Science Seminars

Nanomanipulation-Coupled to Nanospray Mass Spectrometry: A New Tool for Single Organelle Analysis, Liquid Phase Microextraction, and Combinatorial Applications Leading to Applications in Metabolid Disease and Cancer Research

The advent of small volume analysis with mass spectrometry has opened the door to investigation of micron and submicron analytes, and bringing the sampling directly to the interest. The nanomanipulator is a multistage bioworkstation consisting of a four-positioner system that has been directly and indirectly coupled with nanospray mass spectrometry. This coupling allows for new significant applications developments in the areas of single organelle analysis and combinatorial library sequencing. The option to employ multiple end-effectors simultaneously is ideal for cellular probing. Single organelle analysis has been demonstrated with cotton seed samples. Single lipid bodies were stained with BODIPY and extracted from within the cells into a suitable spray solvent. Analysis of an extracted lipid body was subsequently completed by nanospray mass spectrometry, and the results showed the distribution of triglycerides present. Recent work has also proven the nanomanipulator to be a rapid and efficient tool for one-bead-one-compound, also known as split-mix, combinatorial peptide library analysis. In this experiment, a peptide sequence coated on a 130 um resin bead was extracted using a suitable spray solvent. The resulting extract was then collected and analyzed, and tandem mass spectral data allowed the elucidation of the peptide sequences. The translational resolution of the nanomanipulator, which is beyond the optical limit in both coarse and fine modes, allows for fine precision and control of the tools required for probing in these types of experiments. We also highlight the suitability of LPME –MS (Liquid Phase Microextraction-Mass Spectrometry) by extracting and analyzing molecules from three different types of samples—1) visualization of an extracted fluorescent dye, 2) non-polar polymer additives in vertebrate serum, and 3) controlled acid-catalyzed hydrolysis of triacylglycerols within industrial refined cottonseed oil demonstrating the ability to perform chemical reactions within nanospray capillaries. Coupling of the nanomanipulator to nanospray mass spectrometry has been extremely beneficial due to the small (300 nL) volume and sample mass (300 attograms) required.

All of these developments are leading to a tool that would allow the researcher to perform cradle-to-grave analysis without leaving the microscope. This greatly reduces prep and gives the researcher better control over the experiment. We have started to use this instrument to tackle puzzling questions about metabolic disease and cancer cell heterogeneity.