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PNNL Team Develops Antibody-Free SRM-MS Method for Low-Abundance Biomarker Quantification

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Researchers at Pacific Northwest National Laboratory and the National Cancer Institute have developed a new selected-reaction monitoring mass spectrometry-based technique capable of quantifying proteins at low picogram-per-mL levels without the use of immunoenrichment.

According to Wei-Jun Qian, a PNNL researcher and one of the project's leaders, the method could prove particularly useful as a targeted pre-verification step, helping fill the gap between protein biomarker discovery and clinical validation.

The technique, called PRISM, combines reversed-phase liquid chromatography for protein enrichment with online SRM-MS monitoring of internal standards to enable highly sensitive, multiplexed quantification of target analytes without the use of affinity agents.

As part of a study published this week in Proceedings of the National Academy of Sciences, the researchers used the method to quantify prostate-specific antigen levels in clinical serum samples, obtaining results equivalent to those observed using a standard clinical ELISA. They also quantified bovine carbonic anhydrase, bovine β-lactoglobulin, and E. coli β-galactosidase, all at levels of 100 picograms per mL or lower.

The PRISM workflow uses high-resolution reversed-phase capillary LC to separate a digested peptide sample into 96 fractions, which are deposited into a 96-well plate. During this LC run, the researchers also perform online SRM-MS monitoring of heavy-isotope-labeled synthetic peptide internal standards spiked into the sample.

Via this online monitoring, they are able to track the elution times of the target peptides, enabling them to determine which targets are in which of the 96 fractions. This determination allows them to select only the fractions containing peptides of interest for subsequent nano-LC SRM-MS analysis – upping overall throughput while still maintaining high sensitivity.

"The problem with targeted proteomics is that you have to fractionate the sample into too many fractions, so your overall throughput is quite low," Qian told ProteoMonitor. "We fractionate [samples] into 96-well plates, and traditionally you would have to analyze every well, which would be very slow."

"Our idea was to add an internal standard," he said. "We monitor that internal standard so we can know exactly where the target is so you can analyze only the fraction [containing the target]."

Additionally, these target fractions can be pooled for the ultimate SRM-MS analysis, further upping throughput. According to Qian, results from a follow-up study suggest that a 96-well plate can be combined into 12 fractions without a significant loss in sensitivity. Given that researchers can analyze roughly 50 proteins per fraction, this means they can likely quantify between 600 and 700 proteins per run, he said.

The technique's low picogram-per-mL sensitivity is comparable to that of immunoenrichment-based SRM-MS techniques like SISCAPA, which require antibodies to the target proteins.

Because of the extensive chromatography involved in the process, however, PRISM – even with its ability to pool multiple fractions – has considerably lower throughput than immunoenrichment methods. According to Qian, the technique would likely max out at around 10 samples per day.

At that level of throughput, the method "isn't in the ballpark" of what is needed for validation or clinical work, Leigh Anderson, CEO of SISCAPA Assay Technologies, told ProteoMonitor, noting that "this tradeoff of number of analytes versus cost/time is the classical discovery/verification pivot point."

Anderson, who was not involved in the PNAS work, said, however, that the technique was "certainly valuable from a discovery perspective and perhaps early verification [perspective]." Qian, similarly, said that he saw the technique's most obvious value as a workflow to bridge the gap between biomarker discovery and validation steps.

High-throughput, antibody-free targeted mass spec assays typically don't offer the picogram-per-mL sensitivity levels needed to detect the low-abundance proteins that many researchers think could prove to be the most effective biomarkers. Antibody-based mass spec assays like SISCAPA and conventional immunoassays can offer the needed sensitivity, but generating quality affinity reagents can be time consuming and expensive.

PRISM, Qian said, could help researchers winnow down the hundreds of protein markers they identify in discovery experiments to a more manageable ten or twenty targets for which they can then develop antibodies.

"We think this is a very good technique for pre-verification, because in cancer biomarker discovery you can have hundreds of potential biomarkers," he said. "We can screen them to say what candidate biomarkers should move on to the next stage. We start with a limited sample set but with sufficient sensitivity… and then you can reduce your targets down from 100 or 200 to 10 or 20."

Qian added that the technique could also prove useful in systems biology research where scientists are looking to elucidate large protein signaling networks.

"Often with a signaling pathway or network you have a lot of proteins that you want to do absolute quantification for, but you don't need to run thousands of samples," he said. "If you want to just do several hundred samples, [for instance,] I think this method is doable."

In addition to its high sensitivity, the technique also offers solid reproducibility, Qian said, with most assays having coefficients of variation of ten percent or lower.

For the PNAS study the researchers used a Thermo Fisher Scientific TSQ Vantage instrument. Qian said that the technique might demonstrate increased sensitivity on a newer triple quadrupole machine, but, he said, it would likely be a small bump, perhaps "two- to three-fold."

Looking forward, Qian said that the method's sensitivity would "probably be difficult at this moment to improve significantly" but that the researchers are working on refining the PRISM workflow, particularly with regard to improving its multiplexing capabilities.

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