May 15, 2008 (Vol. 28, No. 10)
Proteome Sciences Believes that Its TMT Technology Surmounts Biomarker Bottlenecks
Proteome Sciences (www.proteomics.com) recently concluded a worldwide licensing deal for its Tandem Mass Tag (TMT®) technology with Thermo Fisher Scientific (www.thermofisher.com). The agreement came just a few months after the firm received broad U.S., European, Australian, and Canadian patents covering the isobaric tandem mass tagging concept and the TMT product family.
By relying on Thermo Fisher’s sales channels for volume catalog products, Proteome Sciences says that it will be able to focus on custom-labeling services and products.
Isobaric tandem mass tagging is a reproducible, highly accurate method that allows both comparative and absolute MS/MS-based quantitation of proteins and peptides in biological samples. Allowing the evaluation of differential protein expression in up to six samples in a single experiment, the technology is set to shake-up the quantitative analysis of proteins for biomarker research and applications, according to Ian Pike, Ph.D., CBO.
In 2002, the company acquired Xzillion, which held the basic isobaric tandem mass tagging IP and expertise. This take-over gave Proteome Sciences a dedicated proteomics facility in Frankfurt, which is responsible for ISO-certified production of the TMT products.
The TMT technology has been developed as part of the company’s ProteoShop® toolbox of gel-based and gel-free proteomics technologies. The range of products and services include 1-D/2-D separation, gel-free separation with isobaric/isotopic tagging, mass spectrometry including LC/MS/MS and MALDI-TOF-TOF, bioinformatics, and assay development including both immunoassays and mass-spectrometry methods.
Proteome Sciences aims to leverage the TMT technology in a number of ways, comments Dr. Pike. “The most immediate source of TMT-derived revenues will no doubt be from the licensing deal we just signed with Thermo Fisher Scientific for the worldwide distribution and marketing of TMT reagents.”
“We are also using the TMTs internally for biomarker discovery, validation, and assay-development projects in partnership with clients. We anticipate seeing a rapid growth in the industry’s drive to develop companion-diagnostics or theranostics. The potential of biomarkers to improve the development and use of new medicines is being increasingly recognized by industry, regulators, clinicians, and patients,” Dr. Pike notes.
The company’s in-house research is focused on the discovery and commercialization of biomarkers for the CNS field. In particular, the firm concentrates on Alzheimer’s disease and stroke, areas in which Proteome Sciences has already out-licensed several markers to major players in the diagnostics sector.
Biomarker Assay and Validation
“The literature is awash with putative biomarkers that could feasibly help speed disease diagnosis, aid decision-making in patient-specific prescribing, and monitor drug response,” Dr. Pike points out. “Unfortunately, very few of even the most promising biomarkers have made it to the market or into clinical use.
“The sticking point between biomarker identification and clinical utility at least partly relates to the lack of assay and validation strategies for these newly identified biomarker candidates. We wanted to develop a technology that could address the majority of issues holding back this area of proteomics research.
“The approach had to allow biomarker discovery work to interface easily with subsequent assay development and biomarker validation and facilitate the development of multiplexed assays for panels of multiple biomarkers without the need for additional investment in dedicated instrumentation. We believe the TMT technology fulfills all these requirements.”
Nuts and Bolts of Technique
The TMT concept exploits the fact that as soon as biological samples are mixed, all the same peptides or proteins originating from the different samples will have exactly the same fate in subsequent procedures, explains Peter Schulz-Knappe, Ph.D., CSO. “This conserved ratio drastically reduces the coefficient of variation following sample manipulation and so improves the precision of subsequent measurements.
“In tandem mass tagging, we label each sample with one of up to six chemically identical tags prior to mixing the samples. Because these tags are identical in chemical structure, the conserved ratio of labeled proteins is retained in the mixture even after procedures such as freezing/thawing, gel electrophoresis, absorption, or MS. And because each intact tag also has an identical mass, a single MS run, for example, would still only generate a single peak for each protein, as identical proteins originating from each sample retain the same overall mass and charge irrespective of which tag they have been given.”
The chemical structure of a tandem mass tag combines an amine-reactive ester, which attaches the tag to N-terminal amines and lysine side chains, with a mass normalizer and mass reporter. Between the normalizer and reporter is a cleavable linker, which breaks during MS/MS. The mass reporter is split off and measured by the mass spectrometer.
The tagged proteins can only be differentiated through MS/MS fragmentation, a process which splits the mass reporter ion from the protein-tag complex. The reporter ion is measured by the mass spec instrument, and because the reporter ions on each type of tag have different masses, they each generate a different peak. The peak height/peak integral for each reporter therefore denotes the relative amount of protein originating from each of the labelled samples.
Proteome Sciences offers a number of TMT options. TMTzero™ is composed of a single tag with an overall mass of 224 Da and generates a reporter ion of 126 Da. TMTduplex™ comprises a set of two 225 Da tags, both chemically identical to TMTzero, but each containing a single 13C isotope on one or the other side of the cleavable linker to generate mass reporters of 126 Da and 127 Da. Similarly, TMTsixplex™, for comparing up to six samples, consists of a set of six 229 Da tags, each with four differentially placed 13C isotopes and one 15N isotope to generate mass reporters of 126–131 daltons.
Standardization and Multipoint Calibration
Proteome Sciences has also developed isobarically labeled reference materials to facilitate the standardization of proteomics studies. Under this approach an identical amount of TMT-labeled biological reference is added to each individual sample allowing relative quantification of a large number of proteins across all study samples. Since the biological reference can be used by multiple laboratories, tightly controlled cross-study and cross-laboratory comparisons are possible.
The use of reference materials allows samples to be studied individually (i.e., without mixing the samples) and still retain accuracy through calibration of results, Dr. Schulz-Knappe explains.
“To gain even further sensitivity, a multipoint calibration curve can be generated, allowing the measurement of absolute concentration of a candidate peptide or protein. In this instance, the reference material is treated to deplete high-abundant proteins and digested into tryptic peptides. For a four-point calibration curve, four aliquots of the depleted reference material would each be labeled with a different TMTsixplex label and mixed in a known ratio, for example, 1:2:4:8.
“The two remaining TMTsixplex tags are then used to label two samples, which are added to the experiment. A typical LC-MS/MS workflow would allow researchers to focus on one particular protein of interest and read out the absolute quantities of peptide by comparing the peaks of the sample reporter tags with those of the four calibration peaks of the reference reporter tags.”
“The TMT technology is integral to our in-house biomarker discovery and validation efforts in the CNS field,” Dr. Pike adds. “With TMT we can derive fast, flexible, and cost-effective workflows based on the ability to do absolute quantitation of previously unidentified proteins. Many of the proteins we are finding have no immunoassay tests available.
“Yet, with TMT we can quickly move from discovery to the validation of 20–50 candidates on the mass spectrometer and based on these results move almost instantaneously using the same reagents to a 5–10 marker multiplex assay suitable for clinical application. Indeed, the concept of clinically useful, mass spectrometry-based in vitro diagnostics is, we believe, a likely prospect in the not too distant future.”
Near-future product-development plans focus on variations of Proteome Sciences’ TMT family of tags, for example, to increase plexing rates and add further reactive functionalities for protein attachment, as well as on a variety of additional reference materials and kits for quantitative multiplex analysis of entire groups of biomarker,” Dr. Schulz-Knappe reports. “We envisage that panels of 10 to 50 proteins can be quantified within one experiment in order to answer complex medical questions such as response to drug treatment alongside presence/ absence/staging of disease.”
“Ultimately,” he adds, “biomarker assays using TMT have the potential to be adopted on a worldwide scale, from which we firmly believe Proteome Sciences can generate considerable revenue and returns.”