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bioRxiv · 10.64898/2026.06.14.731917

RubiSpec-MS: Determination of Rubisco CO 2 /O 2 Specificity Factor using Liquid Chromatography-Mass Spectrometry

Wu, A.·Zhao, Y.·McDonald, J.·Kumar, M.·Wilson, R.·Shoulders, M.
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Abstract

O_LIThe specificity factor(SC/O) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a key kinetic parameter quantifying the ratio of carboxylation to oxygenation reactions. Current methods to measure SC/O are expensive, labor-intensive, require specialized expertise, and/or rely on radiolabeled compounds, hindering Rubisco research. C_LIO_LIWe present RubiSpec-MS (Rubisco Specificity by LC-MS/MS), a method to determine the SC/O of purified Rubiscos using liquid chromatography-coupled mass spectrometry (LC-MS/MS) for separation and sensitive detection of non-radioactive reaction products. C_LIO_LIWe benchmark RubiSpec-MS using multiple model Rubisco enzymes. We find that the assay accurately reproduces corresponding literature values for each enzyme and performs similarly in a side-by-side comparison using an established radiometric method. C_LIO_LIRubiSpec-MS provides a faster, more accessible, and highly parallelizable method to measure Rubisco SC/O using equipment common in academic core facilities and industrial laboratories. C_LI

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Wu, A., Zhao, Y., McDonald, J., Kumar, M., Wilson, R., Shoulders, M.. 2026-06-15. RubiSpec-MS: Determination of Rubisco CO 2 /O 2 Specificity Factor using Liquid Chromatography-Mass Spectrometry. https://doi.org/10.64898/2026.06.14.731917

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