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Robert J. Cotter, Ph.D.

Robert J. Cotter, Ph.D. Robert J. Cotter, Ph.D.

Research Program

The aim of our TCNP technology project is to develop new mass spectrometric instrumentation and analytical methodology to improve our capabilities for the elucidation of lysine modifications, particularly acetylation and ubiquitylation. One specific aims has been to develop chemical derivatization methods that will improve the structural (fragmentation) information available from MS/MS spectra. A new method for producing N-terminal sulfonation derivatives of peptides (1) that results in the exclusive formation of y-ions has provided a particularly effective approach to determine ubiquitylation sites (2,3). Following the digestion of ubiquitylated proteins with trypsin, the terminal Gly-Gly sequence from ubiquitin remains attached to modified lysine residues, the ubiquitylated peptides are recognized from the attachment of two N-terminal sulfonate tags, and the location of the site can then be determined from the y-ion spectrum. This methodology has been used to locate the ubiquitylations sites for CHIP (C-terminal Hsc70-interacting protein) (4).

Instrument development has been a major activity in the Cotter laboratory. A tandem time-of-flight mass spectrometer developed in the laboratory (5) has been used for the identification of acetylation sites in the hyperacetylated p300 HAT domain (6,7). The structural analysis of histone acetyltransferases, and their analogs, has been a collaborative effort with Philip Cole (TCP-4). The Cotter laboratory has also been involved in the development of a bioaerosol mass spectrometer for biological agent detection. When completed, this instrument will be used in the TCNP effort for single-cell and single organelle analyses.

The Cotter laboratory collaborates with many of the other investigators, in particular: with Akhilesh Pandey (TCP-4) for the structural analysis of phosphopeptides, and with Shelley Berger (Wistar Institute, DBP-2) for the determination of methylation and acetylation sites in histones. In addition, the laboratory is now developing MS/MS approaches for SUOMylated proteins.

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