Targeted Mass Spec
Immunoassays are widely used to measure protein biomarkers in patient blood, but Steven A. Carr, Ph.D., director of proteomics at The Broad Institute of MIT and Harvard, observed that useful antibody reagents do not exist for the vast majority of proteins.
In biomarker discovery today, “thousands of proteins are being identified with often several hundred or more of these observed to be highly differentially abundant between cases and controls. The problem is that there has been no technology, other than immunoassays, to establish if these protein biomarker candidates are detectable in patient blood and that they hold up as potential markers when a few hundred patients are studied,” Dr. Carr noted.
One of the main reasons so few protein diagnostics have been developed is the lack of reagents and technology to build the credentialing information needed, he explained. “Making a new sandwich immunoassay is an expensive and time-consuming proposition, in part, because you need two antibodies per protein candidate to guarantee specificity. Companies are generally unwilling to make an investment in developing reagents for unproven candidate biomarkers.”
In response to this need, Dr. Carr’s team is developing targeted assay methods employing mass spec to screen and quantify low-abundance proteins in plasma. “My lab is using multiple reaction monitoring (MRM) MS as the technology to bridge the gap between discovery and small numbers of proteins worthy of true clinical validation,” he said.
“Peptides derived from each protein candidate of interest are used as surrogates for measuring the change in abundance of the target protein across multiple samples. Quantification is done by spiking synthetic peptides synthesized in a heavy, but stable isotope-labeled form. These peptides are distinguished from the native peptides on the basis of their masses, but they are otherwise identical to the peptides we want to measure.
“The ratio between the observed abundance of the natural peptide and its heavy-labeled counterpart gives us the precise relative quantitative information we require for candidate verification. The peptides we choose to monitor derive from the lists of those we have observed in MS-based proteomic discovery experiments. However, our candidate biomarker proteins also come from the literature and non-MS experimental paradigms such as microarray experiments.
“In these cases, proteomic experimental data may not exist so we use computational approaches to predict which peptides for each protein are likely to give the highest MS response and provide the most sensitive MRM-MS assay. Unlike immunoassays, MRM-MS technology is also highly multiplexible. The method is also far more sensitive than conventional MS methods.
“While we are working hard to develop and apply these methods for biomarker verification in our own studies, another aim is to insure that any lab that would like to use these methods can do so, and that the results obtained will be sufficiently reliable and reproducible. So, a considerable amount of effort is being put into simplifying the process to ultimately allow full automation of sample processing, data acquisition, and analysis, much like clinical measurements are carried out today. ”