Meeting the Challenges of Large Molecule Bioanalysis: Demonstration of an Automated & Standardized, Kit-Based 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 sample preparation. This is especially true for protein quantification workflows which employ the common bottom up approach using enzymatic digestion, SPE peptide level clean-up, and analysis of the resulting peptides. This workflow is complex and time consuming, containing many segments, and introduces variability. Furthermore, given the workflow complexity, method development time and expertise needed are significant. Thus, there is a strong need for simpler, more standardized workflows.  These would ideally employ generic, kitted methods that provide a “recipe” and the reagents and implementation on an automated liquid handler to streamline the workflow. This work aims to provide a practical and fully automated kit-based sample preparation approach for the digestion and subsequent peptide purification for accurate protein quantification.


Commercially available protein quantification and SPE clean-up kits were employed to digest and purify the biomarker C-reactive protein (CRP) and multiple monoclonal antibody-based drugs in plasma. Sample digestion and SPE clean-up was performed using a commercially available liquid handler and supplied script. LC-MS/MS quantification was performed using a low dispersion UPLC system coupled to a high performance tandem quadrupole mass spectrometer (ESI+). A sub-2µm particle, wide-pore C18 column and formic acid in water and acetonitrile mobile phases were used for chromatographic elution.


This standardized and automated approach yielded excellent quantitative performance with linearity of calibrators > than 0.99, QC accuracies between 85-115% and mean % CVs< 15%, indicating a reproducible and accurate method

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