David M. Bunk National Institute of Standards and Technology Office: 301-975-5071 FAX: 301-975-0685 E-mail: david.bunk@nist.gov Job Title: Research Chemist Ph.D. in Analytical Chemistry from Texas A & M University

Speaker: David M. Bunk, Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, MD 20899

Topic: Proteomics Approaches to the Determination of Proteins for Clinical and Food Analysis - Video (running time 00:54:28) ^*

Place: Building 549, Auditorium, NCI at Frederick, Frederick, MD

Time: Tuesday, December 10, 2002, at 2:00 PM

Abstract: The development of reference materials and methods at NIST for important clinical proteins and transgenic proteins introduced into genetically modified (GM) foods requires analytical methods with high sensitivity and selectivity. In both cases, the protein of interest is usually present in low abundance among a high abundance of other proteins present in the sample matrix. For example, the concentration of human troponin I, present in serum after a heart attack, is approximately 1 ng/L, while serum albumin is present in the same sample at approximately 50 g/L. Measuring this one protein among more than 50 billion other protein molecules is quite an analytical challenge. Clinical methods for troponin I measurement use antibodies to perform this measurement. However for the types of measurements developed and performed at NIST, using an antibody in a reference method could produce substantial measurement bias based on the specificity of the antibody used. Another approach is needed.

Current research in proteomics offers new techniques and strategies that may be applied to the analysis of protein-based clinical markers and proteins in foods. The proteomics approach for the analysis and quantification of proteins differs from traditional approaches in that measurement is done at the peptide level and extensive purification is not used. For the proteomics analysis of the transgenic protein ESPS Synthase in GM soybeans, all of the proteins present in a soybean extract are enzymatically digested into peptides. This complex mixture of peptides is subsequently analyzed by multidimensional chromatography coupled with mass spectrometry. Through this approach, several peptides originating from the ESPS Synthase can be detected which are not present in protein extracts from non-GM soybeans. Using proteomics, GM and non-GM soybeans can be differentiated without using an antibody-based assay.

Quantitative proteomics has been demonstrated using approaches such as isotopically coded affinity tags (ICAT). The ICAT approach has been used predominately for relative quantitation such as the measurement of increased/decreased protein expression in diseased cells relative to healthy cells. For absolute quantitative analysis, only limited examples using a proteomics approach can be found in the literature. Current research is being performed at NIST to explore the quantitative potential of proteomics. With the proper isotopically labeled internal standards and selective chromatographic separations and mass spectrometric-based detection, accurate and precise determinations of low abundance proteins in serum or food extracts should be possible.

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