1. Abstract Our objective is to develop next generation isobaric labeling reagents to greatly expand the utility of multiplexed quantitative proteomics and its applications in clinical and pre-clinical biomedical research. The isobaric labeling strategy involves a set of chemical reagents for tagging peptides and other biomolecules with a stable isotope-encoded barcode. The power of the approach derives from the fact that each tag within a multiplexed set contains the same number of heavy isotopes, with the barcode being encoded by the placement of these isotopes within either the reporter ion (RI) or balance region of each tag. During the analysis, the measured intensities of the RIs are proportional to the relative abundance of the analytes. This ability to mix and analyze multiple samples simultaneously without increasing LC-MS complexity preserves analytical depth, reduces experimental variability, and increases both sample preparation and analytical throughput regardless of the number of samples mixed together; this is unique to isobaric labeling among other quantitative proteomics techniques. These advantages are extending the boundaries of proteomics into more profound areas of primary and clinical research, with incredibly powerful and far-ranging applications reported in areas such as drug target identification, biomarker discovery, and temporal regulation of proteome dynamics. While isobaric labeling has been established as an accurate, reliable, and sensitive quantitative technique?-?, there is a definitive need for improvement in isobaric multiplexing capacity. The 10-fold multiplexing of current isobaric labeling reagents? limits experimental design when replicates are required for statistical significance or when sample sizes are large. Our strategy employs a novel series of isobaric tags that spontaneously forms two reporter ions per reagent as opposed to one reporter ion per reagent in existing commercially available reagents. The addition of a second reporter ion provides the ability to encode additional barcoding information, via heavy isotopes, which ultimately expands the capacity of multiplexed proteomics. Following the synthesis of these novel isobaric reagents, we will assess the accuracy, precision and utility of these reagents against controlled whole proteome mixtures. This will also include the synthesis of reagents that exceed the current multiplexing capacity of 10. Finally, we will assess the ability to incorporate barcoding information into a third reporter ion which can provide additional quantitative precision or multiplexing capacity. The ability to increase the multiplexing capacity has a number of key advantages. One, as more samples can be analyzed in parallel, the application of quantitative proteomics to large cohort studies becomes more amenable. This facilitates the inclusion of quantitative proteomics to highly valuable high-throughput analyses. Second, the stochastic under-sampling inherent to traditional discovery based proteomics experiments is greatly reduced or eliminated providing more robust characterization of proteomes and deeper biological insight. Finally, the ability to multiplex more than 10 samples per experiment permits experimental designs that can include more replicates for statistical power or higher resolution longitudinal time-course experiments.
Quantitative proteomics is an emerging tool for both pre-clinical and clinical research and development, but the limited multiplexing capacity hampers widespread utility. Our unique innovation greatly improves the throughput of quantitative proteomics providing new avenues for accurate, sensitive and reproducible interrogation of clinically relevant biology.
