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.