(200-19) Rapid absolute quantification of abundant proteins using LC-MS/MS: Rubisco as a case study

Body of Abstract: Quantification of enzymes and other proteins in plants is a critical aspect of plant molecular biology research. Accurate measurement of protein levels enables researchers to better understand the roles of specific proteins in various physiological processes, such as photosynthesis, signal transduction, defense mechanisms and facilitates the study of plant responses to abiotic and biotic stresses – all of which contributes to the development and application of resilient and sustainable agricultural practices. Here, we present a method for the rapid quantification of abundant proteins via ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a high-resolution, high-accuracy Orbitrap mass spectrometer. As a case study, we applied the method to rapidly quantify the rubisco protein in leaves across multiple phylogenetically diverse plant species (poplar, switchgrass, Arabidopsis, pennycress, sedge, and Sphagnum). The LC-MS/MS-based quantification method employs targeted parallel reaction monitoring (PRM) and quick HPLC separations to analyze rubisco-specific peptides across species. Though this method capitalizes on the overabundance of rubisco relative to other proteins present in leaf tissue, it can easily be expanded to include other highly abundant proteins or can be applied to protein enrichments via affinity purification. For each plant species, we analyzed 5 biological and 3 technical replicates for all physiological measurements. These included LC-MS/MS-based rubisco quantification, total chlorophyll, total protein, water percentage, and hyperspectral imaging. Sphagnum, Arabidopsis, and pennycress had the highest percentage of water in the leaf tissue (80-90%). Sphagnum and sedge had the lowest total protein (1.068 mg/g of tissue and 0.368 mg/g of tissue, respectively). Protein and peptide recovery varied across species. Preliminary data on poplar showed rubisco ranged from 2-6pmol/mg of tissue dependent on sample harvest time. When paired with other physiological metrics, this rapid, accurate, quantitative measurement can provide additional molecular information important towards a more comprehensive understanding of plant physiology.