Genome databases have enabled mass spectrometry (MS) to become the pre-eminent tool in proteomic scale studies, but the quantitative capabilities of MS methods in the analysis of complex samples remain a topic of active research. We are developing both experimental methods and informatic algorithms to quantitatively address several key problems in molecular and cell biology: protein folding, siRNA knockdown efficiency, and foreign-sourced protein in an animal. For protein folding of many proteins within a mixture, we extend to detailed time-temperature kinetic studies our recent 'Cys Shotgun' labeling methods that take advantage of ionization-surviving, fluorescent dyes which facilitate tracking (1). In LC-MS/MS studies of several hundred of the most prominent proteins in a cell lysate, Cys that are buried are labeled moreso with denaturant at low temperature, so that thermal activation energies demonstrate a reasonable correlation to solvent exposure in a number of crystal structures. Quantitative assessments are put to the test in siRNA knockdown of protein (2) by comparing to densitometry-based immunoblotting, showing that MS agrees within about 25% but presents an advantage of also revealing likely expression changes of non-targeted proteins. The species-specificity of genomes further translates to an ability to estimate, for example, how many human cells engraft in a mouse tissue, which is a key question in stem cell and cancer models. For any given protein, we find species-specific tryptic peptides are often about 30% of detectable peptides, which means that human-to-mouse protein (wt/wt) is detectable at a small percentage, as we verify with mixture studies. Quantitative MS is thus proving useful for a wide range of cell and molecular specific studies. References: (1) Johnson et al. Forced unfolding of proteins within cells. Science 317: 663-666, 2007. (2) Kim et al . Polymersome delivery of RNAi and antisense oligonucleotides. Journal of Controlled Release 134: 132�140, 2009.

Quantitative mass epectrometry proteomics in studies of folding, knockdown, and xenografts

239th National Meeting (2010)

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