Single-cell proteomic analysis of glucosinolate-rich S-cells in Arabidopsis thaliana

Single-cell analysis is essential for understanding the processes of cell differentiation and metabolic specialisation in rare cell types. The amount of single proteins in single cells can be as low as one copy per cell and is for most proteins in the attomole range or below; usually considered as insufficient for proteomic analysis. The development of modern mass spectrometers possessing increased sensitivity and mass accuracy in combination with nano-LC-MS/MS now enables the analysis of single-cell contents. In Arabidopsis thaliana, we have successfully identified nine unique proteins in a single-cell sample and 56 proteins from a pool of 15 single-cell samples from glucosinolate-rich S-cells by nanoLC-MS/MS proteomic analysis, thus establishing the proof-of-concept for true single-cell proteomic analysis. Dehydrin (ERD14_ARATH), two myrosinases (BGL37_ARATH and BGL38_ARATH), annexin (ANXD1_ARATH), vegetative storage proteins (VSP1_ARATH and VSP2_ARATH) and four proteins belonging to the S-adenosyl-l-methionine cycle (METE_ARATH, SAHH1_ARATH, METK4_ARATH and METK1/3_ARATH) with associated adenosine kinase (ADK1_ARATH), were amongst the proteins identified in these single-S-cell samples. Comparison of the functional groups of proteins identified in S-cells with epidermal/cortical cells and whole tissue provided a unique insight into the metabolism of S-cells. We conclude that S-cells are metabolically active and contain the machinery for de novo biosynthesis of methionine, a precursor for the most abundant glucosinolate glucoraphanine in these cells. Moreover, since abundant TGG2 and TGG1 peptides were consistently found in single-S-cell samples, previously shown to have high amounts of glucosinolates, we suggest that both myrosinases and glucosinolates can be localised in the same cells, but in separate subcellular compartments. The complex membrane structure of S-cells was reflected by the presence of a number of proteins involved in membrane maintenance and cellular organisation.