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New Perfinity Approach Allows for Single-tube Immuno-MS Sample Prep


NEW YORK(GenomeWeb News) – Proteomics sample prep firm Perfinity Biosciences has developed a single-tube immuno-mass spec process that could significantly streamline and speed up such workflows.

Called QuicPrep, the approach lets researchers perform both target immunoenrichment and trypsin digestion in a single tube, reducing the liquid handling steps involved and bringing the time required for such sample prep down from around two days to as little as three hours, Kevin Meyer, Perfinity's executive vice president and CSO, told GenomeWeb.

"Historically [for immuno-MS assays] you would have to do [antibody] capture off one set of beads, followed by elution, neutralization, reduction, alkylation, digestion, [and] solid phase extraction," he said. "[QuicPrep] eliminates the need to perform lots of those steps and enables you to perform the key steps all in a single tube."

Use of immuno-mass spec methods has been growing in recent years, particularly as researchers focused on clinical biomarker work aim to use mass spec for targeted quantitation of proteins. While some proteins can be measured using mass spec alone, quantitation of lower abundance proteins often requires immunoenrichment upfront of mass spec analysis to achieve the required sensitivity.

Additionally, enriching target proteins before mass spec analysis results in cleaner samples with less interferences, which allows the use of shorter LC gradients and, consequently, higher throughput – a key consideration for clinical assays.

As Meyer noted, however, immuno-MS workflows are fairly tedious. And while some players in immuno-MS development like Leigh Anderson, developer of the SISCAPA immuno-MS method, and Perfinity itself have worked to develop robotics systems for fully automating such workflows, many researchers do not have access to such tools at the moment.

Beyond that, Meyer said, immunoaffinity and mass spec are, traditionally, two relatively separate areas of expertise.

"You traditionally have a lot of people with immunoassay backgrounds having to work with groups that are coming from the LC-MS space or vice versa – people from LC-MS having to learn the ligand-binding techniques," he noted. "This just makes it easy for these techniques to be adopted by basically anybody."

"In our lab here we had a summer intern who hadn't yet even graduated from undergrad running immunoaffinity workflows with [a] high degree of sensitivity and reproducibility," he said.

Perfinity has traditionally targeted the clinical research and pharma markets with its products, but Meyer said he thought the QuicPrep would draw significant interest from researchers working on biomarker validation experiments.

Carmen Fernandez-Metzler, president of pharma analytic services firm PharmaCadence Analytical Services, told GenomeWeb that after a few months working with the product, she expected the company would use it in place of roughly 80 percent of their conventional immuno-MS workflows.

The method's speed was key to its appeal for PharmaCadenc, Fernandez-Metzler said, noting that it has brought the sample prep time required down from two days to three hours.

This is particularly useful from the perspective of method development, she noted.

"If I'm doing method development, it lets me quickly get an answer and adjust my method development, whereas the other way I had to wait three days," she said. "So this is a big help in that respect."

Eliminating many of the manual sample handling steps involved in immuno-MS workflows could also improve reproducibility, though Fernandez-Metzler said that she and her colleagues had not used the approach long enough to generate data addressing this question. She noted, however, that they had gotten good linearity on their calibration curves with the method.

Also key to reproducibility is good trypsin digestion, which, Meyer said, the method achieves through use of Perfinity's immobilized trypsin, which both significantly speeds up digestion and eliminates the enzyme's autolytic activity, which can lead to differential decay of peptides during digestion and a consequent lack of reproducibility.

Meyer said that Perfinity's decision to pursue the QuicPrep approach was in part informed by past work in the PCR field.

"We took a lot of inspiration from the PCR space where they figured out how to do these one-step PCR workflows where they heat-activated the enzyme," he said. "So we thought that if we can do the same thing on the proteomics side it will simplify the [immuno-MS] workflow in a similar manner."

Key to the process was engineering the digestion and immunoaffinity steps such that each would be active under different conditions, allowing the two steps to proceed in the same tube but at different times, and to achieve high recovery from each step.

Immuno-MS workflows either enrich at the protein level, pre-digestion, or at the peptide level, post-digestion. Traditionally, Perfinity's immuno-MS tools, including its Perfinity Workstation, which automates immuno-MS sample prep, have focused on protein-level enrichment. The initial version of QuicPrep likewise is intended for protein-level enrichment, but, Meyer said, the technology could also be used for peptide-level enrichment, as in SISCAPA assays.

"We anticipate having a variety of workflows capturing a variety of things over time, whether it's classes of proteins or peptides, the flexibility is there," he said.

Meyer said that thus far, the approach has worked as well as conventional multi-step immuno-MS workflows in all the cases in which they've tried it. However, he noted, given the multitude of research projects using immuno-MS, it's difficult to say if it would perform equivalently in all cases.

"Whenever you are making a tool for this space, it is always the diversity of the workflows that is challenging," he said. "So we don't anticipate there being any downsides, but there are so many labs working on so many different projects it's impossible to predict."