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Improving Technology, Increasing Researcher Interest Push Imaging Mass Spec Toward 'Tipping Point'

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Buoyed by increased researcher interest and new vendor offerings, imaging mass spectrometry is approaching a "tipping point," Vanderbilt researcher Richard Caprioli told ProteoMonitor this week.

One of the leaders in the field, Caprioli said that improvements in instrumentation and workflows are making the technique more accessible to non-experts, with particularly strong interest coming from pharmaceutical firms and the histology and pathology areas.

"It's really beginning to penetrate," he said, noting that many of the requests to speak on the technology are now coming from clinical and medical research organizations. "All of the major [mass spec] manufacturers are making imaging-capable mass spectrometers," which is helping to make it easier "for rank-and-file biologists who are not experts in mass spectrometry" to use the technique.

Indeed, while Bruker has typically dominated the imaging mass spec field – and continues to lead among large vendors – Waters, AB Sciex, and Thermo Fisher Scientific have all explored the technique on their instruments. At this year's American Society for Mass Spectrometry annual meeting in May, Bruker and Waters both introduced new software offerings for imaging mass spec – their ImageID and High Definition Imaging products, respectively (PM 5/25/2012).

During a presentation at the meeting, Bruker president and CEO Frank Laukien specifically highlighted increasing interest in imaging mass spec as "driving a resurgence of MALDI" for MALDI FT-MS, MALDI TOF, and MALDI TOF-TOF.

"Spatial proteomics – retaining biological information in tissues and cells – which is incredibly important, is driving MALDI," he said.

Also at ASMS, startup mass spec firm Simultof Systems announced the launch of three new MALDI mass specs – the SimulTOF 100 Linear system, the two-stage reflector SimulTOF 200 system, and the TOF-TOF SimulTOF 300 Tandem system, the company's first commercial products.

The Simultof instruments "represent a next generation of the [imaging mass spec] technology," Caprioli said, noting that the machines implement innovations developed in his lab through previous work with Simultof prototypes. He added that his lab has ordered one of the new machines and expects delivery by the end of the month.

They "have capabilities you want in an [imaging mass spec] instrument," he said. "Speed, certainly. Ease of use in terms of getting samples in and out. Resolution. I think other [mass spec] companies are also rolling ahead with next-generation [machines for imaging].

Caprioli identified speed as a key area where improvement is needed, noting that his lab had made advances that allowed it to reduce the time for imaging runs that had once taken hours down to a matter of minutes.

Also key, he noted, is improving data analysis tools to provide protein IDs in a more straightforward way. "I think probably the biggest need right now is to get the data mining to the point where … it doesn't just give mass spectra," he said. "And I think this is where a lot of the effort is going on with [vendors] right now. Some of the software coming out is very enabling."

"I want to see the average biologist who doesn't have a lot of mass spec experience be able to put a sample in, outline what they want to look at, push a button, and get an image out on the back end," Caprioli said. "And we're doing that today. So it's not pie in the sky."

He cited the pharma industry as a primary early adopter of the technology, noting that "almost all the big pharmas have imaging mass spec groups." He added that he expected the technique would also make significant inroads in the histology and pathology areas where, he said, it would enable advances in discovery work and highly multiplexed assays beyond the reach of conventional methods.

"One of the advantages of imaging mass spec is that you don't have to target a specific reagent," he said. "You can just hit the areas [of interest] with a laser and see what's there. So, for example, we’ve done some work looking at skin lesions, and what we've found is that the molecular makeup of those skin lesions [as identified by imaging mass spec] provides additional molecular information from what you get using existing histology approaches."

"The second [advantage] I believe will come once validation [of those markers] takes place," Caprioli added. "We're finding that for the diseases, it's not just one protein, but a signature of 15, 20, 30 proteins that make up a particular phenotype signature – and you can't [measure that quantity of proteins] using [conventional] immunohistochemistry. So imaging mass spectrometry offers a completely new paradigm for doing this."

His team is also exploring the technique's utility in a variety of other areas – for instance, in a recent collaboration with fellow Vanderbilt researcher Eric Skaar to combine magnetic resonance imaging with imaging mass spec in order to characterize the immune response in mice infected with Staphylococcus aureus.

In that study, which was detailed in a paper published in the June issue of Cell Host & Microbe, the researchers built a three-dimensional, spatially resolved profile of proteins linked to the immune response to S. aureus, using imaging mass spec to perform unbiased discovery of the molecules involved.

"We were interested in seeing if we could apply [imaging] mass spectrometry to infectious disease, and particularly infections that lead to the formation of pathological lesions that are macroscopically visible," Skaar told ProteoMonitor. "So we could see the lesion and then use imaging mass spectrometry to image changes in protein distribution in response to infection."

"The strength of [the technique] is the sensitivity and specificity of the mass spectrometer, which I think from a discovery standpoint allows us to identify new molecules that are expressed and abundant at sites of infection, and hopefully some of those molecules are important for the outcome of the disease," he said.

Incorporating MRI with imaging mass spec allowed the researchers to combine the resolution of MRI with the molecular information provided by mass spec, Caprioli said, adding that this was helpful in directing the mass spec analysis by precisely identifying features of interest – in this case the pathological lesions.

MRI provides "a high-resolution image that can be represented as a rendered volume … where you can see 3D structures," he said. "So you're honing in – you have an idea of where you need to look with the mass spectrometer to get information" relevant to the disease being studied.

The study identified several proteins linked to immune response. However, Skaar said, the effort was meant primarily as a proof of concept. The researchers didn't make the actual protein IDs using the imaging platform, but rather identified masses of interest and then used that information to make the protein IDs via LC-MS/MS analysis of homogenized and fractionated tissue sections.

Skaar said, though, that Caprioli's lab has had success doing in situ digestion and protein identification directly on the imaging mass spec platform, calling it "a real positive and immediate advance that is going to happen."

Caprioli likewise noted his team's advances in identifying proteins directly in the tissue being imaged. "We have developed and published [on] in situ digestion in microspots in the tissue," he said. "For small molecules you can identify things directly off the tissue. For larger molecules, you first have to do a digestion and then you can sequence and identify the proteins."

He added, however, that for very large molecules, "you're probably better off taking a microregion [of tissue] and extracting it" for analysis via LC-MS/MS. We're not 100 percent of the way there yet. But for a lot of it, we know how to perform the analyses."

Identification of infection-specific protein biomarkers would be one obvious application of the workflow described in the Cell Host & Microbe paper, Skaar said. His team, though, is now aiming instead to bring a third form of imaging – in vivo luminescence of gene expression – into the mix in order to simultaneously monitor the response of the host and the infecting bacteria.

"What we want to do is identify proteins with altered abundance at sites of infection and then see how bacteria change their gene expressi[on] in response to those changes in protein abundance," he said. "The hope is to develop a 3D image of the infection … all in the context of the anatomy of the animal."

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