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This project will: develop new micro-machined devices for the separation of proteins and couple these devices to high performance mass spectrometers for protein identification.

Primary Faculty co-Advisors:

• Terry Bricker, Biological Sciences Department (Protein Chemistry)
• Pat Limbach, Chemistry Department (Mass Spectrometry)
• Steve Soper, Chemistry Department (Micro-instrumentation/Chemical Separations)

Off-campus Participant: Jon Amster (University of Georgia)

Technical Proposal: "Biochemistry is a technique-driven science. One deals today with those problems that today's technology gives one a chance of solving, not necessarily with those problems one would like most to solve."(1) The goal of this project is to develop new analytical instrumentation and protein analysis methodologies. The scientific advances which will result from this project revolve around the development of new micro-instruments for separation and purification of proteins and the interfacing of these devices to high performance mass spectrometers. These micro-instruments are ideally suited for the separation and purification of small amounts (one-billionth of a gram or less) of biomaterials and will result in a dramatic improvement in analysis throughput, thereby allowing for a number of informative studies to be performed to rapidly elucidate the function of various proteins. The technical and procedural developments from this project will result in a new paradigm for biomolecule analysis.

Protein Characterization. Elucidation of the functional properties and structural organization of membrane protein complexes is one of the central objectives of current biochemical investigation. Biological membranes are involved in virtually every aspect of cellular organization and activity. One of the most intriguing aspects of membranes is their role in energy transfer in photosynthetic organisms. Light energy, which is the product of a most violent physical process, fusion, is transformed into biological energy equivalents utilized by the photosynthetic cell. The photosynthetic process provides both the carbohydrate which lies at the base of virtually all food chains and, as a byproduct, all of the atmospheric oxygen. Recently, much effort has been directed towards understanding the structure, function, and assembly of the membrane protein complexes involved in the photosynthetic light reactions. Despite much progress, the mechanisms involved in protein processing, membrane insertion, cofactor assembly, and regulation of photosynthetic electron transport remain poorly understood.

In this project, we will examine the functional proteomics of the thylakoid lumen from both higher plants and cyanobacteria. This subcellular compartment is very poorly understood and until recently had only been cursorily examined. Preliminary results from the Bricker laboratory and others indicate that, while more than one hundred protein components may be present in the lumenal compartment, the functions of less than fifteen of these have been identified. We propose to isolate and identify the proteins located in the thylakoid lumen in both higher plant (Arabadopsis) and cyanobacterial (Synechocystis 6803) systems. These model systems are particularly appropriate for these studies as the genome of Synechocystis 6803 has been sequenced and that of Arabadopsis will be completed within the next two years (about 40% of this genome is currently known). We will then systematically delete the genes encoding these proteins in the cyanobacterial system. These experiments will allow us to form working hypotheses as to the function of these lumenal components, which will then be tested by rigorous molecular and biochemical approaches.

Initially, the lumenal proteins will be isolated from higher plant and cyanobacterial thylakoid membranes. The Bricker laboratory has already developed efficient procedures for the isolation of these components from higher plants (2) and is currently extending these techniques to cyanobacterial thylakoid membranes. The proteins of these lumenal preparations will then be identified as described below which will permit the determination of the genes encoding the individual protein spots. Once the genes encoding the lumenal proteins have been identified, insertional and/or deletion mutagenesis will be performed in Synechocystis 6803. These techniques have already been implemented in the Bricker laboratory. Mutant screening will allow a preliminary assessment as to the functional lesion induced by the mutagenesis and will provide a first glimpse as to the function of these lumenal proteins.

Micro-instrumentation and Mass Spectrometry While the specific target of this research project will be to characterize lumenal proteins, another major component will be to fabricate the appropriate micro-instrumentation which will allow for the purification of protein mixtures, such as those provided by the thyalkoid lumen, prior to their identification and structural characterization by mass spectrometry. The lumenal proteins, which will be isolated in the Bricker lab as discussed above, provide an excellent model for us to test the performance of our micro-machined devices and to train and educate our students in the areas of protein chemistry, micro-instrumentation, and structural analysis. As only approximately 100 lumenal proteins will be present, the level of complexity compared to that of another model system, such as Eschericia coli, is reduced, thereby allowing us to examine an important and relevant biological system without prematurely attempting a system that is too complex.

The fabrication of micro-instrumentation using photolithographic techniques has permeated into the molecular biology and chemistry arenas and has already had a profound impact on instrument development and assay methodologies. The principal advantages associated with these devices include: (a) the instrument footprint is small; (b) the micro-fabricated device can be multiplexed easily so that several analyses can be completed simultaneously; (c) complex patterns of channels for fluid manipulation and mixing can be fabricated; (d) the potential for integrating various components onto the device is high; and (e) the sample requirements are small.

Sample preparation, which will be a key step in our protein analysis methodology, has been neglected to a great extent by a number of research groups developing microfabricated devices. The difficulty in developing miniaturized devices for sample preparation is the low volume requirement associated with these devices, creating potential difficulties in transferring the sample from one device to another. That difficulty is somewhat reduced in our application, due to the low sample amounts of proteins to be characterized. As structural analysis using mass spectrometry requires fairly high concentrations (µM –nM), reducing the volume during the sample preparation step actually results in higher concentrations which can be delivered directly to the mass spectrometer.

We will design and construct two micro-machined devices for lumenal protein purification. The first will be based on standard electrophoretic (i.e., charge) separation. The goal here will not be to develop a replacement for 2-dimensional polyacrylamide gel electrophoresis (2D PAGE), but to optimize the interface between the protein isolation, protein purification and mass spectrometry steps. The second device to be investigated will be a more elaborate micro-machined system which will contain several different sample purification modules. The modules to be investigated will allow separation of proteins and peptides based on their chemical and physical properties. In addition, modules which contain immobilized proteases (enzymes which digest proteins), can be constructed which will allow us to generate peptide maps of the proteins of interest in-line prior to direct mass spectrometric analysis.

Mass spectrometry has long been a preferred method of analysis due to its speed, sensitivity and ability to physically separate (by mass) complex mixtures. Recently, a number of uses of mass spectrometry in gene and protein studies have been proposed. Although mass spectrometry is theoretically well-suited for such applications, a number of technical and scientific concerns have limited its use in these areas. To overcome many of these concerns, we will focus on the development of an appropriate interface between micro-machined devices and the mass spectrometer which will permit the structural characterization of small amounts of biomaterials.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a popular analytical method for the characterization of peptides and proteins. This technique involves combining the analyte of interest with an untraviolet light-absorbing molecule (the matrix), co-crystallizing this mixture on a sample plate, and analysis of via desorption and ionization using an ultraviolet laser. Electrospray ionization mass spectrometry (ESI-MS) is a complementary method to MALDI-MS, and involves flowing a solution containing the protein or peptide of interest through a needle held at a high electric potential. The aerosol produced by this process, which contains charged droplets of the analyte, is then focussed into the mass spectrometer. Both MALDI-MS and ESI-MS are proven technologies for peptide and protein analysis; the Limbach lab has extensive experience with these technologies.

The Limbach lab, in conjunction with the Soper lab, will focus on developing appropriate interfaces between the micromachined sample preparation devices and the MALDI or ESI mass spectrometer. We propose to develop a unique sample deposition process that will permit the sample to be placed on the MALDI sample plate with the appropriate matrix solution directly from the micromachined device. Our approach is based on a prior macroscale method of sample deposition.(3) For the ESI-MS studies, we will investigate a variety of different interfaces which will couple the micro-machined devices to the ESI mass spectrometer.(4) Preliminary work in this area is already in progress in the Limbach lab and we have identified that the physical coupling of the ESI transfer device (which is a fused silica capillary) to the micromachined device is an area in need of more study. Dr. Soper's group generates micromachined devices from poly(methylmethacrylate), PMMA; hence, unique coupling methods are available that cannot be had with the traditional glass-based micromachined devices.

The students involved in this research project will be exposed to a variety of interrelated technologies and disciplines. The students will learn classical protein chemistry techniques, will be exposed to new instrumental methods of analysis, and will have the ability to design an experimental protocol that is optimized for the analysis of low amounts of proteins isolated from our organisms of interest. By combining the protein chemistry with the technology developments, these students will be able to develop more efficient preparation or analysis methodologies, and these students will develop an understanding and appreciation of the role of technology in biochemical advancements.

Number of IGERT apprentices to be recruited and probable home departments: Two--one from Chemistry & one from Biological Sciences.

Consistency with the Macromolecular Education, Research & Training theme: The project requires its students to understand proteins, synthetic polymers used for microfabrication, and advanced methods for characterizing these macromolecules. The MS-I and MS-II courses are particularly valuable, for one must learn the state of the art in equipment and methods before advancing it.

How does the project form a vector cross-product of existing research themes by the participants?

Existing research directions. Bricker's group has been involved in the structural characterization of membrane protein complexes for fifteen years Limbach's group has focused on developing new analytical mass spectrometric approaches for the analysis of complex biomolecule systems. Bricker and Limbach have recently begun a collaborative project aimed at developing new mass spectrometric approaches for the analysis of hydrophobic peptides. During that collaborative project, these investigators have found that analysis bottlenecks include the lengthy sample preparation steps and the poor sensitivity of conventional sample preparation techniques. Soper's group has focused on fabricating micro-instrumentation using PMMA instead of traditional glass-based substrates. His group has also used these devices for high-throughput analysis of DNA for sequencing and diagnostic applications.

New research direction. This project will bring together for the first time this diverse group of investigators to develop new methodologies and technologies from the "ground up". The micro-instrumentation will be designed with the specific needs of this research project in mind. The mass spectrometric characterization of proteins and peptides delivered from the micro-machined devices is a naturally extension of the current collaborative work between Bricker and Limbach. This research project will also lead to spin-offs in other areas of biomolecule characterization including new instrumentation for genotyping or other proteomics interests.

How do students benefit from the team-oriented research, beyond what would be available to them from either advisor separately? Biological mass spectroscopy is a very hot area for employment. The student from biological sciences will gain a far deeper understanding of the mass spectroscopy craft from Professor Limbach then would be obtainable simply by pushing buttons on the instrument. Similarly, students involved in the microfabrication aspects will have to apply them to a complex biological system that they might never consider without Professor Bricker's expertise--and reconcile their results with the large knowledge base for that system.

Briefly describe the support level available to each individual faculty or off-campus participant (i.e., without IGERT) The LSU faculty involved are all independently supported for research in related fields. Together, they hold 8 major grants of about $800,000/year. This ensures a stable environment for the IGERT students, including healthy exchange of skills and ideas with postdocs and graduate and undergraduate students.

Interdisciplinary strengths of the team project: Although Limbach and Soper were both trained as analytical chemists, Limbach has focused on biological mass spectrometry and Soper concentrates on developing new instrument for molecular separations. Bricker's background is in microbiology; he brings the more classical biochemist approach to this project. Bricker and Limbach collaborate currently on membrane protein characterization. These investigations led to this proposed project.

Commitment of faculty & off-campus participants to work side-by-side with apprentices:

Bricker, a full professor within the Department Biological Sciences, also maintains an active personal research program and interacts daily with his laboratory members. He is excited about the possibility of personally working side-by-side on a project of 2-6 weeks with students in the "master-apprentice" model presented in this IGERT proposal. This time commitment would be fulfilled during winter or spring breaks. Soper is currently an associate professor of Chemistry. He has been actively and personally involved in the micromachining and the fabrication of micro-instrument platforms for the past several years as part of NIH-sponsored research. The students involved in the micromachining aspects of the project will utilize the CAMD synchrotron storage ring and related facilities; we are well-equipped to carry out all phases of the micromachining tasks. Soper will also incorporate both students involved in this project into his group's normal group meeting schedule so that the students can present their results in a formal setting. Limbach is presently and assistant professor of Chemistry. His involvement will include training the students on the necessary instrumentation, assisting the students with the micro-instrumentation–mass spectrometry interface designs, and working with the students on the protein characterization studies. This can easily lead to a small project and report with a new apprentice. Outside of the project meetings, Limbach will meet regularly with the students to assess their progress and to guide these students on appropriate upcoming experiments.

(1) "The RNA world" edited by Raymond F. Gesteland, John F. Atkins. Cold Spring Harbor, NY : Cold Spring Harbor Laboratory Press, 1993. 630 p.

(2) Prevost, M., Vu, M., Frankel, LK. and Bricker, T.M., in preparation.

(3) a.Hensel, R. R.; King, R. C.; Owens, K. G., Rapid Commun. Mass Spectrom. 1997, 11, 1785-1793. b. Axelsson, J.; Hoberg, A.-M.; Waterson, C.; Myatt, P.; Shield, G. L.; Varney, J.; Haddleton, D. M.; Derrick, P. J., Rapid Commun. Mass Spectrom. 1997, 11, 209-213.

(4) a. Valaskovic, G. A.; McLafferty, F. W., J. Am. Soc. Mass Spectrom. 1996, 7, 1270-1272. b. Mazereeuw, M.; Hofte, A. J. P.; Tjaden, U. R.; Greef, J. v. d., Rapid Commun. Mass Spectrom. 1997, 11, 981-986. c. Bateman, K. P.; White, R. L.; Thibault, P., Rapid Commun. Mass Spectrom. 1997, 11, 307-315. d. Hannis, J. C.; Muddiman, D. C., Rapid Commun. Mass Spectrom. 1998, 12, 443-448.

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