BONCAT in Arabidopsis cell culture LC-MS

ABSTRACT: Determining which proteins are actively synthesised at a given point in time and being able to extract them for analysis is important to understanding plant responses to environmental or developmental stimuli, but there are few verified experimental tools to do this directly or quantitatively. In-vivo labelling with stable isotope labelled salts or amino acids allow the measurement of the synthesis rates of different proteins. However, they do not allow enrichment of newly synthesised proteins, which could increase sensitivity and would enable biochemical features of nascent protein sets to be studied and evaluated in isolation. Here we show that using the methionine (Met) analogue homopropargylglycine (HPG) enables BONCAT (Bio-Orthogonal Non-Canonical Amino acid Tagging) of proteins that are being synthesised in Arabidopsis plants or cell cultures, facilitating their enrichment for analysis using commercially-available click-chemistry kits. Importantly, the sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met-sites throughout the amino acid sequence of proteins and correlation with independent studies of protein labelling with 15N verified the data. We provide evidence that HPG-based BONCAT tags nascent plant proteins in a superior manner to azidohomoalinine (AHA)-based BONCAT in Arabidopsis and show that AHA’s induction of Met metabolism and greater inhibition of cell growth rate than HPG likely limits AHA incorporation at Met sites in Arabidopsis. HPG-based BONCAT provides a verifiable method for determining which plant proteins are being synthesised at a given time point and enriches new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis.

INSTRUMENT(S): 6550 iFunnel Q-TOF LC/MS

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Root, Cell Culture, Leaf, Shoot

SUBMITTER: Nathan Tivendale  

LAB HEAD: A. Harvey Millar

PROVIDER: PXD023170 | Pride | 2021-06-23

REPOSITORIES: Pride