Friday, September 19, 2014

Home

Research in the Ong Lab for Quantitative Biology

Mass spectrometry (MS)-based proteomics is a uniquely powerful and versatile tool in biology as it allows unbiased, comprehensive and sensitive detection of proteins in complex mixtures. With the ability to identify thousands of proteins in a single experiment, MS-based proteomics makes it easy to generate lengthy protein catalogs, but qualitative comparisons of lists of proteins is less informative. Instead, the ability to measure abundances of specific proteins and observe these changing over time in response to a defined perturbation is extremely powerful. Such information can be obtained with quantitative proteomics, which greatly enhances the power and utility of MS-based methods.

We use chemical labeling methods, like iTRAQ or metabolic labeling with SILAC, to quantify changes in protein abundance, enabling functional assays to compare protein expression levels in perturbed and control cell states. In SILAC, proteins from two cell populations labeled with normal isotope abundance or stable isotope labeled amino acids are observable in the same mass spectrum and distinguishable by their respective "light" and "heavy" peptide signals. This transforms the proteomic experiment into a format akin to a microarray experiment: when samples are mixed in equal proportions, signals from both populations are detectable unless the absence of either the light or heavy member is a direct result of the experimental perturbation. With this method, protein expression changes can be modeled and significant changes called with high confidence. Issues related to stochastic sampling of control-experiment pairs that plague classical proteomic approaches are avoided altogether. Along with dramatic improvements in the speed and sensitivity of MS instruments over the last decade, these quantitative methods have enabled impressive proteomics studies like the comprehensive identification of proteins in sub-cellular organelles like mitochondria and nucleoli, and quantification of subtle changes in whole proteomes induced by microRNA overexpression.

Our laboratory focuses on the development of novel approaches to study functional roles of protein complexes; particularly the dynamics of their formation and recruitment, abundances and post-translational modifications in cellular signaling, growth and proliferation.

 

New paper comparing SILAC and stable isotope dimethyl labeling

Our new paper is out in the Journal of Proteome Research! We analyze mixtures of SILAC-labeled and stable isotope dimethyl-labeled proteins and peptides from unfractionated lysates or pull-downs in single LC-MS runs, allowing the direct comparison of quantitative accuracy and recovery of peptides. We also describe a StageTip stable isotope dimethyl labeling protocol that can be applied to in-gel digests of proteins excised from SDS-PAGE gels. This provides an easy way to incorporate stable isotope labels after the fact.  

Heavy Methyl SILAC book chapter

Our new Heavy Methyl SILAC book chapter just came out in print in the new Humana Methods in Molecular Biology series. Rock on... \m/ 

New paper on identifying mono- and poly(ADP-ribosylated) sites

We have a new paper out with our close collaborators, the Leung lab at JHU. Casey spent several months in the lab in 2012 to learn some mass spectrometry and proteomics. Here's the sweet fruit of her labor - just out in accepted manuscripts at JPR. The title is: "A phosphoproteomic approach to characterize protein mono and poly(ADP-ribosyl)ation sites from whole cell lysate."

 

Page 1 of 3