Mass Spectrometry Facility

NCRR National Research Resource in Mass Spectrometry, Biomolecular Mass Spectrometry and Proteomics

Contents:

Introduction

This Mass Spectrometry Facility is one of several national technology centers focused on advanced mass spectrometry in biomedical research supported by the National Center for Research Resources of the NIH. This program was initiated at University of California, Berkeley in 1973 and moved to University of California, San Francisco in 1978.

The Center's purpose is to develop and promote the effective usage of leading techniques of mass spectrometry in biomacromolecular research. Our particular goals revolve around solving challenging problems in protein biology or proteomics that impinge on molecular abnormalities that underlie human disease.

Our investments in the development of new methods and technologies are driven by the unmet challenges and research opportunities at the current forefront of biological and clinical research. Some priorities are chosen from among those expressed by our collaborative colleagues about their needs. We also anticipate what additional tools will be required to maintain state of the art methodology. This involves developments in complex separations, protein identification and quantitation methodology, and bioinformatics. These tools enable more advanced studies in human proteomics, including gaining a comprehensive knowledge of epigenetic modulation of protein function.

This resource enjoys broad multidisciplinary faculty participation (Burlingame, Bradshaw, Babbitt, Chalkley, Guan, Medzihradszky, and Trinidad) with major research interests in protein and glycobiology, cell signaling networks, bioinformatics as well as mass spectrometry including Fourier transform ion cyclotron resonance (FTICR) mass spectrometry technologies.

Background

Unparalleled advances in macromolecular mass spectrometry continue at a rapid pace and are providing ever more powerful tools to unravel and define the protein composition of cells on a global scale[1,2]. Indeed, complex suites of proteins can be studied in concert as well as comparatively in their functional biological context through recently available multiplexed tools for quantitative studies of protein dynamics[ 3 ].

Our resource vision is focused on fostering integration of the complementary technologies required to address the diverse questions in proteomics - from low volume sample handling (and robotics) to high throughput mass spectrometry. The development of high capacity bioinformatics tools is essential for processing and management of mass spectrometry experiments on the large scale required for effective proteomic and dynamic studies of mammalian cells, subcellular organelles, protein machines and immunoprecipitates[4].

Current technologies are focused on the identification of unknown proteins[5-10], the structural characterization of modified proteins[11-20], and the separation, identification and quantitation of complex mixtures of proteins[3,18,20].

Collaborative Projects

The collaborative projects being pursued are of key scientific significance and are almost always carried out with biological or clinical investigators. As already noted, many collaborative projects drive core methodology, technique and software development. Collaborations usually represent a mix of multi-disciplinary, inter-dependent efforts involving sample handling, adaptation of techniques that require experience for their optimal use, and development of new software to enable the analysis of data and facilitate its comprehension. Other collaborations require acquisition of more advanced instrumentation and investment in its further tailoring or optimization to meet particular kinds of analytical needs. Some require the development of instrumentation that will provide critical characteristics that are not commercially available.

Many questions and opportunities require identification of posttranslational modifications at the macromolecular level[21], usually in the context of structural factors that govern non-covalent molecular recognition, binding and communication. Hence, many new opportunities involve deciphering the molecular determinants of cell phenotype and phenotypic differences. Since protein abundance and physiological function are not in general directly represented by expression of message, gaining an understanding of cell and tissue function or dysfunction requires a comprehensive knowledge of differential protein expression and its temporal modulation by, for example, exposure to extracellular stimuli, progression through the cell cycle, etc. Many times these changes (modulation of protein activity or function) involve differential occupancies effected by multiple changes due to posttranslational modifications[11].

Current Research Portfolio

However, as indicated above we are pursuing a longer-term major initiative aimed at development of the necessary technologies to deal effectively with studies of epigenetic processes prevalent in many aspects of cell biology that are presently poorly understood on a global proteomic scale[21-24]. These include phosphorylation, O-GlcNAcylation, sulfation, acetylation, e-aminolysine mono-, di- and trimethylation, ubiquitination and sumoylation[23]. In some cases these covalent modifications function as docking sites in signaling cascades[21, 25-27]; in others, less well understood, they occur as clusters of different modifications recognized by multidentate effectors[24, 28-31] that are mostly unknown proteins and/or protein complexes.

The considerable challenge is to define posttranslational occupancies and stoichiometries for each modified protein isoform[11 ]. The methods of choice usually involve some form of robust tandem mass spectrometry as well as, in some cases, application of new capabilities based on non-ergodic energy deposition using Fourier transform ion cyclotron resonance instrumentation[11]. The recent discovery of electron capture dissociation(ECD) has provided a fundamental new technique that can be further developed and optimized to pursue this goal by permitting de novo sequencing of covalently modified proteins directly. To solve this problem we have implemented SWIFT on a commercial FTICR-MS system to enable selection of specific ion charge states for electron capture of isoform specific components of histones mixtures[32-34].

We are continuing to pursue both optimization and acquisition of the most advanced technologies to enable tackling these new opportunities as well as to prepare for future challenges in biomedical research.

Finally, we are working on solving current inadequacies or bottlenecks while we plan to take advantage of the new instrumentation and methodologies that will most likely permit major changes in our experimental strategies.

Some recent major changes in capability revolve around the scale of capacity in both expertise and technology required to meet the challenges, opportunities and needs in biomedical proteomics. These opportunities are particularly compelling, since the human proteome is to a large extent, qualitatively unknown. In addition, knowledge of quantitative differences in protein posttranslational occupancy levels will be required in many cases. This situation requires continued development and refinement of micro sample handling capabilities with mass spectrometric technologies and major development of data management and bioinformatics tools to help meet these major challenges.

Protein Prospector and Software Development

In addition to the pioneering development of ProteinProspector software tools over the last decade, the Laboratory Information Management Systems (LIMS) and computer infrastructure continues to enable the 'automated' analysis and management of very large datasets that are becoming characteristic of proteomic experiments.

Thus, in fact, much of our effort has focused on the refinement of robust tandem mass spectrometry and data management platforms for high capacity protein and phosphoprotein characterization and quantitation. The new capabilities of Protein Prospector has allowed us to process large datasets (> 100 LC-CIDMS runs) and has laid the foundation for further development of core methodology and automation to meet the accelerating scale of collaborative needs in the biomedical research enterprise[4]. In addition, we have produced a new web version (currently under beta testing) for LC-MSMS analysis that produces report summaries tailored to analysis and comprehension of very large data sets[4]. In addition, a software tool called SearchCompare permits inter-comparison of results on large data sets from related experiments.

For relative quantitation analysis of related samples, software tools are required to extract the quantitative information and present in a succinct, yet comprehensive format. Protein Prospector is a very flexible tool for this type of analysis, and has been used for quantitative studies using a number of different isotopic labeling strategies [3, 35, 44, 45].

This improved capacity has proven invaluable for studies of components of protein digests bearing low stoichiometry and other modifications (especially phosphorylation [12,17,19,20,35], acetylation [11,12,14], methylation [11-13], O-GlcNAcylation [18,35,36], ubiquitination [15], the discovery and characterization of aliphatic protein sulfation [16] and chemical protein-protein crosslinks based on formaldehyde [37] or bifunctional reagents[38,39]).

These projects represent the beginning of an important trend towards tackling more complex, labor intensive projects in cell biology that would have been previously intractable on a global scale without the remarkable new power of the ProteinProspector processing capability[3,19,20]. In truth, these kinds of important projects are not instantaneously soluble even by the most powerful methods of mass spectrometry. However, it is now technically feasible to obtain a comprehensive knowledge of the identity of the suites of protein players up front for any given research project[3,19,20,40]. This early knowledge permits an investigator to formulate further research questions and experiments focused on the unknown, unanticipated or most promising components of his research.

Rapid development of further robust bioinformatics tools in ProteinProspector is essential for mining and exploiting the information in the genome and protein databases.

Emerging Themes

This resource is moving forcefully into development of methodologies that will facilitate the definition macromolecular interactions and their modulation by extra-cellular stimuli. This focus was initiated through experience gained during comprehensive studies of nucleocytoplasmic trafficking where binding constants for GST fusion pull downs are in the micromolar range or stronger[41]. We have recently begun to invest major effort in development of strategies that permit studies of weaker in vivo machines as well as protein recognition and communication cascades. This involves formaldehyde cross-linking in vivo in studies of binding partners of the TAP probes in protein components in the ras pathway protein [7,8]. This knowledge will open new views of the casts of players carrying out intra- and inter-cellular processes.

All such studies require an ability to define suites of proteins and their posttranslational variants in the range from several to hundreds per experiment. Information processing, comprehension and interpretation are critical. The development of strategies for integration of such global knowledge into a better understanding of the cell and human biology is essential.

It is self-evident that we have entered a revolutionary new era, technically and intellectually, involving collaborative problems in the mainstream of biological and medical science of our time, seeking an understanding of their common molecular denominators. How do macromolecular entities such as phosphoproteins, histones or chromatin remodeling complexes carry out their roles in cellular homeostasis, gene expression or silencing, cell division, physiological recognition, immunochemistry, communication and regulation? What are the progressions of transformations at the molecular level that are responsible for biosynthesis, transport, targeting, message transmission and feedback of such high molecular weight, structurally and topologically distinct, diverse and yet highly specific biological substances? What are the macromolecular interactions that govern non-covalent assembly of macromolecular machines?

The latest mass spectrometry has the inherent analytical potential, versatility and power to help answer these types of questions [42,43]. What ingredients will bring these potentialities into daily realities to address such problems?

Our mission is to be able to provide the appropriate interdisciplinary expertise to develop interconnectivity between the biological scientists and the mass spectrometrists centered around the particular major macro-molecular classes at issue: proteins, their covalently modified forms and nucleic acids. We must provide the interdisciplinary expertise required to realize the interconnectivity essential in exploiting the present instrumental embodiment of the state-of-the-art while catalyzing the innovation necessary to bolster further developments in instrumentation that will meet imminent requirements.

As a facility in the heart of a premier biological/medical university we have always been faced with the fact that a significant fraction of the salient, important biological problems are always just beyond the immediate reach of expertise (mass spectrometric and/or biological) or the most advanced presently available mass spectrometric instrumentation - no matter how sophisticated. But today's real challenge becomes tomorrow's feasibility and the next day's enhanced repertoire, which ultimately provides other bioscientists and clinicians with answers to similar analytical or structural problems.

The impact of this mass spectrometry resource on biomedical research and research training continues to focus on making both instrumental methodology and expertise in its utilization available to a wide variety of biochemical and biomedical scientists, clinicians and their co-workers. The resource disseminates to researchers who lack the interest, knowledge or capability to independently take advantage of mass spectrometry to solve problems encountered in their own fields. This has been made possible by the long-term support of resource scientific and technical expertise by the unique NCRR Biomedical Research Technology Research Resource Program.

Hence, this resource is able to provide extensive knowledge and experience drawn from our interdisciplinary scientific and technical faculty and staff in helping prospective research scientist or clinician users on a one-to-one basis with their research questions and problems.

There are of course projects in the chemistry, chemical biology and biochemistry research community where the research directors themselves have the knowledge base to bring their problem to the point of its being amenable to some type of mass spectrometric analysis, but the vast majority of scientists and research directors need the continued involvement of resource expertise, as well as use of the facilities, in the planning and execution of their experiments, in order that the results from mass spectrometric effort can be obtained and interpreted in a meaningful manner. This demand for our expertise is growing at an exciting rate as more biomedical investigators are introduced to the dramatic new capabilities of mass spectrometry.

Outlook

Mass spectrometry technologies are poised to play the key role in efforts to gain a comprehensive knowledge of human proteomics. There is accelerating awareness that mass spectral strategies can provide crucial information to areas such as the challenging problems associated with the elucidation of protein signaling cascades and networks, temporal modulation of protein machine assembly and disassembly and their posttranslational regulation. The utility and accessibility of the technology has begun to influence the design of experimental protocols in fields that previously mass spectrometry had been only minimally involved with. Since excellent technology breeds high quality experimentation, this trend will certainly continue at an accelerated pace and the expertise of the resource will be increasingly called upon for intellectual and technical assistance.

The primary challenge that remains ill-defined today is a lack of a clear strategy worldwide to handle the inherent complexity and full dynamic range of protein expression and its dynamic posttranslational modulation. But at this juncture, no suite of technologies other than those based on mass spectrometry is more critical or important to accelerating and sustaining the advance of gaining knowledge of the protein machinery underpinning biomedical research. This has led to explosive growth in new opportunities to elucidate the molecular size, heterogeneity and function of components of all biological systems[1,2]. It is particularly suited to characterization of mixed biopolymeric species involved in biological function and dysfunction, providing essential knowledge that is required to eventually understand human health and disease. Through discovery and sequencing of new proteins and defining their covalent regulation by new intact sequence mapping (ECD methodology), key physiologically relevant epigenetic products of the human genome are being unraveled rapidly with unprecedented precision[11] , yielding important information on physiological structure/function correlations.

References

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[16] Medzihradszky KF, Darula Z; Perlson E, Fainzilber M, Chalkley RJ, Ball H, Greenbaum D, Bogyo M, Tyson DR, Bradshaw RA, Burlingame AL. "O-Sulfonation of serine and threonine: mass spectrometric detection and characterization of a new posttranslational modification in diverse proteins throughout the eukaryotes." Mol Cell Proteomics. 2004 May; 3(5):429-440. [Pubmed]

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[40] Casati P, Zhang X, Burlingame AL, Walbot V. "Analysis of leaf proteome after UV irradiation in maize lines differing in sensitivity." Mol Cell Proteomics. 2005 Nov; 4(11):1673 - 1685. [Pubmed]

[41] Allen NPC, Patel SS, Huang L, Chalkley RJ, Burlingame AL, Lutzmann M, Hurt EC, Rexach M. "Deciphering networks of protein interactions at the nuclear pore complex." Mol Cell Proteomics. 2002 Dec; 1(12):930-946. [Pubmed]

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[44] Guerrero C, Tagwerker C, Kaiser P, Huang L. "An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network." Mol Cell Proteomics. 2006 Feb;5(2):366-78. [Pubmed]

[45] Hirsch J, Hansen KC, Sapru A, Frank JA, Chalkley RJ, Fang X, Trinidad JC, Baker PR, Burlingame AL, Matthay MA. "Differential impact of low and high tidal volumes on the rat alveolar epithelial type II cell proteome." Am J Respir Crit Care Med. (submitted)

The UCSF Mass Spectrometry Facility (A.L. Burlingame, Director) is supported by the Biomedical Technology Resource Centers Program of the National Center for Research Resources, NIH NCRR RR01614

last modified Tue Aug 22 10:59:37 2006