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Bio-Organic Biomedical Mass Spectrometry Resource

A. L. Burlingame, Ph.D.

Center Overview

The National Bio-Organic, Biomedical Mass Spectrometry Resource Center, supported by the NIH National Center for Research Resources, provides both scientific and technical expertise in mass spectrometry and proteomics as well as the indispensible state-of-the-art high-performance, tandem mass spectrometric instrumentation. The facility is a world leader in proteomic analysis focused on addressing daunting challenges posed by poorly understood molecular-level underpinnings of human protein biology. In addition, it provides a service for small molecule analysis.

Significant instrumentation in the facility includes three QSTAR quadrupole orthogonal time of flight instruments, and three vintages of the LTQ-hybrid platform: an LTQ Velos Orbitrap instrument with electron transfer dissociation (ETD), an LTQ-Orbitrap XL with (ETD) and an LTQ-FT linear ion trap FT-ICR instrument equipped with the ability to perform electron capture dissociation (ECD). The Center also has a 4700 Proteomic Analyzer MALDI tandem time of flight instrument; as well as a QTRAP 5500 hybrid triple quadrupole linear ion trap instrument.

The major research focuses within the Center are the analysis of post-translational modifications, including phosphorylation, O-GlcNAcylation, acetylation, methylation and ubiquitinylation as well as their co-occurrence and possible cross-talk on many protein classes, especially in chromatin. In addition a growing need requires dealing with modulation of PTM site specific changes during cell stimulation by drugs or inhibitors. Thus development of methods for multiplexed quantitative comparative analysis of protein and post-translational modification levels is on-going. The program also continues to develop one of the leading suites of tools for analysis of mass spectrometry proteomics data, Protein Prospector. The current web-based release allows unrestricted searching of MS and MSMS data from the orthogonal CID and ETD energy deposition techniques, as well as the ability to perform comparative quantitative analysis of samples using isotopic-labeling reagents. It is the only freely-available web-based resource that allows these types of analyses.

Impact on Human Health

Human cells and tissues are comprised of a genetic encyclopedia, and corresponding proteins sometimes referred to as the machinery of cells. The measurement of an individual's genes is now a solved analytical problem.

In stark contrast, most translated proteins are not physiologically active until they are covalently converted to an active form. This can be achieved by specific backbone cleavages (proteolysis), their covalent alteration at one or more sites by adding and removing a variety of small molecular entities at specific locations in a protein's sequence, or their participation in non-covalent complexes. Several of these possibilities may be required sequentially or in concert to meet a myriad of the cells' physiological needs and maintain overall homeostasis.

Through the last decade mass spectrometry has emerged as the pre-eminent technology having sufficient inherent power and versatility to drive global biomedical research aimed at unraveling the molecular-level complexities of human protein biology.

Hence, current mass spectrometers work to accelerate our abilities to pursue the global detection, definition and eventual understanding of the molecular details involved in the function of the machinery of cells for normal states as well as for dysfunctional disease states.

The long-term objectives are to gain a comparative, comprehensive knowledge of human protein biology for healthy and disease states. The success of such studies requires research using this technology, since it can "see, pinpoint and define" these dynamic alterations. The information obtained will reveal the presence of any defective proteins causing disease states and be able to determine the molecular nature of their defect(s). This knowledge reveals then the array of possible protein targets that will accelerate the design of strategies for the development of therapeutic interventions.

On yet another front, many viruses represent world wide health challenges. In collaboration with the NIH NIGMS HIV Accesssory and Regulatory Complexes (HARC) Centers we have invested considerable effort in detecting and identifying the human proteins that interact with the 18 open reading frames of HIV. These studies have revealed several hundred new human proteins (Nature, in press).

In summary, the biomedical research community requires state of the art mass spectrometers to detect, define and measure this daunting array of structural changes to the cell's proteins that determine their various functions and dynamics. Repetitive large capital investment for new instrumentation is required to stay at the technological forefront and is virtually necessary on an annual basis. Thus such investments are best made within national Centers where the expertise and analytical power can be optimized and then shared among a large segment of the biomedical research community.