Angew. Chem. Int. Ed. 51, 10386–10389 (2012)

Credit: © 2012 WILEY

Determining the distribution and concentration gradient for a metabolite inside live cells can lead to a better understanding of its turnover, transport and compartmentalization properties. Electrospray mass spectrometry is widely used for analysing metabolites; however, most techniques for preparing a sample from a specific organelle require the organelle to be separated from the rest of the cell prior to chemical analysis, using an approach such as non-aqueous extraction. Now, Akos Vertes and co-workers at The George Washington University in the US, have combined microdissection with laser-ablation-electrospray-ionization mass spectrometry to detect metabolites localized in a subcellular compartment without the need for complicated sample preparation.

To demonstrate the potential of their method, the team analysed the metabolites inside plant cells from an Allium cepa (onion) bulb. In the first step, a microdissection needle cuts a hole in the cell membrane while leaving the organelles intact. Only a single cell is cut open during dissection and the neighbouring cells remain undamaged. In the next step, an optical fibre with a 15-μm tip is used to direct a laser IR pulse at a specific region. The pulse creates a plume of ejected material that is ionized and characterized using electrospray mass spectrometry to identify the metabolites present. The sharpened tip of the optical microfibre is a similar size to the cell nucleus, which enables the team to selectively ablate it — although some cross contamination with the surrounding cytoplasm is inevitable. Aiming the optical fibre solely at the cytoplasm allowed for the selective analysis of the metabolites present outside the nucleus.

Using a combination of multi-variance statistics and a comparison of the relative intensities of the peaks, it was possible to pick out metabolites that were present in different concentrations in the cytoplasm and the nucleus. The mass spectrometry data also enabled a putative identification of the metabolites. Hexose and some secondary metabolites were found to accumulate primarily in the cytoplasm, while some amino acids are more common in the nucleus.