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. Our laboratory makes extensive use of two Applied Biosystems API QSTAR(tm) Pulsar i QqTOF mass spectrometers for the characterization of complex biological samples obtained from collaborative research projects. In agreement with ABI (Framingham, MA) and Sciex (ON. Canada), we continue to investigate the performance of these instruments and we continue to work with ABI on software development for this instrument and data analysis. Currently, our focus is on establishing routine capillary LC-IDA-MS/MS for proteomics and glycomics applications, in addition to characterization the large variety of biological samples that are submitted to the MS Resource. Use of automated IDA MS/MS functions are continuously being optimized for typical protein digests, lipids and carbohydrates, and batch parameter files have been generated for ease of use by the members within the laboratory. Processing scripts have been optimized for use on workstations for data analysis. A Waters capLC was installed in 2004; sensitivity can be excellent but frequent plumbing problems have interfered with high throughput use of columns having diameters less than 100 um. The use of the smaller diameter column assemblies offers a significant increase in sensitivity, enabling samples of low concentrations or limited quantity to be analyzed. Consequently, the optimum operation of the combined system should offer an alternative, improved approach for analysis of important biological samples. The current phase of this project can be simply divided into two principal aims; primarily the optimization of the Waters CapLC system, followed by method development studies to complement the current research undertaken within the MS resource. Following substantial instrument optimization, current method development is being pursued for proteomics-based studies, including phosphorylation analysis. Future directions for method development include the analysis of other post-translational modifications, including glycosylation, and carbohydrate samples.
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