Kristy J. Reynolds University of Maryland Office: 301-405-8618 FAX: 301-405-8615 E-mail: kjr17@hotmail.com Job Title: Graduate Student Ph.D. in Analytical Chemistry (Expected May, 2003) from the University of Maryland

Speaker: Kristy J. Reynolds, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742

Topic: Development of the Proteolytic ¹⁸O Labeling Method for Comparative Proteomics and Its Application to Acquired Drug-Resistance - Video (running time 00:55:10) ^*

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

Time: Tuesday, March 11, 2003, at 2:00 PM

Abstract: As the sequencing is completed of many species' genomes, the study of the protein compliment to the genome, or the proteome, has emerged as a dynamic field of research. A common approach to characterizing changed states is comparative proteomics, in which the relative amounts of proteins present in two or more samples are compared. In order to determine the relative amount of the proteins present, a proteolytic method for ¹⁸O labeling can be used. Briefly, pools of proteins are enzymatically digested in parallel in H₂¹⁶O and H₂¹⁸O. In the latter pool two atoms of ¹⁸O are incorporated into the carboxyl-terminus of each new peptide. Comparative proteomic studies can be performed by mixing the unlabeled peptide pool (generated in H₂¹⁶O) and the isotope labeled peptide pool (from H₂¹⁸O) and analyzing the peptide pairs by mass spectrometry. Relative quantitation information is derived from ratios of the isotope pairs. Tandem mass spectrometry experiments provide sequence information to identify the proteins.

This method is being applied to detect differences in regulation or modification of proteins in the cytosolic fraction of drug susceptible and doxorubicin resistant human breast cancer MCF-7 cell lines. Breast cancer is a disease affecting millions of women every year. Common treatments for this disease use apoptosis inducing drugs. One of the most commonly used drugs is doxorubicin. Development of resistance to this drug is a major factor limiting successful treatment of this and other cancers. The development of doxorubicin resistance, and drug resistance more generally, is not fully understood.

The proteolytic ¹⁸O labeling method has matured and is applicable to complex protein samples. The application of this strategy to fractionated cytosolic protein pools of drug susceptible and doxorubicin resistant cell lines revealed several proteins which were up-regulated in the doxorubicin resistant line. Several of these proteins (superoxide dismutase, thioredoxin, metallothionein, etc.) have roles in detoxifying oxidative stress in a cell. Several immune response and chaperone proteins were also up-regulated in the doxorubicin resistant line.

____________________

^* Video viewing minimally requires the latest free version of RealPlayer® and a 56 Kbps dial-up bandwidth.