282345 An Innovative LC-MS/MS Workflow for the Characterization of Combinatorial Post-Translational Modifications

Tuesday, October 30, 2012: 9:15 AM
Fayette (Westin )
Peter A. DiMaggio Jr., Department of Chemical Engineering, Imperial College London, London, United Kingdom and Nicolas L. Young, National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL

Extensive genetic screens and biochemistry experiments over the past 20 years have revealed that modifications present on DNA and histones function in concert as docking or exclusion sites for a diverse family of proteins and protein-protein complexes that possess the activity necessary for turning the genes on or off.   Indeed, many of subunits of these chromatin-associating protein complexes contain multiple putative chromatin-binding domains, thus allowing them to simultaneously recognize a particular subset of modifications to localize the activity of the complex.  This apparent necessity for multiple points of contact between chromatin modifications and effector proteins suggests a highly specific regulatory checkpoint for gene activation or repression.  As a result, there has been a tremendous surge in the demand for technologies that can analyze combinatorial sets of histone post-translational modifications (PTMs). Although mass spectrometry is a natural starting point for addressing such a problem, traditional LC-MS/MS platforms (i.e., reversed-phase separation and CID fragmentation) are not efficient in their ability to resolve and fragment intact modified proteins, and existing alternatives suffer from lack of throughput and reproducibility.

In this work, we present a unified experimental and computational LC-MS/MS-based workflow for the high-throughput characterization of combinatorial protein modifications.  At the center of this methodology is a customized chromatography based on weak-cation exchange HILIC that provides unprecedented resolution of PTM isoforms and is directly compatible with mass spectrometry, thus allowing for the online acquisition of electron transfer dissociation (ETD) tandem mass spectra for PTM localization.  A significant challenge in this workflow is the ability to interpret the resulting LC-MS/MS data, which consists primarily of 'mixed' tandem mass spectra containing fragment ions from several co-eluting modified forms that are isobaric (i.e., same exact precursor mass).  Existing methods for analyzing mixed tandem MS operate exclusively on a spectrum-by-spectrum basis to determine the co-eluting species present.  Here we present an innovative approach that simultaneously interprets all of the mixed tandem MS at once by directly incorporating important chemicophysical relationships derived from the chromatography into the analysis. We demonstrate the ability of this approach to produce reliable and robust tandem MS identifications over a wide dynamic range of PTM isoforms, even in the absence of 'complete' fragmentation.   Several cell models will be used to illustrate the utility of the method for interrogating biological function.

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