Every day we gain a better understanding of how cells work. In the past 20 years, new tools to examine gene expression and function have illuminated many different mechanisms that guide all aspects of cellular behavior. However, to fully understand normal cellular functions and how they malfunction in disease, we need more in-depth information about the many proteins our genes produce. Which proteins are being produced? How are they modified? What is each protein’s ultimate function and how do they interact on a system-wide level? New technologies in the proteomics facility at Sanford-Burnham are providing reams of data that could help answer these and many other questions.In a room full of advanced technology, the Thermo LTQ-Orbitrap Velos mass spectrometer system stands apart. The system has been part of the proteomics toolbox for about a year and has proven its value identifying proteins several times over. Dr. Laurence Brill, director of Advanced Proteomics in Sanford-Burnham’s Proteomics Facility, notes that the Velos system is 10 times more sensitive and three times faster than previous machines, but there’s a lot more to the core’s success than the excellent equipment. “We use very stringently applied analytical methods that take years to develop and refine,” says Dr. Brill. “We are thinking very carefully about the goals and biology of each assay and making them reproducible from run to run.”
The process begins with good samples, continues through the complex assay process and finishes with intense data crunching. The Excel spreadsheet for a sample can contain 250,000 lines and 50 columns. The core works with colleagues in bioinformatics to develop the computer scripts to address this intense level of detail.
All this data provides a wealth of information about which proteins are in a cell and how those proteins are being modified. For example, Dr. Dieter Wolf’s laboratory partnered with Dr. Brill and the proteomics team to identify 4,600 proteins in fission yeast – nearly every protein in the yeast cell. The Guy Salvesen laboratory is using proteomics tools to characterize protein cleavage events in a cell – thousands of which occur at any given time, as proteins are activated, inactivated or perform certain functions. This information might help them identify biomarkers for a variety of diseases.
In another study, changes in protein abundance and phosphorylation, a process that tags proteins with a small chemical group to alter their functions, were characterized during human embryonic stem cell differentiation. For this project, Dr. Brill and Dr. Junjie Hou worked with Dr. Evan Snyder’s group to identify 10,000 phosphorylated proteins in pluripotent and differentiating stem cells. By analyzing the differences between them, the team can better understand the mechanisms that cause a stem cell to become a neuron.
“We may be the only core facility in the world that can do that level of phospho-proteome analysis with that type of sensitivity,” says Dr. Brill.
In fact, the core has been recognized for the quality of their work. Not long ago, Dr. Reudi Aeberold, one of the pioneers in proteomics, visited the facility and validated much of what they are doing.
But there is no resting on laurels. In addition to developing new methods to improve results, researchers in the facility are setting their sights on even more difficult problems, for example glycosylation, the process by which proteins are modified with sugars.
“It would be helpful to understand all the modifications being made to proteins,” says Dr. Brill. “That would give us a better picture of cellular function in both health and disease.”