Todd D. Williams, Ph.D. - US grants

This request is for a Hybrid Mass Spectrometer to be part of the Mass Spectrometry Laboratory at the University of Kansas, serving the Departments of Chemistry, Medicinal Chemistry, Pharmaceutical Chemistry, Pharmacology and Toxicology, KU Medical Center and the centers for Biomedical and Bioanalytical Research. The primary need is for increased fast atom bombardment (FAB) capability. This has been an established service lab with growing capability and sample load for 10 years. A total of thirty groups currently use the lab and at 7/12 through the fiscal year the lab has run 1690 samples and will easily reach 2500 by July. This work is being carried out on three facility instruments,a Varian-NUT CH5, a Nermag quadrupole GC/MS system, and a high resolution VG ZAB HS. The Nermag and the ZAB are used for the bulk of the samples. The CH5 (bought in 1970) is obsolete as a FAB instrument. The Nermag is currently saturated in use by probe and GC experiments. The ZAB has reached near saturation with routine exact mass and FAB analysis. Starting in July of 1989 the demand for FAB analysis has increased 6 times from the previous year. FAB work is expected to double again as peptide molecular weight experiments are done in support of four groups just beginning to use mass spectrometry for peptide characterization. The peptide projects involve the identification and localization of modified amino acids in small peptides. The MS/MS capability of the hybrid will be used to provide the sequence information not available in the FAB experiments. The unit mass precursor and fragment ion resolution obtainable with a hybrid in CAD experiments will be vital for peptide projects involving bromine and deuterium labeled peptides. Two groups are currently synthesizing coordination compounds and have been the major FAB users for confirmation of intermediates and final products. Both groups have had mixed success in obtaining molecular ions from their metal containing samples. Frequently demetallated species are observed in the FAB spectra. Similar compounds have been characterized by Field Desroption (FD). The use of FD for coordination compounds provides a soft ionization method that avoids the complications of matrix chemistry.

DESCRIPTION (provided by applicant): The University of Kansas Mass Spectrometry Laboratory (MSL) proposes to build an open access mass spectrometry system capable of supporting research in the synthesis of organic compounds. Users would be NIH-funded researchers primarily in the departments of Chemistry, Medicinal Chemistry and Pharmaceutical Chemistry. Support is requested for the purchase of two systems, a GC/MS and an ESITOF instrument, each with automated sample introduction. The ionization methods would be El or CI on a GC/MS instrument with a quadrupole analyzer and electrospray with a Time of Flight analyzer. The intention is to make available a set of instruments that an investigator could easily use by presenting a sample in an auto-injector vial, logging in, selecting a "basic" description of an experiment then launching the spectral acquisition. The result would be a spectrum, automatically printed, presenting the major ions detected from the sample. Time from sample introduction to spectra is minutes. Spectra will be saved electronically for later review and a workstation will be available to re-analyze files. There are 18 active synthetic groups in Chemistry, Medicinal Chemistry and Pharmaceutical Chemistry. Seven of the most active will present a broad structural diversity among samples ranging from low molecular weight intermediates through reasonably complex drugs (mw 600-1100) to coordination compounds, synthetic peptides and the occasional modified protein. The instruments will be managed by the permanent staff of a successful core facility. The primary motivations to create this capability are four fold: 1) increase sample through-put capacity without adding staff; 2) decrease turnaround time with automatic sample introduction; 3) lower per analysis cost to investigators and; 4) replace a 23 year old instrument. The addition of new instruments is important to maintain recent sustained growth in the research environment.

Recent advances in proteomics have provided tools for protein characterization that allow sampling of proteins in the important biological context of protein complexes. We propose to apply high sensitivity and high dynamic range protein characterization by mass spectrometry to identify proteins, detect posttranslational modifications and quantify relative and absolute protein levels. A hypothesis of this proposal is that quantitative measurements of protein abundances in tissue or cell culture samples will reveal roles of the target proteins. Further, the complexes are at the cell-cell or organelle-organelle interface and thus are part of a membrane micro domain. The lipids in these domains organize proteins, serve as signal reservoirs, donate or trap reactive oxygen species products and are intimately involved with the function of the proteins. A second hypothesis is that the difference in lipid profile of the micro domains with age, or manipulation, will reveal details of the protein lipid relationship. Aim 1) to identify proteins isolated in protein complexes obtained with immuno- or affinity extractions, a) Use MALDI TOP and TOF/TOF to rapidly confirm the ID of proteins and guide sample preparations, b) Use LC/MS/MS on the LTQ-FT to improve protein identification coverage with capillary HPLC and nanoUPLC. c) Improve information content from LC/MS/MS experiments on the LTQ-FT or QTOF instruments using MALDI search engines on LC/MS1 "survey" data, d) Develop an alternative protein ID validation strategy. Aim 2) To quantify stoichiometry and detect post translational modifications in protein complexes, a) Use LC/MS/MS on LTQ-FT or QTOF to extend protein coverage and find modified peptides. b) Use LCMS on LTQ-FT or QTOF for quantitation by SILAC, 16O/18O labeling, or spiked internal standards c) Improve data handling from LC/MS. Aim 3) Profile lipids in raft or membrane preparations from membranes at different ages, a) Profile sphingo- and phospholipids using head group specific MS/MS scans, b) Improve the quantitation and data handling in sphingolipid profiling. We hope to identify proteins/lipids that are most relevant to the aging process and oxidative stress using feature selection algorithms. In addition, our lipid and protein data will be used in a systems biology context to identify biological networks and pathways affected by aging and oxidative stress via mapping the proteins/lipids to known pathways.

Recent advances in proteomics have provided tools for protein characterization that allow sampling of proteins in the important biological context of protein complexes. We propose to apply high sensitivity and high dynamic range protein characterization by mass spectrometry to identify proteins, detect posttranslational modifications and quantify relative and absolute protein levels. A hypothesis of this proposal is that quantitative measurements of protein abundances in tissue or cell culture samples will reveal roles of the target proteins. Further, the complexes are at the cell-cell or organelle-organelle interface and thus are part of a membrane micro domain. The lipids in these domains organize proteins, serve as signal reservoirs, donate or trap reactive oxygen species products and are intimately involved with the function of the proteins. A second hypothesis is that the difference in lipid profile of the micro domains with age, or manipulation, will reveal details of the protein lipid relationship. Aim 1) to identify proteins isolated in protein complexes obtained with immuno- or affinity extractions, a) Use MALDI TOP and TOF/TOF to rapidly confirm the ID of proteins and guide sample preparations, b) Use LC/MS/MS on the LTQ-FT to improve protein identification coverage with capillary HPLC and nanoUPLC. c) Improve information content from LC/MS/MS experiments on the LTQ-FT or QTOF instruments using MALDI search engines on LC/MS1 survey data, d) Develop an alternative protein ID validation strategy. Aim 2) To quantify stoichiometry and detect post translational modifications in protein complexes, a) Use LC/MS/MS on LTQ-FT or QTOF to extend protein coverage and find modified peptides. b) Use LCMS on LTQ-FT or QTOF for quantitation by SILAC, 16O/18O labeling, or spiked internal standards c) Improve data handling from LC/MS. Aim 3) Profile lipids in raft or membrane preparations from membranes at different ages, a) Profile sphingo- and phospholipids using head group specific MS/MS scans, b) Improve the quantitation and data handling in sphingolipid profiling. We hope to identify proteins/lipids that are most relevant to the aging process and oxidative stress using feature selection algorithms. In addition, our lipid and protein data will be used in a systems biology context to identify biological networks and pathways affected by aging and oxidative stress via mapping the proteins/lipids to known pathways.

Project Summary/Abstract Proteomics research at the University of Kansas (KU) is limited by antiquated mass spectrometry instrumentation. This proposal requests $600,000 to acquire a new state-of-the-art Q Exactive HF mass spectrometer (1) coupled to an EASY-nLC 1200 chromatographic system for proteomics operations within the KU Mass Spectrometry Laboratory (MSL). The mass spectrometer combines a prefilter, a high performance quadrupole, and an ultra-high field Orbitrap analyzer. The primary rational for the requested instrument is that its analytical performance will enhance the research of NIH-funded scientists from six department at KU: Molecular Biosciences, Pharmacology and Toxicology, Medicinal chemistry, Chemistry, Pharmaceutical Chemistry and Ecology, and Evolutionally Biology. Additionally, as proteomics service of the KU MSL have expanded beyond the KU Campus, the requested instrument will impact the biological and health-related research of investigators at neighboring institutions such as Kansas State University (KSU), and the University of Kansas Medical Center (KUMC). Including these institutions brings the total number of regular users to 15. This proposal highlights several specific proteomics applications for each NIH-funded user that leverage the essential features of the requested instrument. The requested instrument will serves as an analytical workhorse for a wide variety of health-related research projects with the common focus of protein quantitative analysis in complex mixtures. Examples include, 1) quantitative protein up and down regulation in cancer cells deficient of Hsp90 using SILAC technology with the focus for low abundant kinases, 2) identification of SUMOylation substrates in primary hepatocytes under stress conditions that typically cause inflammation; 3) identification of partners in protein-protein interaction machinery relevant to mitochondrial neurodegeneration in Alzheimer's disease using mouse models.