Meeting the Challenges of Large Molecule Bioanalysis: Demonstration of an Automated & Standardized, Kitbased Workflow for LC-MS/MS Protein Quantification

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Paula Orens, Mary Lame, Mike Pollier, Wei Fu, Steven Calciano, Erin Chambers
Waters, Hamilton Company
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Other Symposium
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Protein Quantification

In any bioanalytical or clinical research assay, one of the greatest sources of variability arises from the sample preparation. This is especially true for protein quantification workflows, which employ the common bottom up approach using enzymatic digestion and analysis of the resulting peptides. This workflow can be complex and time consuming, containing many segments-each with multiple steps introducing variability. The multitude of possible options within this workflow, method development time, and the level of expertise needed are significant, typically requiring a skilled and experienced scientist. The difficulty in implementing these assays is further aggravated by the complexity of the troubleshooting required when analytical goals are not met. At the same time, an assay is expected to meet acceptable accuracy and precision guidelines and provide reliable, reproducible results to make critical research decisions. Thus, there is a strong need for simpler, more standardized workflows. These would ideally employ generic, kitted methods that provide a universal protocol, all necessary reagents and implementation on an automated liquid handler to streamline the process, maximize productivity, reduce errors and improve analytical method performance. Thus, facilitating implementation by less experienced scientists. This work aims to provide a practical, broadly applicable strategy to simplify and streamline the protein bioanalysis workflow. This solution incorporates an automated, kit-based approach with pre-measured reagents and a universal protocol, to accurately and reproducibly quantify proteins in plasma

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