Eukaryotic Post-Translational Modifications

Project: Research project

Project Details

Description

DESCRIPTION (provided by applicant): In the field of mass spectrometric protein analysis, there is an ongoing shift to use higher mass accuracy instruments and higher mass polypeptides for analysis. Our laboratory has been developing all three major aspects of analysis of complex mixtures of intact proteins for the past seven years, and now seek to mature this technology yet further. Working in both hypothesis-driven and discovery modes, we will improve the front end handling of intact proteins, establish a 12 Tesla LTQ FT mass spectrometer, streamline data collection, and expand data processing tools for expanded use by the community of mass spectrometrists. We will develop this technology by applying it to yeast as a model of ischemia, with 14N/15N metabolic labeling providing protein pairs for detection of differential PTMs (not just protein expression). Many believe that Top Down MS is best applied to specific protein targets, with high PTM or genetic diversity. Working in this mode on Apo-A1 from human blood will allow detection of environmental and genetic variation in the human population and those patients with bad hearts. We continue to explore new avenues in chromatin biology to generate unique data on the combinations of PTMs present on histones and other chromatin-related proteins in the human nucleus as they proceed through the cell cycle. We will also focus on modified non-histone proteins such as High Mobility Group proteins and p300 to further the knowledge base regarding the PTM profiles of these proteins with recently established mammalian cell culturing facilities for a continuous pipeline of samples. We will also apply our platform to human leukocytes in a population of local blood donors, aimed at discovering how genetic and post-translational complexity can occur together (e.g., allele-specific PTM patterns). With advanced separations and high-throughput online LTQ FT analysis we are also preparing to undertake large-scale profiling and identification of human proteins. The 12 Tesla LTQ FT will be the first such instrument in the world, and will be described in detail in the text below. Few laboratories are dedicated to tandem mass spectrometry for high mass species, and we have been active in this area since 1999. We now seek continued support to further accelerate the maturation of mass spectrometry and thereby provide cell biologists and clinician-scientists with unique data on the molecular level variation of proteins.
StatusFinished
Effective start/end date8/1/107/31/12

Funding

  • National Institute of General Medical Sciences (7R01GM067193-08)

Fingerprint Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.