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U Victoria-Genome BC Team Develops 97-Protein MRM Panel for Dried Blood Spot Analysis

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NEW YORK(GenomeWeb) – Researchers at the University of Victoria-Genome British Columbia Proteomics Centre have developed a multiple-reaction monitoring mass spec assay for simultaneously quantifying 97 proteins in dried blood spots.

The protein panel, which was detailed in a paper published this month in Molecular & Cellular Proteomics, is one of the largest to date to use dried blood spots as a sample source.

To date, the bulk of dried blood spot work has focused on small molecule research, such as pharmacokinetic assays. However, interest is growing among proteomics and protein biomarker researchers, as well, driven by the convenience and potential savings the sample format offers.

In addition to the University of Victoria-Genome BC team, a number of proteomics researchers and companies are pursuing dried blood spot analysis including SISCAPA Assay Technologies' Leigh Anderson and protein diagnostics firm Healthtell.

Typically consisting of microliter volumes of blood spotted and dried on filter paper, dried blood spots can be stored and shipped without refrigeration. Additionally, because these samples require only a finger prick, patients can do them at home without having to go to a doctor's office to have blood taken.

This makes them an ideal format for applications like general population screening, said Christoph Borchers, a University of Victoria researcher and senior author on the MCP paper.

"I don't want to cut out the doctor, but when it comes to population-wide screening, if everyone can do it by themselves at home, that is definitely a great advantage," he said. He noted that studies comparing tests done on dried blood spots taken by patients to those done on blood samples taken in a doctor's office found that the two formats provided largely the same results.

In addition to patient convenience, dried blood spots also promise to be a much more economical sample format than conventional blood draws. The latter, Borchers said, require a cold chain infrastructure that includes rapid shipping on dry ice via services like FedEx.

"That costs a lot of money," he told GenomeWeb. "What we pay for FedEx shipping of samples on dry ice is unreal — hundreds of thousands of dollars."

Dried blood spots, on the other hand, can be stored at room temperature and shipped through standard mail, although Borchers noted that the durability of the sample varies depending on the target protein.

"We've tracked the stability of almost 100 proteins for over half a year," he said. And while the majority remained stable over that time period, some did not.

"Some fall apart within 10 days," Borchers said, adding that 90 to 95 percent of the roughly 100 proteins they studied remained stable at 10 days and a majority of those proteins remained stable at six months.

Another consideration when quantifying proteins from dried blood spots is how to normalize for sample-to-sample variations in volume and hematocrit content.

One approach is to measure the levels of certain plasma-specific and red blood cell-specific proteins to normalize sample-to-sample differences. Another method, developed by sample prep firm Novilytic Laboratories, uses collection cards that use a series of filters to standardize and extract proteins in dried blood spot samples. These cards, named Noviplex Cards, are offered by Shimadzu, which is also working to incorporate standards and antibody pull-downs directly on the cards.

In the case of their MCP work, Borchers and his colleagues found that a simple volumetric approach provided highly reproducible results.

"There are these small, inexpensive devices that you apply to a drop of blood and it sucks up exactly 15 microliters and you can then place that on the filter paper, and we have shown that we achieve [coefficients of variation] around 5 percent [with that approach]," he said.

Running the samples on an Agilent 6490 triple quadrupole, Borchers and his colleagues quantified a total of 169 peptides corresponding to 97 proteins, finding that their dried blood spot measurements largely corresponded with measurements made in whole blood samples.

One disadvantage of the dried blood spot approach, Borchers noted, was a lower sensitivity compared to analysis of conventional blood samples. While in conventional samples the researchers are able to quantify proteins across six orders of magnitude, they were able to cover only five orders of magnitude in the dried blood spot samples.

A possible route to improving the method's sensitivity is immunoenrichment of target proteins or peptides. Borchers said that his team is currently looking into applying their iMALDI approach, which uses antibodies to pull down targets of interest prior to mass spec analysis.

"Hopefully with immunoenrichment we can go deeper into the proteome," he said.

He noted that even before publication of the MCP paper, his group, which offers mass spec services and kits commercially throughUniversity of Victoria-GenomeBCspinout MRM Proteomics, had received inquiries from potential customers about dried blood spot work.

For instance, the Bill & Melinda Gates Foundation has expressed interest in measuring certain proteins in dried blood spots, he said. "There is already a lot of interest in dried blood spots. So it's not like we were just building something and seeing who would come."

MRM Proteomics has an ongoing marketing and distribution agreement for protein quantitation kits with Cambridge Isotope Laboratories and, Borchers said, he and his colleagues are interested in turning their dried blood spot panel into a kit that could be sold under that agreement.

He added that they could develop customized kits looking at specific subsets of the proteins in the 97-protein panel – apolipoproteins, for instance. The full panel includes a number of US Food and Drug Administration-approved protein biomarkers, he said.

Development of custom subsets would allow for shorter assays more amenable to large-scale clinical projects, Borchers said.

"Instead of running 30-minute [cycles], we can run like 5-minute [cycles] and go into high-throughput proteomics," he said. "Dried blood spots are a good vehicle for providing lots of samples inexpensively, and now we need to make the LC-MRM-MS analysis time less expensive so we can really view thousands of samples."

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