Josip Blonder, M.D. SAIC-Frederick Office: 301-846-7211 FAX: 301-846-6037 E-mail: blonder@ncifcrf.gov Job Title: Senior Scientist Doctor of Medicine from the University of Rijeka, Croatia

Speaker: Josip Blonder, SAIC-Frederick, Frederick, MD 21701

Topic: A Simple Solution-Based Method for Targeted and Global Membrane Proteomics Using µLC-MS/MS Analysis

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

Time: Tuesday, October 14, 2003, at 2:00 PM

Abstract: The analysis of hydrophobic integral membrane proteins, which mediate many vital cellular and physiological processes, is a challenging analytical problem for mass spectrometry (MS) based proteomics in terms of sample preparation, separation and identification. This is mainly due to challenges related to their special solubilization requirements. Extensive glycosilation of eukaryotic integral membrane proteins and their relatively lower expression make the issue even more complicated. An optimal sample preparation method for targeted MS based analysis of highly enriched individual integral membrane protein should permit facile and complete sequence mapping, preferably without the use of detergents or cyanogen bromide, that adversely affect both, downstream separations and effective MS analyses. Also, protein digestion should be carried out using preferably, specific enzymatic proteolysis (e.g. trypsin). Further more, such technique should permit isolation, extraction, solubilization, separation, and identification of proteins and/or peptides from complex protein mixtures permitting the global proteomic analysis of integral membrane proteins regardless of their hydropathy profile. Over the past several years, a number of various solution-based methodologies have been developed for MS-based membrane proteomics using either matrix-assisted laser desorption ionization MS or electrospray ionization using µLC-MS/MS. Each of these approaches possesses certain disadvantages, such as the use of detergents, protein precipitation, extensive sample handling or lack of specific enzyme constrain. Hence, there is still a need for improved techniques for the identification and characterization of integral membrane proteins. The objective of this work was to develop a simple and robust method for targeted and global membrane proteomics. The present technique, unlike other methods is detergent and/or cyanogen bromide free and permits efficient extraction, solubilization and tryptic digestion of membrane proteins employing sonication in organic/aqueous buffered system. We used the present approach to characterize the primary structure of highly enriched prototypical membrane protein, bacteriorhodopsin by 1D-µLC-MS/MS. Further; we validate the applicability of this methodology for global proteomics, using multidimensional µLC-MS/MS analysis of human epidermis and mouse NK cells.

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