The Joy of Measurement

By Eric Sauter

John Yates's undergraduate work was in zoology—and then he took organic chemistry. After he was introduced to mass spectrometry, there was no turning back.

"The first time I saw a mass spectrometer, I was attracted to it," said Yates, who is now a professor in the Department of Cell Biology at The Scripps Research Institute, "although I have a hunch that part of this was because there was a small computer attached to it and computers were still new. But mass spectrometry was a great way to analyze molecules—you get good answers, very accurate and precise. I did my Ph.D. work with someone who was trying to sequence proteins using mass spectrometers—which wasn't easy back then—and I've been with it ever since."

Yates calls mass spectrometry "the bathroom scale of science" because scientists use it to measure the mass of molecules. The technique works by first converting the molecule into an ion, placing the ions in a mass analyzer where they are sorted according to their mass to charge ratios and then the amount of the ions at each respective mass to charge ratio is measured. Mass spectrometry can also be used to identify unknown molecules and to reveal their structural and chemical properties.

Even though the technology dates back to the turn of the last century, in recent years mass spectrometry has undergone something of a revolution, as dramatic advances in the technique—some of them originating from the Yates lab—have enabled scientists to measure larger and more fragile molecules.

Pioneering Technologies

Take proteins, for example.

"Proteins are not easy to sequence or analyze," Yates said, "because they tend to appear in tiny amounts and have a range of chemical properties. But in the late 1980s and early 1990s new technologies for the ionization of whole proteins became available. The result is that now we can ionize any large molecule we want."

Key among these advances was the Yates lab's development in 1993 of a technique to identify proteins using peptide mass fingerprinting, a method using sequence databases and mass spectrometry data to identify proteins rather then sequence them.

Many of the technologies pioneered by the Yates group come with a sense of humor, a nice touch considering the seriousness of what they've accomplished in a relatively short period of time.

For example, Yates was the lead inventor of SEQUEST (pop culture note: seaQuest was a sci-fi television series that ran between 1993 and 1996), a computer algorithm that automatically correlates tandem mass spectrometry data to amino acid sequences in protein and nucleotide databases.

The SEQUEST program was modified to run on a Beowulf Cluster called Shamu, a modification of the original algorithm that allows the software to be quickly run on large datasets using computing clusters. Another piece of related software, GutenTag, identifies peptides by sequence tagging. The GutenTag software was a kind of private joke between a pair of graduate students, one of them from Germany. "The name works," Yates said. "A good tag finds a sequence in the database."

For those truly complex protein experiments, Yates developed Multidimensional Protein Identification Technology or MudPIT, a tool that his laboratory continues to use extensively. MudPIT combines a multidimensional chromatographic separation system, a kind of chemical colander that is used to separate complex mixtures into their components, with a tandem mass spectrometer.  The tandem mass spectrometer creates fragmentation spectra (MS/MS) for peptides in the mixture, and hundreds of thousands of MS/MS spectra can be collected during an automated MudPIT analysis in a 24-hour period, which then uses the SEQUEST software to match spectra with peptide sequences in a database.

"MudPIT was named by a postdoctoral student of mine," Yates said. "It gave us a bit of trouble when we first put it in a paper. A reviewer called the name juvenile and wanted us to change it."

Previously Unknown Proteins

No one is complaining anymore. Because it was through the use of tools like MudPIT that Yates and another Scripps Research scientist, Professor Larry Gerace, were able to identify 62 previously unknown proteins in the inner nuclear membrane of the human cell, a number of which were associated with some rare degenerative muscle and nerve diseases, such as congenital muscular dystrophy, Limb-Girdle muscular dystrophy, and spinal muscular atrophy, as well as several forms of the neurodegenerative Charcot-Marie-Tooth disease.

Using a process called subtractive proteomics, Yates and Gerace analyzed the nuclear membrane components by subtracting off contaminant proteins found in an analysis of the endoplasmic reticulum, which accounted for more than 40 percent of the membrane proteins and came up with the final list of 62 new proteins, out of a total of 2,071.

That was in 2002. In 2006, Yates and a long-time Scripps Research collaborator, Professor William Balch, used MudPIT to identify key protein chaperones that play critical roles in the development of cystic fibrosis. The results of that study could lead to new therapies for other protein-based diseases as well, including Alzheimer's disease, Parkinson's disease and variant Creutzfeldt-Jakob disease, the human form of mad cow.

The Saliva Project

Currently, Yates is involved with the Human Saliva Proteome Project, a multi-site, multi-disciplinary project with scientists from the University of California, Los Angeles; the University of California, San Francisco; and the University of Rochester.

Yates has completed his basic mass spectrometry analysis to create a draft of the saliva proteome, which, he says, identifies the majority of the proteins in human saliva. The idea behind the project was the development of tests that could help monitor health status, disease onset and progression, and even treatment outcomes through noninvasive techniques, such as spitting in a cup.

"The basic question we set out to answer was what proteins are present in saliva," Yates said.  "Instead of looking at whole saliva (like when you spit on the grass, which can be contaminated with food and bacteria), we collected saliva directly from the parotid gland on the side of the mouth, the largest of saliva-producing gland and two others, the submandibular and sublingual glands under the tongue."

His research has identified approximately 1,500 proteins, although the number and types vary slightly from person to person and between men and women. Yates spoke about his findings in February at the annual meeting of the American Academy for the Advancement of Science.

"What's going to be most interesting is when we finally get a clearer understanding of what functions saliva serves in maintaining oral health," said Yates. "Clearly there's some kind of balance between the micro flora in the mouth and saliva and health, because if you eliminate the entire micro flora or saliva, severe problems develop."

Yates's proteomic work is just the first step in a series of projects under the umbrella of the National Institute of Dental and Craniofacial Research. The next step lies in a more diagnostic direction. Once it's known what proteins are in the saliva of healthy people, scientists can determine how these differ in various disease states. 

A Turnkey System

For Yates, the success of his work over the last decade comes from studies like these, but also from the fact that, thanks to his work over the last decade or so, mass spectrometry has become a turnkey operation, available to pretty much anyone who wants to use it. No special ticket required.

"With tandem mass spectrometry and computer software, we can now easily identify peptide sequences and the proteins they come from," he said. "That has made it possible for non-experts, those without formal training, to do highly sophisticated proteomic experiments. This has always been the goal.  We're big believers in the technology, its power and potential. So we take a lot of satisfaction from the fact that others are now seeing what we always knew was there."

Yates himself came to Scripps Research from the University of Washington in 2000. Over the course of his career, Yates has received the American Society for Mass Spectrometry Research Award, the Pehr Edman Award in Protein Chemistry, the American Society for Mass Spectrometry Biemann Medal, The Christian B. Anfinsen award from the Protein Society, The Herbert A. Sober Memorial Lectureship from American Society for Biochemistry and Molecular Biology, and the Human Proteome Organization's Distinguished Achievement Award in Proteomics and has published over 350 scientific articles.

"Scripps Research has been a great place to work," Yates said. "Its strength in cell and molecular biology has meshed well with proteomics and its wonderfully creative people come up with new questions and ideas for collaboration that challenge the technology. One of the priorities of our laboratory is to continue to advance the technology to address new and unusual biological questions and then use it to help raise the science around us."

Along with the saliva project, Yates has embarked on a new quantitative proteomics strategy to study the developmental changes that occur in rat brains from newborn to adult, which he believes could lead to new approaches to study diseases such as schizophrenia and depression.

But ultimately, his work comes back to measuring weight and to how broadly and efficiently that can be accomplished.

"About 12 years ago, a scientist studying a protein complex could identify one sequence of one component of that complex in a year," he said. "Now we can do that inside of 24 hours and for all of the components of the protein complex. We're trying to advance the complexity of the systems we're able to study, from proteins to organelles and whole cells, and then into complex structures like brain tissues. All I can say is that it's working so far… We're getting back good answers."

Send comments to: mikaono[at]scripps.edu

"The first time I saw a mass spectrometer, I was attracted
to it," says Professor John Yates, whose work has helped revolutionize the technology. Photo by BioMedical Graphics.