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

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