spectrometry meeting in Nashville, Tenn. two weeks ago. While Applied Biosystems and New Haven, Conn.-based start-up Agilix offered up multiplexed labeling reagents, Waters pushed the use of its new Q-TOF for label-free quantitation. Each of these companies is hoping to provide easier and faster techniques for multiple samples, and to cash in on biomarker validation.
Going Multiplex Means Going Isobaric
ABI’s new iTRAQ reagents, which the company introduced informally in February and then formally launched at ASMS, are isobaric — so they show up only in MS/MS runs — and are multiplexed to allow for absolute or relative quantitation of four samples at once (see PM 3-5-04, 5-28-04). They are available with software that is compatible with ABI instruments, and are “great for biomarker discovery, identification, and quantitation,” Tony Hunt, ABI’s director of systems integration, proteomics, said at ASMS. They also are well suited for biomarker validation, time-course studies, and affinity pull-downs, he said. Unlike ICAT, the labels are amine-binding and therefore bind to all peptides, instead of just those with a cysteine residue, as ICAT did — but they are only used for peptide binding, not for protein binding. John Haley, of OSI Pharmaceuticals, told ProteoMonitor last week that he was using the reagents for time course studies on receptor tyrosine kinase signaling pathways in the presence of inhibitors and activators, and praised the sensitivity and simplicity of the system. He described using the 4-plex function to look at four different time points, mixing the samples together, and then feeding them into a nano-HPLC-Q-STAR system for analysis.
Enter Agilix. The five-year-old, 40-employee spin-off from Yale University until now has been known primarily for its GenCompass genome analysis array, based on technology licensed from Paul Lizardi at Yale. But at ASMS, the company informally introduced isobaric tags that it claims allow for the absolute or relative quantitation of 29 samples at once, and that, like iTRAQ, will also be targeted for such applications as biomarker validation and time-course studies. While Hunt said that ABI is “still in the research phase” of developing reagents that can multiplex more than four samples, Agilix “can make another set of 29 very easily by a small mass shift in the parent composition,” according to Darin Latimer, vice president of engineering at Agilix. “We haven’t yet found people who can make use of 100 labels, but 100 labels is easily within our scope,” he said.
Latimer co-developed Agilix’s labels, called i-PROT, with Brian Chait of Rockefeller University, in 2000. Chait, along with Steven Carr, director of proteomics at the Broad Institute, brings star power to Agilix’s scientific advisory board. The iPROT labels are made up of polyglycine backbones with cleavable aspartic acid-proline bonds, and reactive groups that can bind to various amino acids, Latimer said, depending on how the chemistry is designed. Samples can be labeled either pre- or post-digestion, so they are compatible with both top-down and bottom-up experiments, according to Latimer. As with ABI’s iTRAQ, the labels are isobaric, so they don’t show up as separate peaks in the MS phase. But during MS/MS fragmentation, the tag cleaves the aspartic acid-proline bond to yield different masses that can be resolved on the m/z spectrum.
Latimer said the system would work with any tandem mass spec, although Agilix has primarily used MALDI instruments. The company has also developed in-house software to analyze the complex results, which “is not as scary as it looks — we make use of a modification of something that has existed for awhile in the industry.” Latimer added that use of the labels would be compatible with the next version of Mascot.
At ASMS, Agilix presented data from a biomarker validation study the company conducted on rheumatoid arthritis samples, in which it labeled the proteins in samples from seven patients with seven different labels, ran them out on a 2D gel, and then digested them and ran them on a mass spec to look for differential expression. Latimer said that the experiment could just as easily have been done with an LC system or other standard front-end technology. The talk “created quite a buzz around these labels, so we have a number of entities who have expressed an interest in working with us on a collaborative basis,” Martin Mattessich, CEO of Agilix, told ProteoMonitor. “These tend to be very large, well-funded entities, including larger pharmaceutical companies.” He said that in addition to forming these sorts of collaborations, “we have a very fine interest in democratizing this technology to the greater marketplace, [but] we don’t see ourselves necessarily as an end marketer to that greater population.” Mattessich said the company is looking for “channel partners” — perhaps an instrument company — that could market the labels to the larger marketplace.
The sort of marketplace that Agilix will be going for is similar to the one ABI is going for. In addition to biomarker validation studies, Mattessich said the labels would be ideal for time-course pharmacokinetic data — where “you need more than two, three, four time points” — as well as for “bar code” labeling of vectors. “We can now insert those into plasmids, vectors, or genomes through a knock-in and then follow those proteins that we’ve knocked in,” he said.
While Agilix’s claims are impressive, however, the company may have a hard time convincing customers that a 29-plex method from a small start-up is as reliable as a 4-plex method from an established company like ABI. “It would be good to see some real results, so I remain skeptical,” Matthias Mann, director of the Center for Experimental Bioinformatics at the University of Southern Denmark, told ProteoMonitor in an e-mail message. Mann acknowledged, however, that the isobaric approach could theoretically accommodate the level of multiplexing that Agilix claims — whereas for labels that differentiate in an MS spectrum, “multiplexing with more than five labels would tend to make the spectra too complex.”
Another issue that could come up, Mann said, is a limitation to the amino acid labeling capabilities. Agilix’ multiplexed tags, unlike ABI’s, bind to specific amino acids, and so do not label every peptide the way iTRAQ does. In that sense, they are similar to ICAT — except that they are capable of labeling amino acids other than cysteine, Latimer said. “If we under an experiment come up with a preference to label lysine rather than cysteine, one would simply change the reactant attached to our label to allow us to label lysine rather than cysteine,” he said. “Whatever existing chemistries are out there, we can very easily incorporate into these labels.” But Mann said that beyond lysine and cysteine, options are fairly limited. “One can easily label cysteine, the N-terminus, and lysine. In all other cases, one usually gets a lot of side products,” Mann said.
But Mattessich expressed plenty of confidence that i-PROT would catch on. “As soon as [people] recognize the power of being able to make more measurements at one time, and what that does for the data, and what that does for their experimental design, I think there will be a stampede to get into this type of a system,” he said.
Waters claimed at ASMS that it has the first commercially available technology that uses peak intensity, combined with exact mass measurements, to do both qualitative and relative quantitative analysis of two or more samples on a mass spec run, without the use of labels. Mark McDowall, marketing director for the Micromass Mass Spectrometry Technologies Center in Manchester, UK, said that Waters’ new Q-TOF-based Protein Expression System, with its accompanying software, uses intensity-based methods similar to those that small start-ups Caprion Pharmaceuticals and SurroMed use. Researchers Dick Smith and Andrew Emili (see PM 5-14-04) have also worked on similar methods, and Steven Gygi published a paper in Nature Biotechnology in February that described an intensity-based identification method. McDowall said Waters is targeting biomarker analysis as well as affinity pull-down applications for its Q-TOF system, and that the strength lies in the flexibility of the system and the ability to both identify and quantify in the same run. “You can either identify everything and quantitate everything, or you can find a subset [of peptides] where the quantitation is different, and then only identify those,” Tim Riley, vice president for proteomics business development at Waters, said at ASMS.
McDowall said that the benefit of shirking labels was that you can “analyze the sample in its native biological state.” And while intensity-based methods of the variety that Waters is marketing do only relative quantitation and are generally intended for comparison of two samples, removing a labeling step — and the expense of buying them — may be a convincing argument for intensity.