This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cell responses to environmental cues are controlled by key regulatory proteins whose localization, activity, and abundance are often modulated rapidly by one or more post-translational modifications (PTMs). PTMs including phosphorylation, acetylation, methylation, ubiquitination, sumoylation, nitrosylation and O-GlcNAc-ylation have fundamental roles in regulating signal transduction, gene expression, and metabolic pathways. Although PTM to a few specific proteins have been detected in response to various hormonal and stress-related cues, only recently have systematic approaches been developed to identify such modifications at a global level. The ability to monitor changes in PTMs at a global level would greatly accelerate the discovery of the initial events in signal transduction mechanisms; however, many technical challenges remain before we can routinely identify important PTM events in complex samples. These challenges include decreasing both the dynamic range and complexity of the samples to match the analytical ability of the current generation of mass spectrometers. One way of achieving this goal is to purify peptides containing a specific modification. This approach has had limited success but is effective for several modifications. An example is the purification of phosphopeptides using immobilized metal affinity columns (IMAC). The major problem with this approach is only the isolated peptides are monitored. To obtain a complete picture of the event under study many separate experiments must be preformed. If the experimental samples are expensive or difficult to obtain this may not be feasible. Additionally, it may be difficult to compare different biological samples given the short treatment times necessary to study the initial events in signal transduction. We are exploring the possibility of combining protein and peptide purification methods with advanced mass spectrometric instrumentation to analyze a number of PTMs as well as changes in protein abundance using a single sample.
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