Julie A. Leary - US grants

The long term goal of this project includes the development of analytical methodology for sequencing and determining linkage position of underivatized oligosaccharides which are coordinated to various metals. Delineation of gas phase mechanisms and fragmentation pathways of these oligosaccharides, which is equally important, will be investigated using Tandem Mass Spectrometry (MS/MS, and MS/MS/MS). Semiempirical calculations will also be utilized as a tool for predicting gas phase conformation of these metal-coordinated compounds. Fast atom bombardment ionization in combination with tandem mass spectrometry of lithium- coordinated oligosaccharides has already been used to unambiguously differentiate the linkage position of isomeric di-, tri-, and tetrasaccharides of the glucose moiety. Extensions of this method to branched glucose and glucosamine oligomers, glycolipids and cerebrosides will be undertaken as well as extending the mass range of analysis to 3,000 Dalton. Tandem mass spectrometry of the multi-lithiated oligosaccharides will be investigated as well as probing the coordination chemistry of several other metals (Mg, Ca, V, Mn, Fe, Co, Cu, Ag, Zn) to these sugars. Extensive labeling studies will be carried out to probe the chemistry of dissociation of these oligomers. Semiempirical calculations of the oxygen-linked and carbon-linked oligosaccharides will provide information which will allow us to determine the most likely site(s) of metal coordination.

The long term goal of this competing renewal is the development of methods that allow for the analysis of various enzymatic processes using mass spectrometry. The major areas proposed herein include: 1) Kinetics and catalytic mechanism determination for phosphohexose-isomerases. 2) Kinetics, substrate determination, and mechanism determination of tyrosylprotein sulfotransferases. This will also include a new procedure for unambiguous determination of sites of sulfation on natural substrates. 3) Investigation of substrate and inhibitor interactions with a newly discovered human endosulfatase, Hsulf-2 Each of these areas employs electrospray ionization (ESI)mass spectrometry (MS)using either an LTQ ion trap or an FTICR instrument to measure various kinetic parameters such as Km, Vmax, Ki, kcat and specificity constants. It is proposed that kinetic parameters can be calculated using ESI-MS thus obviating the need for chromophores as required by traditional spectrophotometric techniques. Equally important is the fact that kinetic information, either previously intractable or difficult to measure, can be gleaned using the MS based methods outlined herein. The problem with analyzing sulfated biomolecules is well recognized in mass spectrometry. The proposed research will alleviate those problems associated with loss of sulfate upon electrospray ionization and MS/MS in the positive ion mode. Similarly, methods are proposedfor obtaining biological data on phosphoisomerase systems; one of which is highly difficult to classify kinetically and mechanistically. . .

[unreadable] DESCRIPTION (provided by applicant): The College of Chemistry, at the University of California-Berkeley, is seeking funds for the purchase of a MALDI-TOF mass spectrometer. This instrument will be housed in the Mass Spectrometry facility and will be available to students and staff on a 24 hour, 7 days per week basis. The facility is in critical need of new state-of-the-art instrumentation in order to meet the growing demand for biomolecule and polymer analysis of its NIH funded researchers. Although the facility has been fortunate to secure good instrumentation in the past, many of the current mass spectrometers are becoming quite old and do not meet the needs for high molecular weight dendrimers, oligonucleotides, proteins and carbohydrates. The research of six faculties are listed below, each of who would benefit greatly from an in house, open access MALDI-TOF. Five of these researchers have established NIH funding, while one is the newest assistant professor in the department. These six groups, alone, include 81 postdoctoral associates, graduate and undergraduate students who will benefit directly from this equipment. [unreadable] [unreadable]

The broad goal of this competing renewal focuses on studying carbohydrate:protein, as well as protein:protein non-covalent interactions. In particular, we propose to investigate the binding and interaction of glycosaminoglycans (GAG) with chemokine ligands of the N-terminal G-protein coupled receptors (GPCR), CCR2 and CCR5, as well as exploring possible ternary complexes of all three using Q-TOF/Ion Mobility Mass Spectrometry. Binding of the GAG with chemokine will be explored via incubation of various GAG libraries with the chemokines of CCR2 and CCR5. Specific binders will be identified using our GAG compositional analysis. Chemokines will be expressed and purified in house. Collisional cross section measurements will be made for any of the non-covalent complexes identified, thus providing information about conformation change upon binding. Additional Surface Plasmon resonance and MS kinetic binding studies along with thermal calorimetry will provide important information about the binding strength. The intent is that a detailed understanding of these interactions will allow for effective targeting of the chemokine system for therapeutic benefit. The major areas proposed are interwoven in such a way that methods development, biotechnology and application to biologically relevant systems are the cohesive elements that bind the proposed research together.

The development and implementation of the mass spectrometry core is essential to the research of the listed[unreadable] principal investigators. In particular the core will provide mass spectral analysis using the proposed bottomup[unreadable] and top-down methods employing both Q-TOF technology as well as FTICR high-resolution analysis.[unreadable] Both a top-down (intact protein identification) and a bottom-up (protein digestion) approach will be used to[unreadable] identify proteins of the various ribosomal complexes. Differences in post-translational modifications will be[unreadable] determined for ribosome complexes which have been subjected to infection by various viruses (hepatitis c,[unreadable] dengue, etc). eif3 factors associated with the ribosome will be analyzed and investigated for the presence of[unreadable] phosphorylation. Both the high-resolution capabilities of the exisiting FTICR and the sensitivity and flexibility[unreadable] of the Q-TOF, in combination with well established sample preparation methods such as IMAC, will allow for[unreadable] the unambiguous determination of protein constituents and associated post-translational modifications.

The development and implementation of the mass spectrometry core is essential to the research of the listed[unreadable] principal investigators. In particular the core will provide mass spectral analysis using the proposed bottomup[unreadable] and top-down methods employing both Q-TOF technology as well as FTICR high-resolution analysis.[unreadable] Both a top-down (intact protein identification) and a bottom-up (protein digestion) approach will be used to[unreadable] identify proteins of the various ribosomal complexes. Differences in post-translational modifications will be[unreadable] determined for ribosome complexes which have been subjected to infection by various viruses (hepatitis c,[unreadable] dengue, etc). eif3 factors associated with the ribosome will be analyzed and investigated for the presence of[unreadable] phosphorylation. Both the high-resolution capabilities of the exisiting FTICR and the sensitivity and flexibility[unreadable] of the Q-TOF, in combination with well established sample preparation methods such as IMAC, will allow for[unreadable] the unambiguous determination of protein constituents and associated post-translational modifications.

[unreadable] DESCRIPTION (provided by applicant): The University of California at Davis seeks funding for an accurate mass, high resolution mass spectrometer. The Thermo Electron Orbitrap instrument has been identified as the best instrument for the campus Satellite Mass Spectrometry Facilities (CSMSF). The two satellite Facilities, which comprise the CSMSF, are available 24hrs/day, 7 days/week to campus researchers. Currently there are no high resolution mass spectrometers on the Davis campus for small molecule analysis. There are several PI owned high resolution instruments (FTICR) however, these are not available to the general research community on campus. An FT in the Genome center is used exclusively for proteomics. The Orbitrap can be easily integrated into the existing CSMSF which is composed of three other instruments all of which are operated on a 24/7 basis. This open access system has worked remarkably well and the Orbitrap would finally bring high resolution mass measurements to the general scientific campus community. Demonstration samples run on the Orbitrap (see data in proposal) have shown it to be very reliable, very accurate and easily operated by graduate students and postdoctoral associates. The lack of cryogens makes this instrument particularly attractive and will serve as a robust high resolution, student operated instrument. A mechanism is in place to train and oversee student researchers and Professor Leary's group is available for consultation, advice and data interpretation. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The U.C. Davis Campus Mass Spectrometry Facilities (CMSF) is seeking funds to replace a 13 year old obsolete LCQ Ion Trap. This instrument is used for LCMS and is no longer supported by Thermo-Fisher. Down time is extremely high and thus sample analysis and turnaround time have been severely impacted. The anticipated replacement instrument is a Waters Xevo Q-TOF with interfaced UPLC. Five major users have been identified from the Departments of Chemistry, Molecular and Cellular Biology and Neurophysiology;all are supported by NIH.

DESCRIPTION (provided by applicant): The U.C. Davis Campus Mass Spectrometry Facilities (CMSF) is seeking funds to replace an 11 year old, obsolete AB 4700 MALDI-TOF/TOF. This instrument is used for high-throughput protein, peptide, glycan and oligo analysis by all our major users and is anticipated to soon be no longer supported by AB Sciex. The anticipated replacement instrument is a Bruker UltrafleXtreme. Seven major users and one minor user have been identified from the Departments of Chemistry, BioMedical Engineering, Animal Science, Med: Dermatology, Med: Hematology & Oncology and all are supported by NIH.