Sample prep firm Labcyte said this week that it has received a $1 million Phase II small business innovation research grant for work on developing the company's acoustic liquid handling system as part of MALDI mass spec workflows.
The award, which follows a $196,000 Phase I SBIR grant the company received in November 2011, will fund a collaboration between Labcyte and researchers at Stanford University and the Fred Hutchinson Cancer Research Center exploring the use of MALDI mass spec for verification and validation of breast cancer biomarkers.
Labcyte’s technology uses sound waves to create and manipulate minute liquid droplets, which, in the context of MALDI mass spec allows for spotting extremely small amounts of sample on MALDI matrices. Generally speaking, the smaller the sample spot, the more homogeneous it will be, which leads to improved assay precision and sensitivity.
Using Labcyte's Portrait 630 Multi-spotter for the first phase of the project, researchers led by Mark Stolowitz, director of the Proteomic Core Facility at Stanford’s Canary Center for Cancer Early Detection, generated MALDI mass spec assays to nine different ovarian cancer markers. According to Stolowitz, for eight of those markers, the assays performed with coefficients of variation of between 4 and 6 percent. The other had a CV of between 10 and 15 percent.
Additionally, the assays, which used peptide immunoenrichment upfront of the mass spec analysis, demonstrated sensitivity on par with similar SISCAPA multiple-reaction monitoring mass spec assays. According to Stolowitz, use of the Portrait 630 device offered a roughly 1.5-fold increase in signal-to-noise.
Bolstered by these and other similar findings, MALDI mass spec has in recent years emerged as a potential platform for clinical proteomics. Generally speaking, the field has viewed multiple-reaction monitoring mass spec on triple quadrupole instruments as the likeliest route for mass spec-based proteomics to move into clinical applications, but with MALDI demonstrating that it can overcome its traditional issues with precision and sensitivity, the technology's relative simplicity and high throughput make it an attractive alternative.
Indeed, Stolowitz told ProteoMonitor, one goal of the second phase of the project is to perform a head-to-head comparison of MALDI and triple quad-based MRM mass spec assays. For that his lab plans to collaborate with Fred Hutchinson researcher Amanda Paulovich, who has done extensive work on MRM-MS assay development.
"We're going to use the same antibodies and the same peptides, and the [MRM assays] will be run in [Paulovich's] lab and the MALDI will be run in mine, and then we hope to publish that direct comparison," he said.
The primary focus of the project's second phase will be automating the Labycte-assisted MALDI workflows developed in the first part of the effort, Stolowitz said. "We've just acquired an Agilent AssayMAP Bravo liquid handling robotic platform, and we're going to be able to use that to automate all of the upfront right through the stage where we use the Labcyte technology to spot [the sample]."
This work will take significant advantage of previous efforts using the Agilent AssayMAP Bravo to automate sample prep for MRM workflows.
"Agilent has already published an application note on using [the platform for MRM work], so that's our starting point,” Stolowitz said, noting that he expected that around 75 percent of the MRM automation work would be transferable to the MALDI effort.
In the second phase, the researchers are moving from ovarian cancer markers to breast cancer markers for which they will use antipeptide antibodies developed by Paulovich.
"Since [our assays] are dependent on anti-peptide antibodies [for target enrichment] and [such antibodies] remain in relatively limited supply, we've entered into a collaboration with [Paulovich] because her lab has developed a large library of breast cancer-related anti-peptide antibodies," Stolowitz said.
The project aims to analyze 16 different markers in quadruplicate, simulating the simultaneous analysis of 64 markers.
The researchers are also moving to a different mass spec platform, switching from the AB Sciex TOF/TOF 5800 they used in the first phase to a SimulTOF 200 MALDI-TOF from SimulTOF Systems.
The SimulTOF machine features a 5 kHz laser, making it the fastest MALDI mass spec currently on the market and five times faster than the AB Sciex instrument, Stolowitz said. The Stanford researchers can on their current platform analyze 96 patient samples per hour. With the SimulTOF, that time should drop to around 10 to 12 minutes per 96 samples, he said.
Recent research exploring MALDI for clinical proteomics work has looked into using Bruker's Microflex LT instrument, the system used in the company's MALDI Biotyper platform, the notion being that because the MALDI Biotyper has already received regulatory approval in a number of countries for clinical microbiology work it could prove an easier route into the clinic for conventional protein biomarker assays.
Stolowitz agreed that these regulatory approvals did make the Microflex an interesting machine and noted that for researchers looking to move quickly into the clinic "that is probably the best choice of instrument." However, he said, his work with Labcyte is focused more immediately on developing a platform for biomarker verification and early validation, and, as such, he preferred the performance advantages afforded by the SimulTOF machine.
"We feel there is a real unmet need in the gap between biomarker discovery and actual validation studies," he said. He noted that while validation analyzing thousands of patient samples is still typically done by ELISA, he believes "there is an opportunity for [a platform well suited to] looking at hundreds of patient samples at the verification stage of the platform."