N. Leigh Anderson, Ph.D. Founder SISCAPA Assay Technologies

Proteins and peptides are becoming increasingly important as therapeutic agents and indicators of disease. Their ultimate success in this role rests on our ability to quantitate them both accurately and rapidly, especially from complex matrices. Though traditional immunoassay-based quantitation assays are widely used in this role, they are hampered by a lack of absolute structural specificity, as well as long and laborious assay development, multiplexing limitations, and difficulty with internal standardization.

Analysis via mass spectrometry addresses these limitations. MS-based methods allow for exquisitely specific target identification, with the potential to incorporate stable isotope–labeled standards for very precise quantitative performance. Mass spectrometry is also uniquely able to measure many things without interference among them, using methods that are rapidly configurable.

Enrichment Plus Automation

A patented technology known as SISCAPA— Stable Isotope Standards and Capture by Anti-Peptide Antibodies—combines the power of very high specificity anti-peptide immunoaffinity capture with the precision of MRM mass spectrometric analysis to boost the sensitivity and throughput of protein bioanalysis. The method, which characterizes proteins by purifying and examining their unique component peptides, enables target enrichment of 100,000X or greater directly from crude sample digests, with commensurate improvements in assay sensitivity. Importantly, the “add-only” format of this assay method is also superbly amenable to automation, opening the potential to process great numbers of samples in a highly reproducible manner.

Less Variability, Less Waiting

A stable automated liquid handling platform presents an ideal avenue for bringing the SISCAPA method into the automation space. The platform needs to be flexible enough to allow users to move beyond simply writing protocols that automate existing plate-based preparative steps; the technology should open new ways to intuitively connect individual applications into an integrated, powerful workflow.

LC cycle time presents an additional technical hurdle for throughput in these applications. By transitioning the SISCAPA method from nanoflow to normal flow methods, LC cycle times have been reduced from ~20 minutes per injection down to 3 minutes, with no loss in resolution. This allows processing of as many as 400 samples a day, matching the throughput capacity of high-quality MS analysis.

The Agilent Bravo Automated Liquid Handling Platform provides the precision of 96-tip dispensing and the benefit of an intuitive user interface. SISCAPA implementation on the Bravo has been further enhanced through development of custom software that simplifies the user experience.

The Next Frontier

The Agilent RapidFire MS system carries the potential to make today's throughput limitations a thing of the past. By enabling high-speed QQQ-MS analysis with no LC step at all, such systems allow MS injections as rapidly as one every seven seconds. Since samples that have undergone SISCAPA enrichment are extremely clean and free of background matrix, they can be analyzed using RapidFire with a five-plex analysis on a 96-well plate executed in about 10 minutes.

Further information about SISCAPA-MS analysis on the Agilent Bravo: http://www.chem.agilent.com/Library/applications/5991-4120EN.pdf   

Further information about the automated SISCAPA workflow: http://www.siscapa.com/the-siscapa-workflow.html

N. Leigh Anderson, Ph.D., is founder of SISCAPA Assay Technologies.

For more on MS, be sure to check out "Versatile Automated Sample Preparation for High-Throughput Peptide Analysis."

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