SAN DIEGO – Triple quadrupole-based mass spec methods have in recent years dominated the clinical proteomics discussion. However, at the Association for Mass Spectrometry's fourth annual Applications to the Clinical Lab meeting, held here this week, there were signs that MALDI-TOF mass spec is making a comeback as a potential platform for peptide quantitation and other clinical applications.
A number of researchers presented on MALDI-MS methods, including several who discussed adapting workflows traditionally based on multiple-reaction monitoring mass spec to MALDI platforms. Additionally, Marvin Vestal, founder of mass spec firm Virgin Instruments, introduced three new MALDI-TOF instruments that he claimed would represent "a significant improvement in how we do time-of-flight mass spectrometry."
Vestal, the former president of mass spectrometry platform R&D at Applied Biosystems and developer of the first commercial MALDI-TOF mass spec system, told ProteoMonitor this week that Virgin plans to launch the trio of new instruments – a high-end MALDI-TOF-TOF, a high-resolution linear instrument, and a lower cost linear instrument – at this year's American Society for Mass Spectrometry annual meeting.–.
The high-end instruments will feature a five-kHz laser and voltage as high as 30 kV at the ion source and 30 kV post-acceleration, Vestal said, which will provide high speed, sensitivity, accuracy, and resolving power. The instruments' key advance, he noted, is their ability to provide simultaneous space and velocity focusing to reduce the effects of ions' initial position and velocity, which enhances their resolution and sensitivity.
Despite triple-quads' current status as the instrument of choice for clinical proteomics, Vestal said he believes MALDI-TOF could ultimately prove a faster, cheaper, simpler, and more sensitive technology.
Arizona State University Biodesign Institute researcher Randall Nelson agreed that Virgin's MALDI-TOF instruments could prove promising for clinical research. In a presentation this week he noted that in preliminary work using the new machines, Vestal's team had been able to analyze proteins isolated via Nelson's mass spectrometry immunoassay, or MSIA technology, at a rate of five minutes per 96-well plate.
"Our current machines take about 2 hours to do that," he said.
Christoph Borchers, director of the University of Victoria Genome British Columbia Proteomics Centre, presented research using MALDI-MS to measure plasma renin activity – a marker used in the screening and diagnosis of secondary hypertension. Using a peptide immunocapture method called iMALDI, Borchers' team developed a PRA assay that produced comparable results to the conventional radioimmunoassay used for PRA measurements, but with improved specificity, a faster analysis time, and without the use of radioisotopes.
Proteomics researcher and SISCAPA Assay Technologies founder Leigh Anderson is likewise exploring MALDI for protein quantitation. During his presentation on his stable isotope standards and capture by anti-peptide antibodies, or SISCAPA, technique, he said that he was interested in using MALDI for the method – which typically relies on MRM-MS – because it would eliminate time-consuming chromatography steps, thus upping throughput.
Anderson referenced recent research by Morteza Razavi, a PhD candidate in the lab of University of Victoria professor and SISCAPA Assay Technologies co-founder Terry Pearson, on quantifying protein biomarkers linked to prostate cancer using SISCAPA linked to MALDI-MS (PM 12/2/2011). In particular, Anderson noted, Razavi was able to address concerns that MALDI instruments suffer from high coefficients of variation when used for quantitation.
MALDI’s reputation for high variability stems from relative quantitation experiments people have done that produced CVs of 30 percent or 40 percent, Anderson told ProteoMonitor in an interview last month discussing the work. “But it turned out that people had not really systematically investigated how good quantitation would be if you have stable isotope-labeled internal standards [as in SISCAPA],” he said.
MALDI-TOF platforms have smaller dynamic ranges than triple quads, and typically don’t offer the high level of sensitivity and specificity that MRM assays provide, but, Anderson said, Razavi’s work suggests that in many cases SISCAPA’s antibody capture step effectively generates a pure peptide analyte, enabling more effective use of MALDI-MS.
One drawback that Razavi’s work has not been able to address is the low sensitivity of MALDI-TOF instruments compared to triple quads. His assays are able to measure proteins at concentrations of 100 nanograms per mL and higher, making them useful for quantifying medium- and high-abundance proteins but not low-abundance analytes.
Mark Stolowitz, director of the Proteomic Core Facility at Stanford’s Canary Center for Cancer Early Detection, however, has developed a MALDI-based SISCAPA workflow using acoustic liquid handling technology from sample prep firm Labcyte. Stolowitz told ProteoMonitor in a December interview that the workflow offers sensitivity on par with SRM assays on a triple quad.
Labcyte’s technology, which uses sound waves to create and manipulate minute liquid droplets, is able to spot extremely small amounts of sample, which enables creation of very small MALDI matrices. This reduces heterogeneity across MALDI matrix spots, reducing variability and improving the technology’s limits of quantitation, said Stolowitz, who was not a presenter at MSACL this week.
In November, the National Cancer Institute awarded Labcyte $196,000 for its work with Stolowitz on the MALDI SISCAPA platform. Thus far, he said, his team “can’t detect any significant difference in sensitivity” between MALDI-based SISCAPA assays run on an AB Sciex TOF/TOF 5800 and typical MRM-based assays.
"It's my belief that MALDI is starting a resurgence," Vestal said. "And you can sort of see it [at MSACL]. There's more MALDI here this year than there was last year. It went down [in popularity] and now I think it's past the trough and on its way back up, and I think that with the new technology we're [releasing at ASMS] it's going to accelerate a lot faster."
Vestal attributed MALDI's decline in part to the internal politics of mass spec vendors, citing the case of his former employer Applied Biosystems, which, he said, moved away from investing in MALDI-MS because they didn't want it to compete with their triple-quad products.
Bruker, which prior to its 2010 acquisition of Varian had no triple-quadrupole systems, is currently the dominant player in MALDI technology. Its MALDI Biotyper platform made a strong showing at MSACL, with a number of researchers presenting work on using it for bacterial identification.
In particular, Ken Van Horn of Focus Diagnostics, a division of Quest, presented data on use of the device for identifying bacteria at two of Quest's infectious disease facilities. The instrument, Van Horn said, correctly identified 374 of 450 total isolates – or 83 percent – to the genus and species level; and 40 isolates – or 9 percent – to the genus level only, demonstrating good proficiency in a large reference lab setting.
Several other attendees presented bacterial ID research done on different MALDI platforms, including BioMérieux director of product marketing Nedal Safwat, who showed microbial identification work the company performed using a system based on MALDI-TOF instrumentation from Shimadzu.
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