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Celebrating Al Burlingame’s contributions to mass spectrometry
By David Jacobson / Thu Apr 26, 2012
In his first lab at UC Berkeley, complete with a dirt floor and leaky roof, he looked for signs of extraterrestrial life in meteorites. He compares an earlier mass spectrometry technique to “throwing a rock at a mud puddle and looking at the splash.” Nowadays, Burlingame’s laboratory is housed in 21st century digs at UCSF’s Mission Bay campus where he pries apart molecules using electrons, analyzing the most daunting sub-microscopic complexities of health and disease inside human cells.
Milestones in mass spectrometry
As director of the National Bio-Organic Biomedical Mass Spectrometry Resource Center, Burlingame has helped establish milestones in the application of mass spectrometry (MS) to biomedical research. The facility has been funded by the National Institutes of Health (NIH) and administered by the Department of Pharmaceutical Chemistry, UCSF School of Pharmacy, since 1978.
Burlingame helped jumpstart the use of MS to sequence proteins, determining the order of their amino acids that, in turn, determine the structure and function of those molecules underlying health and disease. This work led him to the analysis of the complex and rapidly occurring chemical changes to protein molecules that alter their function—called post-translational modifications, or PTMs—which can even control the expression of our genes.
Another milestone will be celebrated Friday, April 27, 2012, as Burlingame’s colleagues, collaborators, former graduate students, and postdoctoral fellows gather to celebrate his contributions to the MS field—and his 75th birthday this month—with a full-day symposium at the David J. Gladstone Institutes building on the UCSF Mission Bay campus.
The line-up of speakers is filled with scientific collaborators who brought Burlingame biological challenges over the decades, thus driving his advancement of the applications of mass spectrometry:
Stanley Prusiner, MD, Nobel Laureate and director of the UCSF Institute for Neurodegenerative Diseases.
Prusiner worked with Burlingame in the early 1990s to sequence parts of a then-unknown pathogen, an infectious misfolded protein called a prion that causes brain diseases in several mammals, including cows and humans.
Gerald Hart, PhD, director of biological chemistry at Johns Hopkins University School of Medicine.
In the early 1980s, Hart discovered a common and important protein modification by a sugar (dubbed O-GlcNAc) that plays roles in diseases ranging from diabetes to cancer. Two decades later, Burlingame and facility colleague Robert Chalkley, PhD, a faculty member in the UCSF School of Pharmacy, were the first to use a process called electron capture MS to determine precisely where the sugars attach to proteins. Burlingame and Chalkley are now developing ways to identify these PTMs comprehensively in a cell.
Ralf Schoepfer, PhD, professor of pharmacology and molecular neuroscience at University College London.
Schoepfer and Burlingame have collaborated on tracking the quantities and locations of PTMs on proteins at the receiving end of nerve signals, in a nerve cell region called the postsynaptic density.
Kevan Shokat, PhD, chair of the UCSF School of Medicine’s Department of Cellular and Molecular Pharmacology.
He and Burlingame have collaborated for years on analyzing the protein targets, called substrates, of hundreds of different protein kinases. These are enzymes that add phosphates to other proteins in a process called phosphorylation. This common process changes the function of proteins and can go awry in diseases such as cancers.
Applying MS to global biomedical research
The broad reach of Burlingame’s work reflects both his own expertise and the excellence of the global research resource he has led and helped build for nearly 40 years. The facility is now home to more than a dozen mass spectrometers and a staff of 20.
It also reflects the center’s NIH mandate to bring state-of-the-art MS technology to bear on biological challenges, with at least half of the facility's resources to be spent on projects originating outside of UCSF.
During NIH funding reviews, “we don’t get points for just turning the crank on stuff we already know how to do,” says Burlingame. “We get points for finding a problem which looks like it could be tractable and figuring out how to make it tractable.”
Put simply, MS vaporizes and ionizes (adds electrical charges) to molecules in order to identify their chemical compositions and quantities based on a spectrum of their mass-to-charge ratios. Before undergoing analysis, molecules are sorted from the complex chemical stew found in cells and tissues by techniques such as 2-D gel electrophoresis, which places them in a gel and applies an electrical field to separate them by size and charge.
Nowadays, MS is routinely used in biomedical research as a way of measuring the dynamic changes in the protein mix of healthy, diseased, and/or drug-treated cells and tissues, as well as analyzing their significant post-translational modifications (PTMs).
On any given day, the MS facility may help James Wells, PhD, chair of the Department of Pharmaceutical Chemistry, (whose lab is just down the hall) sort out the protein targets most crucial for the normal self-destruction of damaged cells. Or Burlingame may assist neurobiologists at the Weizmann Institute of Science in Israel by analyzing a sample they have sent around the world—a thumbnail-size flask containing signaling proteins that may promote healing in the wake of nerve injuries.
Meteorites, leaky roofs, and advancing techniques
Such applications would have been mere science fiction 50 years ago. Indeed, Burlingame’s career has neatly spanned a pivotal era and contributed to the huge gains in applying MS to biomedical issues.
With almost perfect serendipity, he was seeking a doctoral project at MIT in the early 1960s just when Klaus Biemann, PhD, a progenitor of organic MS, joined the faculty. “Biemann was really the only person in the U.S. who was into it. I went and asked him, ‘You got something I can do?’”
Upon joining the faculty of the UC Berkeley chemistry department in 1963, Burlingame set up his first mass spectrometer in the original Leuschner Observatory (built 1886) in an old machine shop with a dirt floor.
After laying down cement and taking delivery of a brand new mass spectrometer, native Rhode Islander Burlingame discovered amid the rains of his first California winter that “it might as well not have had a roof.”
The observatory location made sense in a way, as Burlingame’s first work was as a chemist in the Space Science Lab with Nobel Laureate and chemist Melvin Calvin, PhD, using MS to analyze carbon-rich meteorites. Burlingame was charged with searching for any traces of extraterrestrial biology that “presumably would be different than the stuff we see on Earth.”
The research drew support from NASA, laying the groundwork for the analysis of returned moon rocks later that decade.
While no molecular extraterrestrials were found, Burlingame was able to advance MS technique with work on computer automation of the identification of mass spectra—the graph lines generated by the relative abundances of different atomic or molecular masses.
Previously, he explains, there were “big rolls of papers and you were on your hands and knees on the floor with a ruler. It was painful.”
Working with Calvin, Burlingame also started using MS to analyze DNA adducts, the binding of chemical metabolites to nucleotides, which leads to mutations during cell division, and thus cancers. His work led to a Guggenheim fellowship at the Karolinska Institute in Sweden where biomedical applications of MS were more common and advanced than in the U.S.
Returning to Berkeley, “the idea was to take advantage of the mass spectrometry methods we’d developed [working with NASA] and apply them to biomedical research problems,” Burlingame recalls. “So that’s why I got this NIH grant that we still have.”
Burlingame began working in the 1970s with several UCSF researchers to develop diagnostic tests for inborn errors of metabolism. These are genetic defects that lead the body to make faulty enzymes that, in turn, lead to failures in metabolizing one substance into another. The idea was to analyze urine samples for metabolites of amino acids.
Limited by the size of the molecules that MS could analyze, Burlingame looked at mere pieces of the components that, in turn, combine in long sequential chains to make protein molecules.
Such research was another opportunity to hone methodology. “The issue was mixture complexity,” Burlingame recalls. “How do we find components in urine?” Today, newborn screening using MS can test for more than 100 such metabolic disorders.
UCSF School of Pharmacy builds its basic science unit
Burlingame was recruited to UCSF in 1978 by the late Jere Goyan, PhD, then dean of the School of Pharmacy, who had the prescience not only to incorporate clinical training and patient care into pharmacy education, but to build a basic science unit in the School’s Department of Pharmaceutical Chemistry. This included the use of computers to search for drugs and technologies like MS and nuclear magnetic resonance spectroscopy to analyze molecular targets.
“In pharmacy, Jere Goyan was the towering figure for his views and foresight,” Burlingame recalls. “He felt it was the School of Pharmacy’s mandate to be the basic science unit of this campus.”
While the department had a nascent MS program under then-department chair John Craig, PhD, Burlingame brought NIH funding, “a lot of hardware and computer stuff developed at Berkeley under NASA auspices, and a way of building a platform to move forward.”
Even before there was space at UCSF for Burlingame to relocate across San Francisco Bay into a basement on the Parnassus campus, he adapted and improved on a then-new technique called liquid sputtering. This was a way of getting peptides (short chains of amino acids that are the building blocks of proteins) vaporized and protonated (adding positive charges to the neutral peptides), and thus able to be analyzed by mass spectrometry.
“It was basically throwing a rock in a mud puddle and looking at the splash,” says Burlingame of that sputtering, which entailed firing a beam of atoms to vaporize peptides floating on the surface of the viscous solvent glycerol.
Burlingame and colleagues improved the technique by using a beam of cesium ions that could be focused, increasing the yield of peptides “splashed off the surface of the glycerol.”
Tackling proteomics before it had a name
Being able to analyze peptides meant that sequencing the larger proteins they compose was within range.
But first Burlingame and colleagues had to develop techniques to apply tandem MS to allow larger ionized molecules to be broken down for repeated cycles of analysis. In tandem MS, a protein is first cleaved into smaller pieces by an enzyme, then it is broken into smaller peptide units for analysis by colliding it with an inert gas and identifying the fragments.
“You ionize this molecule, which is at room temperature, so chemically it’s dead,” says Burlingame. “You can’t do chemistry without activating vibrations and breaking bonds and rearranging stuff. Collisional activation by ramming your ion into a neutral gas is one common way of doing it.”
“So you isolate the intact precursor peptide in MS1 and you do this collision…MS2 records the product ions, so that’s tandem mass spectrometry.”
In addition, computer programs had to be written to compare the collection of peptide masses with databases of known protein sequences, a technique known as peptide mass fingerprinting. The method was vastly sped up by the sequencing of genomes, which provided the chemical blueprints for tens of thousands of proteins.
Under Burlingame, UCSF became a leader in amino acid sequencing of peptides and proteins by MS during an era when the vast majority of researchers were still using a far slower chemical technique called Edman degradation, which could also only sequence a limited number of amino acids.
“We were going to meetings showing you could do sequencing of proteins by mass spectrometry in the ‘80s, and 99% of the people were doing Edman degradations,” recalls Burlingame. “That had been the only way of doing protein sequencing from the beginning of time.”
“We were right by orders of magnitude,” he says.
Such technical expertise was honed by tackling challenges faced by biomedical researchers. In the early 1990s, Burlingame was approached by UCSF cancer researcher Lois Epstein, MD, now professor emerita. She was studying the use of interferon, a protein meant to trigger an immune response, and similar molecules against melanoma, the most lethal form of skin cancer.
Epstein wanted to know how the mix of proteins in the cell changed in response to the treatments. Using the prior techniques, she had identified only three proteins in more than a decade of research. Within about a year, Burlingame’s MS facility had helped her identify about 50.
“That was kind of the beginning of proteomics,” says Burlingame. “Although it wasn’t called proteomics,” until Australian scientist, Marc Wilkins, PhD, coined the term for the study of all the different proteins produced by the cell’s genetic blueprint, as well as their modified forms.
With a characteristic combination of scientific precision and professional modesty, Burlingame is quick to add, “We shouldn’t purport that we were the only people doing this. We’re not, and never were. What we showed before anybody else did was that there was enough sensitivity in tandem double-focusing mass spectrometers to sequence spots on 2D gels.”
UCSF MS: from proteomics to PTMs to the histone code
Nowadays, the UCSF MS facility is a world leader in proteomics, pursuing a path that inevitably involves the advanced analysis of post-translational modifications.
That work has been greatly enabled by more recent technologies at the facility that allow both the vaporization of intact proteins and the use of electrons to surgically pry them apart, preserving post-translational modifications intact as opposed to collisional fragmentation techniques.
Applications remain driven by up to 75 local and global collaborations at any given time, as well as the sort of scientific serendipity that brought Burlingame to MS to begin with.
For instance, at the turn of this century, there happened to be a graduate student at the University of the Pacific, Kangling Zhang, studying histones. These proteins were once thought to be inert spools around which DNA is wound in our cells’ nuclei. Now they are believed to regulate gene expression via complex chemical modifications dubbed “the histone code.”
In need of advanced MS to pursue his thesis, Zhang eventually spent a few years at UCSF’s facility. Burlingame notes, “I didn’t worry very much about what he was doing until he started putting his thesis together. I started reading more carefully and arguing with him about how you do it and whether the data was supporting what he was claiming.”
“I realized that these [modified histones] would be a complex model for looking at the intact molecule, looking at the global occupancy of many post-translational modifications on this same small molecule.”
Sure enough, a decade after Zhang, Burlingame, et al. published papers identifying several histone modification sites via MS. Analysis of the multiple simultaneous modifications of histones has become an on-going focus for the facility in improving analyses of PTMs and their changes over time.
Such research exemplifies Burlingame’s insatiable curiosity. After all, birthday- and career-celebrating symposium aside, he shows no signs of slowing down.
This is a scientist who, in the 1990s, went to London to gain access to an antibody in order to isolate a key cell-surface receptor that interested him, and then set up a mass spectrometry facility for the Ludwig Institute for Cancer Research. He commuted to check up on things, then joined the biochemistry department of University College London—all on the side—until the latter required his retirement at 65.
Reinvigorated for the challenge of complexity
In fact, Burlingame remains driven by “the complexity of human biology,” figuring that “if you include the post-translationally modified proteins then there are probably about 300,000 distinct isoforms, or variations, on 23,000 basic [gene-encoded] scaffolds” expressed in various cell types at various times. And he adds, “The complexity of the PTMs has only been revealed in the last five years and it’s really daunting.” This includes interactions, dubbed crosstalk, between different PTMs on the same protein.
“Scientifically, things are as interesting as they ever have been,” he says. “Mission Bay has got lots of new people, lots of new ideas. It’s a reinvigoration of UCSF.”
“Technologically, mass spectrometry has exceeded all possible expectations,” says Burlingame. “Nobody could have predicted how good it is right now.”
“And much of biology is now concerned with the protein machinery of cells. Most drug targets are proteins of some sort or other. So from a practical point of view, as well as understanding human biology, if we don’t understand proteins and post-translational modifications, we won’t understand human biology. Mass spectrometry will continue to play a major role in that.”
About the School: The UCSF School of Pharmacy is a premier graduate-level academic organization dedicated to improving health through precise therapeutics. It succeeds through innovative research, by educating PharmD health professional and PhD science students, and by caring for the therapeutics needs of patients while exploring innovative new models of patient care. The School was founded in 1872 as the first pharmacy school in the American West. It is an integral part of UC San Francisco, a leading university dedicated to promoting health worldwide.