In proteomics labs, MALDI mass spectrometry has become a household tool for analyzing proteins or peptides in more or less complex samples. But now interest is growing in its use as an imaging tool. Using the laser to directly scan a frozen tissue section, pixel by pixel, researchers can create hundreds of maps from the mass spectra showing the two-dimensional distribution of different proteins. Though tissue imaging MALDI-MS still has some significant limitations, it might soon find its place among other imaging techniques used in drug development and diagnosis.
Imaging mass spectrometry in itself is not new: scientists have long followed small molecules across tissue sections by secondary ion mass spectrometry (SIMS), although it cannot catch peptides or proteins. Others have used MALDI-MS to look directly for peptides in tissue pieces. But it was Richard Caprioli, a professor of biochemistry and director of mass spectrometry at Vanderbilt University, who married MALDI-MS and the scanning approach. Since he and his colleagues published a paper last year in Nature Medicine describing the technique, interest among researchers, drug companies, and instrument makers has steadily risen.
In February, Applied Biosystems began collaborating with Caprioli’s group on several aspects of the instrumentation, including high frequency lasers, but has not decided yet whether it is going to develop a special instrument dedicated to the technique. “It shows potential, but there is still a lot of work to be done,” said Marvin Vestal, a TOF mass spectrometry researcher at Applied Biosystems.
The University of Texas Health Sciences Center in Houston, Caprioli’s former employer, holds the patent on the technology, and has non-exclusively licensed it to at least one party, and others have expressed interest, said Steven Ritter of the university’s office of technology management. Ritter declined to confirm that ABI had licensed the technology.
In the meantime, several drug companies have taken notice. Schering-Plough has been exploring the technique in a collaboration with Caprioli to follow the distribution of drugs in tissue samples, while Novartis added it to its analytical and imaging sciences unit two years ago. Novartis is using the method both to locate specific known peptides, for example amyloid beta peptides in the brain, and to look for changes in protein profiles to find new drug targets or to study drug effects. “The beauty of it is that you really can have all the proteins in [a certain] mass range at once,” said Markus Stoeckli, a mass spectrometry group leader at Novartis and former postdoc in Caprioli’s lab.
Compared to other imaging techniques, like PET, MRI, or immunohistochemistry, one of the main advantages of MALDI-MS imaging is that it requires no labeling or reporter reagent. However, compared to immunohistochemistry, which can zoom in to the subcellular level, its resolution — and some say its sensitivity — is worse. “It’s not a tool which can do everything, it’s not in vivo, but it has its function in the whole imaging field. Combined with the other techniques, it’s a very useful tool,” said Stoeckli. However, he admitted that so far Novartis had not used it successfully to do differential proteomics, in which no prior knowledge of the proteins is required.
What might be the biggest obstacle to using the technique as a discovery tool is the need for additional experiments to identify interesting peaks found in a spot by tissue imaging. At the moment, this requires going back to the tissue, making an extract, separating the proteins by HPLC, and identifying them by peptide mapping or sequencing.
Another significant limitation of MALDI-MS tissue imaging is that it only records a fraction of the proteins present in a sample, currently between 100 and 800 per spot. Some proteins do not ionize well, others are expressed at levels too low for mass spectrometry, and generally, the sensitivity decreases for high molecular weight proteins. “When you look at a spectrum, you have a lot of signals up to about 30,000 Daltons and then just a few up in the higher mass range. That’s probably not what you would expect from the biological distribution of proteins,” said Stoeckli. Another type of protein that has been inaccessible so far is membrane-associated proteins, although Pierre Chaurand, an assistant professor at Vanderbilt''s Mass Spectrometry Research Center, said he and his colleagues have been working on ways to solubilize them using surfactants.
Spatial resolution is another area that might see improvement soon. The laser beam — which determines the size of each “pixel” recorded — can be focused to about 50 micrometers “without too much effort” in commercial MALDI instruments, said Chaurand. Caprioli’s group has already developed an improved laser with a 7 micrometer beam, and others are down to less than one micron, he said.
For practical purposes, what also deserves improvement is the imaging speed, largely determined by the laser repetition rate. It currently reaches 20 Hertz in many commercial instruments, but Novartis has already installed a 300 Hertz laser, bringing the imaging time for 20,000 spots in a small tissue section down to about 20 minutes, Stoeckli said.
With all these technical improvements still in the works, the jury is out on how widely applicable the technique will become. “It’s in some sense too early to talk about what its impact will be,” said Jonathon Sweedler, a professor of chemistry at the University of Illinois who has used MALDI-MS to analyze peptides in subcellular fractions. “If that work continues, this will be a great technique. It has all the right possibilities to make this high impact.”