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

Background :
Developing LC-MS based bioanalytical methods for drug discovery and clinical research can be quite challenging. This is especially true for protein quantification, where complex and time consuming sample preparation is often required. The common bottom-up protein quantification workflow requires many processing steps, and introduces analytical variability. Additionally, developing and optimizing these procedures can be tedious and time-consuming, often requiring a highly skilled scientist. 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 kits were employed to digest and quantify the biomarker C-reactive protein (CRP) and multiple monoclonal antibody-based drugs in plasma. Sample digestion and subsequent 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 triple quadrupole mass spectrometer (ESI+). A sub-2µm particle, wide-pore C18 column and simple formic acid in water and acetonitrile mobile phases were used for chromatographic elution.


Using the commercially available digestion kits, automated versus manual sample digestion performance and accurate protein quantification was assessed. Several unique tryptic peptides resulting from the digestion of C-Reactive Protein (CRP) and inflximab were evaluated and analyzed by LC-MS/MS.  Raw area counts for multiple tryptic peptides from the aforementioned proteins were used to assess comparability and the coefficient of variation (CV), as high CV values are indicative of poor reproducibility and precision. Mean peptide area counts (N=4) from digestion performed on an automated liquid handler were within 25% of those manually digested and intra-assay reproducibility for all tryptic peptides was excellent with CVs ≤15% for samples prepared with the liquid handler and manually. Using this standardized and automated kit-based approach for digestion and SPE clean-up yielded excellent quantification performance for CRP, infliximab and cetuximab with linearity of calibrators > than 0.99, QC accuracies between 85-115% and mean % CVs< 15%, indicating a reproducible and accurate method.

This work demonstrates the high reproducibility, accuracy and precision achievable for protein quantification using a generic kit-based approach and fully automated sample preparation workflow for digestion and peptide level clean-up.  These data suggest that a high degree of standardization can be achieved across analysts, and sites implementing this approach for the development of LC-MS protein quantification methods in support of drug discovery and clinical research.

For Research Use Only. Not for Use in Diagnostic Procedures.

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