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Recent Advances in High Resolution Microscale LC/MS for Proteomic Analysis

presented by

Barry L. Karger
Northeastern University

September 15, 2003

The Scripps Research Institute, W.M. Keck Foundation Amphitheater


Background:

Barry L. Karger is the co-founder and director of the Barnett Institute of Chemical and Biological Analysis at Northeastern University. Dr. Karger?s recent research interests have focused on bioanalysis, including high performance DNA separation and analysis for DNA sequencing, ESI and MALDI mass spectrometric methods for proteome analysis, and new microscale LC columns for high mass sensitivity in LC/MS. He has been an active researcher with more than 275 publications in the field of separation science, with particular emphasis in liquid chromatography and capillary electrophoresis and most recently, genomics and proteomics. He is a member of the editorial boards of numerous journals in his fields of expertise. He is the holder of 28 patents in these fields, a number of which have been commercialized. With Lloyd Snyder and Csaba Horv?th, he is the co-author of An Introduction to Separation Science, a leading graduate textbook. He has trained more than 150 students at the Ph.D. and postdoctoral levels. He organized the first Symposium on High Performance Capillary Electrophoresis (HPCE) in Boston, which has been managed by CaSSS since 1998. Dr. Karger has received numerous honors, including the 2002 Halasz Medal of the Hungarian Chemical Society for Chromatography and Separation Science, the ACS IBC Advanced Technologies and Millipore Award in Separations Science and Technology (1998), and the ACS Fisher Award in Analytical Chemistry (1990).

Abstract:

We are now in the era of the proteomics where the interest is in global analysis of many, if not all, proteins in cells, tissues, etc. At any given time point, thousands of proteins exist in a given cell with 106 levels difference of concentration. This dynamic range in concentration reaches as high as 1012 in plasma. Furthermore, digestion of individual proteins yield, on average, 40 or more peptides. As a consequence, proteome analysis of whole cell lysates, organelles or body fluids such as plasma yield enormously complex challenges. While mass spectrometric instrumentation continues to improve in sensitivity, resolution, mass accuracy and structure elucidation, the front-end sample processing steps prior to the mass spectrometer remain critical. Multidimensional separation is a requirement in order to simplify mixtures eluting into the mass spectrometer. In addition, in many cases, very high resolution LC is needed as a final step into the mass spectrometer. In this talk, we shall overview some of the strategies possible for separation prior to mass spectrometry. We will present results using monolithic columns, which generate high peak efficiency and at the same time allow use of narrow bore columns 25 _m i.d. or less for high sensitivity ESI. We are using such columns for biological samples in which the sample amount is limited, e.g. laser capture microdissection of tumor cells. In a second example, we are employing high resolution LC coupled to (off-line) MALDI-MS for analysis. We will demonstrate several advantages of this approach such as the ability to minimize chemical noise as a result of the chromatographic separation and the ability to optimize the MS/MS analysis for specific regions in the chromatogram of interest. Finally, we will illustrate the use of these and other approaches for quantitative proteomics and biomarker discovery for diagnosis.

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