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MALDI, Other Surface Sampling Methods Continue to Make Inroads in Clinical, Quantitative Proteomics

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While LC-MS/MS workflows using triple quadrupoles and, more recently, Q-TOF and Q-Exactive instruments, have typically dominated discussion of mass spec-based protein quantitation, MALDI mass spec has recently emerged as a potential alternative for such applications.

New sample prep techniques and forthcoming instrumentation should continue to drive the technology onward, said Mark Stolowitz, director of the Proteomic Core Facility at Stanford’s Canary Center for Cancer Early Detection and a MALDI mass spec expert.

At the same time, he added, researchers and vendors are also exploring alternative surface sampling techniques that could bring some of the throughput advantages of the MALDI technique to electrospray instrumentation.

In January, researchers from Bruker and SISCAPA Assay Technologies published a paper in the Journal of Proteome Research in which they used a Bruker autoflex speed MALDI-TOF to perform relative quantitation of peptides with coefficients of variation of between 1 and 2 percent, demonstrating that MALDI-TOF "is very suitable for peptide and protein quantification in the 1-5 [percent] CV range," Bruker director of proteomics Detlev Suckau noted at the time (PM 1/30/2012).

Stolowitz told ProteoMonitor this week that he and his team had since repeated that experiment, independently confirming the results on an AB Sciex 5800 MALDI TOF/TOF. "We ran our analysis on [the AB Sciex machine] and saw comparable results both in terms of sensitivity and precision," he said, noting that they also obtained coefficients of variation of under five percent.

Stolowitz added that his team was able to obtain a roughly 1.5-fold increase in signal-to-noise using an acoustic liquid handling technology from sample prep firm Labcyte. The technology, which the Stanford researchers have incorporated into a MALDI-based SISCAPA workflow, allows them to spot extremely small amounts of sample, reducing heterogeneity across MALDI matrix spots. This cuts down variability and improves the technology’s limits of quantitation.

Labcyte completed the first phase of a Small Business Innovation Research grant in August and is currently waiting for an invitation to apply for a phase II grant, Stolowitz said. Assuming the company does win a phase II grant, he hopes to collaborate with them on several MALDI-related projects, including improving automation of the firm's Portrait 630 Multi-spotter device, which he says could bring spot sizes down to the order of 400 microns from the 600 microns they currently use.

According to Stolowitz, such a reduction could give MALDI mass spec methods an extra log of sensitivity. "In MALDI it's all about bringing that spot size down," he said.

"I think MALDI has huge promise for really high-throughput SISCAPA [assays] and that type of thing," he said, noting that new instrumentation also added to the technology's potential.

In particular, Stolowitz cited the line of new MALDI instruments forthcoming from mass spec start-up SimulTOF Systems, which he said appear to offer significant gains in speed and resolution compared to current instruments.

SimulTOF's top-of-the-line machine, the 300 Tandem, features a 5 kHz laser, allowing it to acquire and process data at roughly five times the speed of current instruments, which typically come with 1 kHz lasers.

The company's mid-range instrument, the 200 Combo, could prove quite competitive, as well, Stolowitz added, noting that the instrument offers more speed than the Bruker ultraflextreme MALDI TOF/TOF along with comparable resolution and sensitivity, but is slated to cost roughly half as much.

SimulTOF plans to start shipping the instruments in the first quarter of 2012. However, Stolowitz noted, as with all new platforms it remains to be seen if the promise matches the reality.

"That's the problem with having the first of anything," he said. "I had the first [AB Sciex] 5800 in North America, and there are always issues to be had if you jump in on the first of anything."

Beyond the advances in MALDI technology, Stolowitz also noted growing interest in non-MALDI surface sampling techniques, such as the desorption electrospray ionization, or DESI, technique developed by Purdue University researcher Graham Cooks and offered commercially by Prosolia, or the liquid extraction surface analysis, or LESA, technique developed by researchers at Oak Ridge National Laboratory in collaboration with biotech firm Advion.

Like MALDI, these techniques allow researchers to take sample directly from a spotted surface, allowing them to skip time-consuming liquid chromatography steps and improve throughput. Unlike MALDI, though, these techniques can be interfaced with electrospray ionization-based systems like triple quad and QTOF machines, which typically still offer higher sensitivity than MALDI instruments.

Stolowitz noted that he recently discussed with Agilent the possibility of applying such a methodology to the company's 6550 QTOF instrument.

"We were talking to them about surface sampling, and there is some interest there in exploring that, particularly [given that] they have this 6550 QTOF," he said. "So I'm trying to talk them into collaborating on putting either a DESI or a [LESA] front end [on it] and looking at what its potential would be as an imaging [mass spec] instrument and also as a surface sampling instrument."

The LESA technology, sold by Advion as part of its Triversa Nanomate platform, has been making inroads with mass spec firms for several years, with vendors including Thermo Fisher Scientific and AB Sciex offering it on their machines.

The roots of the technique go back to 2001, Vilmos Kertesz, an Oak Ridge National Laboratory researcher and, with his colleague Gary Van Berkel, the co-developer of the method, told ProteoMonitor.

"The idea is that you do liquid-solid extraction of the surface," he said. "So the surface is solid, and then you interrogate it with the liquid, and the resulting liquid sample will be analyzed by mass spectrometry."

Kertesz and his colleagues were experimenting with various versions of this technique when they realized that the Triversa Nanomate device – a chip-based nanoelectrospray system – would be an ideal platform for integrating the method.

"In the default configuration the Nanomate device can pick up a pipette tip, aspirate the sample solution into the pipette tip from a vial, and then nanoelectrospray it," he said. "This is the idea behind this device."

"What we realized was that the device can be used to dispense liquid onto the surface you want to analyze and aspirate it back into the tip" for mass spec analysis, Kertesz added. "So it was really easy to put this surface sampling technology onto the market, because the device was already there. They just had to make minor modifications to the device."

Thus far the technique has been used primarily for applications like metabolite screening, but interest is also growing among proteomics researchers.

Indeed, the November edition of the Journal of the American Society for Mass Spectrometry featured a study led by University of Birmingham researcher Helen Cooper using LESA coupled to high-resolution mass spec to diagnose unknown hemoglobin variants in dry blood spots via top-down proteomics.

The researchers used the technique to analyze six dried blood spot samples that could not be diagnosed by current non-proteomic screening methods, suggesting, they wrote, "the power of top-down proteomics for hemoglobin variant analysis in newborn samples."

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