Radioactive multiplexing analytical methods for biomarkers discovery

Abstract

A novel analytical method involves labeling samples with different radioactive labeling agents, mixing and subjecting the mixture to any separation technique, and then differentially detecting and quantifying subcomponents from each sample for comparison. The novel technique exploits the differences in radiation energy or half-life between isotopes to make differential detection and quantification of labels possible. Detailed methods for differential detection and quantification are also described as well as the construction and application of hardware and software to enable and enhance such a process. This method is useful in finding molecular differences between two samples in differential proteomics, phosphor-proteomics, glycomics, metabolomics, transcriptomics, genomics and diagnostic applications.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority of provisional application U.S. Ser. No. 60 / 443,017, titled multiplexing analytical techniques, filed Jan. 28, 2003, the content of which is incorporated herein by reference.BACKGROUND OF THE INVENTION [0002] Many analytical methods require side-by-side or sequential comparison to quantitatively compare differences between two samples. For example, to compare proteins on a Western blot, samples are run on adjacent lanes; to compare small molecules on HPLC, samples are run sequentially one next to the others. This format of analysis requires twice the effort in sample preparation; in addition, variability can be introduced into the system if the treatments of one sample slightly deviated from another. For these reasons, many repetitions plus statistical analysis are required before any differences found become credible. A method that allows two samples to be mixed together for analysis and quantitative co...

AI Technical Summary

Benefits of technology

[0008] This invention provides a novel method for coding individual samples, enabling the samples to be homogenized, testing the samples simultaneously and precisely the same way, then distinguishing and quantifying which molecules belong to which samples. One innovation is in the method that enables quantification of more than one isotope in a mixture. This is accomplished by exploiting the difference in radiation energy between isotopes or the difference in their half-life. By first quantifying total radiation and then quantifying partial radiation passing through a selective blocking screen, the amount of individual radiation from each isotope can be quantified knowing the efficiency of the screen in blocking different isotope. Alternatively, by quantifying total radiation, then storing the sample for radioactive decay (while preserving other attributes) before quantifying total radiation again, the amount of radiation between isotopes with different half-life can be selectively quantified. In addition to the time and cost savings by running two or more samples simultaneously, the increased reliability also provides new possibilities for analysis.

[0010] The ratio of radioactive isotopes in each fraction can be easily monitored. Any fractions whose ratios deviate from a standard ratio can be examined further to identify the exact molecules in those fractions that are responsible. These are the molecules of interest because their levels of abundance vary between the test samples. The molecules can be identified by mass spectrometry and used as biomarkers for drug efficacy or diseased condition. Radioactive labeling with different isotopes also results in same molecules of different mass that can be differentiated and quantitatively compared during mass spectrometry analysis if necessary.

[0012] A further object of this invention is to provide an improved method for genomic and proteomic analysis using radioactive isotopes of different radiant energy level or different half-life to enable simultaneous processing and quantification of multiple samples of DNA, RNA, proteins, and other molecules without damaging or rendering these molecules incomparable or incompatible for analysis purposes, e.g., chemically modifying them. Additionally and importantly, radioactive labeling also provides much higher sensitivity than any other methods of labeling.

Problems solved by technology

While DNA and RNA often can accommodate structural modifications caused by covalent linkage with dyes for DNA array analysis, proteins cannot for protein array analysis.

As a result, the label interferes with the analyte and often prevents an antibody from binding normally to labeled proteins the same way it would to unlabeled proteins.

Furthermore, dye may alter the structure of DNA or RNA significantly, such that proteins, e.g., transcription factors, won't be able to recognize and bind as they would do with native sequences.

Direct detection with devices such as Geiger counter is also possible; however, the cost for making such instruments has limited its use.

Images