Kerry D. Nugent Michrom BioResources, Inc., Auburn, CA Office: 530-888-6498 FAX: 530-888-8295 E-mail: knugent@michrom.com Job Title: President M.S. in Analytical Biochemistry from the University of California at Davis

Speaker: Kerry D. Nugent, Michrom BioResources, Inc., Auburn, CA

Topic: Beyond 2D Gels: Fully Automated MDLC Coupled to Nano ESI-MS/MS for Characterization of Low Abundance PRoteins and Biomarkers

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

Time: Friday, December 3, 2004, at 10:30 AM

Abstract: Although 2D PAGE is currently the most widely accepted technique for separation of complex protein mixtures, it has limitations when trying to identify low abundance proteins in complex proteome samples. With recent developments in automated, data dependent ESI-MS/MS in conjunction with nanoscale HPLC and database searching, simple protein mixtures (tens of proteins) can be quickly identified without the need to purify each protein to homogeneity prior to analysis at the femtomole level. More complex protein mixtures (hundreds to thousands of proteins) still require some degree of fractionation prior to NanoLC-MS/MS of protein digests, since a 1D HPLC separation is limited to resolution of 50-200 components. Orthogonal 2D HPLC methods dramatically improve the resolution of highly complex mixtures of proteins and peptides versus a 1D separation. One approach is to digest the entire proteome sample and then perform orthogonal 2D peptide separations (Mudpit) to simplify the resulting peptide maps. This approach works well for less complex protein mixtures, but is not sufficient when trying to identify low abundance proteins in whole cell lysates. These contain thousands of proteins (and therefore hundreds of thousands of peptides when digested) that can vary in abundance by 6-12 orders of magnitude. An alternate approach is to use orthogonal 2D protein separations to fractionate the proteome samples into simple mixtures containing 10-50 proteins prior to digestion and analysis by NanoLC-MS/MS. 2D protein HPLC can be easily automated to give reproducible separations with quantitative recoveries at femtomole levels. Several schemes for orthogonal HPLC of proteins and peptides have been explored to find optimum conditions for identification of low abundance proteins and biomarkers in complex samples. Future research will also focus on a new multidimensional MS system that may further improve detection of low abundance proteins in complex proteome samples and allow high throughput quantitation of protein biomarkers.