A step by step protocol for absolute quantification of protein using Arabidopsis thaliana transgenic lines carrying NanoLUC-tagged genes v1
Authors: Urquiza-Garcia Uriel , Molina Nacho , Halliday Karen , J. Millar Andrew 
Published: December 2024

Quantification of protein levels in absolute units will allow testing quantitative models of gene regulatory networks. Better quantitative models have application in fundamental plant biology but also in synthetic biology. Models in absolute units can, for example, help guide the design of promoter modifications using CRISPR genome editing technologies, such as base editing. Absolute protein quantification can also be applied to assessing the quality and biological meaning of affinity-constant measurements of transcription factors for DNA- or Protein-Protein interactions. Some absolute quantification methods using targeted proteomics have been described for mammalian cells and plant cells. However, gathering data using such methods is expensive, making it prohibitive for experimental set ups in which dynamic data is required (e.g.: inferring model parameters from time series in chronobiology applications). To overcome these shortcomings, we developed a Nano Luciferase based toolkit and methodology for performing absolute quantifications using recombinant NanoLUC and tagged lines. The steps presented in this chapter are extensively exemplified in the Doctoral thesis “A Mathematical Model in Absolute Units For the Arabidopsis Circadian Clock” (Urquiza Garcia 2018) and preprint (Urquiza Garcia et al. 2024; to be published in Molecular Systems Biology, 2025). Here we described steps to follow and tips for using NanoLUC for absolute quantification of a protein of interest. Introduction In systems and synthetic biology, quantitative data is important for model calibration and testing. In plant biology the largest source of semi-quantitative transcript data generated by RT-qPCR. Which has provided valuable data for quantitative model building and testing. This can be exemplified by many mathematical models of gene regulatory networks (circadian clock, flowering). In contrast, the generation of quantitative protein data has lagged significantly behind. However, having quantitative abundance data of proteins will aid in model refinement by providing the material for challenging model dynamics and parameters for plant systems (Andrew J. Millar, 2016; Andrew J. Millar et al., 2019). The gathering of reliable, dynamic and quantitative protein data remains very challenging. Quantitative Western blots can be used to extract information in absolute units. However, quantitative Western blots can only be performed if the reagents are available (native antibodies, epitope tagged stable lines) and, although possible, there are practical limits with regards to the dynamic resolution that can be achieved with this technique. This leaves vast room for improvement in terms of sensitivity, reproducibility and ease in experimental design. Mass-spectrometry (MS) has been applied for absolute quantification of low abundance protein-like transcription factors in mammalian systems (Simicevic et al., 2013). Further work providing better control on the generation of multiplexed absolute quantification has been reported for animal clock proteins using MS-based Quantification by isotope-labeled cell-free products (MS-QBiC) (Narumi et al., 2016), in which multiplexed production of internal standards for quantification is achieved through Cell-free synthesis using the PURE system (Shimizu et al., 2001). The advantage of this approach is that it does not require the generation of time consuming stable transgenic lines. However, this approach still requires access to expensive equipment and highly trained personnel. So extended time series over different experimental conditions that would be informative for model building and parameter inference can become prohibitively expensive. Therefore a methodology that provides quantitative data of enough quality for easy to access equipment and minimal training would be of high value. Reporter based approaches are important tools in the elucidation of molecular mechanisms. In chronobiology and photobiology the use of Firefly luciferase (FLUC) has served as an invaluable tool. Data generated with this automatable reporter assay boosted the elucidation of key molecular mechanisms governing the emergence of circadian and photobiological responses in plants (A. J. Millar et al., 1995; Ulm et al., 2004). The instability of firefly provides a suitable reporter for tracking changes in transcriptional activity in a relatively straightforward and inexpensive way. However, the instability of FLUC has its drawbacks when studying molecular events that present half-lives significantly larger than the FLUC half-life in vivo. Therefore the use of a more stable reporter was desirable, for which the half-life is mainly determined by the process it is reporting. In particular, we noticed that the NanoLUC technology offers several advantages for tracking protein dynamics in plants (Hall et al., 2012; Urquiza-García & Millar, 2019). Here we describe in detail the process for performing absolute protein quantification, which is publicly available in the Doctoral thesis “A Mathematical Model in Absolute Units For the Arabidopsis Circadian Clock” (Urquiza García, 2018).