NEW YORK (GenomeWeb) – A team led by the lab of Swiss Federal Institute of Technology researcher Ruedi Aebersold has developed a Swath mass spec workflow incorporating new pressuring cycling technology from sample prep firm Pressure Biosciences.
In a paper published last week in the Journal of Proteome Research, the scientists found that adding PBI's MicroPestle tool to a conventional pressure cycling sample prep workflow yielded between 20 and 40 percent more peptides than pressure cycling alone while also upping assay throughput, Tiannan Guo, a post-doc in Aebersold's lab and author on the paper, told GenomeWeb.
The study indicates the workflow could prove useful for a variety of applications including enabling better mass spec analysis of small clinical tumor samples and formalin-fixed paraffin-embedded tissues, Guo said. It also potentially provides a boost for PBI, which has struggled to drive adoption of its pressure cycling technology (PCT).
That technology uses controlled cycles of pressure to break down samples, allowing for improved extraction of molecules such as proteins. In the case of proteomic workflows, which is PBI's primary focus, this can improve the depth and reproducibility of mass spec coverage.
The company's main product has been its line of Barocycler instruments, which create the necessary cycles of high and low pressure. In the recent JPR paper, which PBI scientists co-authored, the researchers employed a new, additional PBI product, its MicroPestle, a disposable, mechanical tissue homogenizer that fits into the sample containers used in the Barocyler instruments.
In a previous paper published last year in Nature Medicine, Aebersold and his colleagues found that integrating PCT with Swath mass spec significantly reduced sample prep time while upping the amount of peptides they could extract from a sample. In the recent JPR paper, they found that adding the micropestle upped peptide yield by another 20 to 40 percent. It also increased throughput by allowing them to process each sample in a single Barocycler sample tube as opposed to the previous workflow, which required two Barocycler slots for each sample. This raised throughput from six to eight samples processed in six to hours to 12 to 16 samples.
Addition of the MicroPestle was particularly useful for hard and fibrous tissues that are difficult to break down and larger pieces of tissue that require more thorough homogenization.
Guo said that the results point towards several uses for the technology, a number of which he and his colleagues are currently exploring.
For instance, he said, the ability to extract more peptides from a given sample means researchers can use smaller samples in their mass spec analyses. This is particularly important for clinical research where in the past certain types of clinical samples like tumor needle biopsies have sometimes been too small to be effectively analyzed by mass spec.
Even in the case of larger clinical samples, Guo said, the ability to analyze small amounts is still desirable given issues of tumor heterogeneity.
"In a real clinical setting, even when we have a relatively big piece of tissue, because of the heterogeneity every piece of the tissue has a different proteome," he said. "So now this technique allows us to really get high-resolution proteomic data for different locations in the tumor."
Use of PCT with the MicroPestle allows the researchers to start from as little as 0.3 mg of tissue, compared to 1 mg without pressure cycling, Guo said, adding that they are currently working on a study using the technology to look at tumor heterogeneity.
Another avenue they are exploring is use of the technology to improve peptide extraction from formalin-fixed paraffin-embedded tissue which is the most common form of clinical sample available but has not traditionally been well suited to proteomic analysis due to the difficulty of extracting a high percentage of the proteins from the fixed tissue. Guo said that he and his colleagues had found that with PCT and the MicroPestle they "can significantly increase the peptide yield" from such samples.
Guo said he sees the PCT-MicroPestle workflow as most useful in more clinically focused research where throughput and reproducibility are more important than achieving maximum proteome coverage.
"In my research we are interested in finding biomarkers from clinical tissues so here we prefer high sample throughput and relatively low proteomic coverage," he said, noting that their PCT-Swath workflow allow the team to run between 20 and 30 samples a day using a 30-minute LC gradient and provided coverage on the order of between 2,000 and 3,000 proteins.
"We could invest [time] in a longer gradient or some fractionation and then get more proteins," he said. "But our philosophy is that we feel that using the same resources we prefer to analyze more samples with limited proteomic depth than a lot of proteins from relatively few samples."
"Our goal is to find the differential proteomic signatures that can be used in the clinic to make decisions, and many low-abundance proteins, while they can be biologically important, in reality in the clinic they are not useful as biomarkers because are not easy to detect reproducibly," he added.
Implementation of the PCT and MicroPestle by a leading proteomics researcher like Aebersold is a boon for PBI's heretofore halting efforts to drive adoption of its technology. In addition to the Aebersold papers, PBI announced at the beginning of the year an exclusive comarketing agreement with mass spec vendor Sciex, under which it is promoting PCT with Sciex's Swath-based proteomics workflows as well as its TripleTOF, QTRAP, and triple quadrupole mass spec systems.
Richard Schumacher, President and CEO of South Easton, Massachusetts-based PBI, told GenomeWeb that these developments, as well as several other recent peer-reviewed publications using the technology, are the result of a shift in strategy the company made several years ago.
"What happened [initially] is that we went out thinking, well, we have the best thing since sliced bread, and it didn't take," he said. "It was very difficult walking into labs and people asking, 'Who else is using it? Show me the papers. Show me the data.' And we didn't have all of that."
"So we went back to the drawing board," Schumacher said. The company revamped its sales approach to focus on driving adoption among leading proteomics researchers like Aebersold.
"This was three or four years ago," he said. "We talked to them about it and ultimately got many of them to start using the system. It took a couple of years for us to get to them and then for them to use it, and now the papers are starting to come."
At the same time, Schumacher said, the company is working to turn the corner financially. Last month the company closed a $6.3 million PIPE financing round with funds coming from investors including PBI's five board members, which Schumacher said allowed it to pay off $3 million in variable-rate debt.
The continuing trend within mass spec-based proteomics toward the clinic would seem to be a beneficial one for PBI, as the company's technology enables the sort of improved throughput and reproducibility as well as lowered sample requirements that are necessary for clinically-focused proteomics research.
Based on the company's full year 2015 results, though, this trend and PCT's increased visibility have not yet meaningfully impacted sales. Revenues for 2015 were $1.8 million, up 31 percent from $1.4 million in 2014, but most of this rise was due to an increase in grant revenue, which rose to $387,700 from $24,594 in 2014. Products and services revenue, meanwhile, was up 4 percent, to $1.41 million from $1.35 million in 2014. Operating loss for the year was up 7 percent to 3.6 million from $3.3 million the year before.