Sensitive counting – Biological quantification at the Proteomics Core Facility
A decade or so ago the quantitative barrier began breaking down through the development of new, highly sensitive technologies and methods. When it comes to counting proteins the premier tool is a type of mass spectrometry called selected reaction monitoring, or SRM, and Gunnar Dittmar is the go-to person at the MDC.
"There are two basic approaches to mass spectrometry," Gunnar says. "'Shotgun' methods take a sort of census of all the proteins present in a cell or sample and allow you to watch how these populations change over time. In addition to this type of global analysis, the MDC's Core Facility for Proteomics offers a more targeted approach, focused on obtaining precise quantitative information about specific molecules, even when they appear in very low amounts."
When he says "low", he means it: Gunnar's personal record is the detection of just ten copies of a protein per cell. This capacity comes from the platform's ability to focus on specific molecular targets using its triple-quadrupole mass spectrometer.
Gunnar launched the facility back in 2007 after heading his own junior group at the MDC. At the time his focus was ubiquitination, ubiquitin-like proteins and the proteasome system: major mechanisms by which cells degrade proteins that are defective or no longer needed. The system is fundamental in enabling cells to alter their molecular content, allowing them to sense new signals or other changes in the environment and respond in new ways. It's also an area in which numbers are important: the rates at which proteins are degraded give insights into the way cells use various mechanisms to coordinate this crucial process. The facility continues to carry out a number of projects related to ubiquitination and the proteasome, in collaboration with Thomas Sommer and other scientists at the MDC.
In the beginning Gunnar had no assistance, but with rising demand for proteomics services he received help in the form of one technician and a postdoc. In the meantime, third-party funding has added five PhD students. "Each is involved in a collaborative project, centered on a biological question in which mass spectrometry plays a fundamental role," he says. The system extends beyond the boundaries of the institute: the facility has partnerships with members of the Signgene graduate school, a shared PhD student with the Technion in Haifa, Israel, and other collaborations.
Ideally, Gunnar says, the facility steps in at an early stage in the development of a project with a group and helps plan experiments so that scientists are able to acquire exactly the data they need. "The methods usually need to be highly customized based on the problem at hand," he says. "Sometimes we have to invent them." He cites a project with Helmut Kettenmann's lab to define the role of a protein called TRPV1 in brain cells and a fatal type of brain cancer called high-grade astrocytoma. With Thomas Blankenstein's group, the facility is working to identify peptides bound to MHC complexes – a key step in the efforts of Thomas and his colleagues to transform human T cells into therapeutic tools to fight cancer.
Claus Scheidereit says that his group's work with the facility has provided deep insights into signaling events centered around the NF-κB family of transcription factors and the cellular mechanisms involved in their regulation. These molecules are crucial nodes in a number of cellular pathways, and their processing and activation depend on the ubiquitin system.
"So far the mechanisms by which NF-κBs and their regulators are processed for activation has been poorly understood," Claus says. "By combining isotope labeling of amino acids with mass spectrometry, Gunnar helped us track concentrations of pathway components very precisely over time. The results were used to test various models developed by Jana Wolf's group, and that process has finally shed new light on important details of these pathways." One result of the project, he says, has been to deliver much more precise targets to manipulate pathway components upstream of NF-κB. That's important because the molecule plays so many roles: in healthy cells, various types of cancer, and also as a modulator of the effects of cancer therapies. The group is now working with Jens von Kries and the campus Screening Unit to intervene at precise points in these pathways – in hopes of developing targeted therapies that will affect cancer cells and leave their healthy neighbors intact.
In terms of technology, a particular focus of Gunnar's group has been automation. "Sample preparations for mass spectrometry are typically very labor-intensive," Gunnar says. "Sample preparation usually requires long rounds of pipetting. We've spent a great deal of effort developing robotics, to the point that we don't prepare anything by hand. We've done this in collaboration with external companies, and other labs have expressed a keen interest in the prototypes we made."
Such common projects have helped the facility acquire equipment, including a hundred-thousand Euro robot on permanent loan from a company. One reason is that while working on cutting-edge scientific projects, a high-quality facility is challenged to push existing technologies and develop them to take on new scientific questions. It's a common theme in the MDC core facilities.
Another important competency of the group is bioinformatics. "Our work involves not only capturing data, but interpreting it – rendering it in a form that a scientist can work with," Gunnar says. "That's another area where talking to people is crucial; there has to be a healthy exchange of ideas to translate a mass spectrometry experiment into something truly useful."
One goal for the future, he says, is to extend the sensitivity of the group's methods even further: ultimately, to the analysis of single cells. "This will heavily depend on advancing current methods of protein extraction," he says. "Over the past five years, we've already seen improvements by a factor of ten. There is still a ways to go – but this is the future of molecular biology."
