Abstract: “Single cell analysis is fundamental in understanding the behavior and physiology of organisms. Aplysia californica (A. californica) is a well-studied organism in learning and memory due to its simple nervous system and large neuron sizes. Previous analyses have successfully detected and quantified 20 proteogenic amino acids and several neurotransmitters in sensory neurons from the pleural ganglia of A. californica. However, applying this to mammalian single cells has proven to be difficult since they have 10 to a thousand times smaller cell volumes compared to A. californica cells. Here, capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) was used due to its ability to analyze small sample volumes and identify metabolites in single rat cerebellar neurons. To apply this technique to mammalian neurons, a sample preparation protocol was optimized with a desalting step to increase analyte signal intensities by using microfluidic separations coupled to ESI-MS. This new approach enables a tenfold increase in sample analysis rate to 2 minutes per analysis, allowing for high-throughput CE-ESI-MS experiments compared to a laboratory operated system. Using the microfluidic system, we successfully detected lysine, histidine, arginine, tyramine, and dopamine, thus expanding upon previous work by developing a protocol for rat neuron analysis. Future plans include applying the developed procedure to single neurons in different regions of the rat brain.”
East Central IL ACS Undergraduate Research Conference
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It is not clear from your presentation what happens to the salts. You apply the desalting solution and then evaporate it off. I have trouble believing the salts evaporate with the solvent, so what happens to the salts?
Hi Linda, I apologize for the confusion. In my workflow, after the desalting solution is added, the samples are placed into the centrifuge. I then transferred 40 µL of the supernatant into a new set of tubes since the salts will have settled. The samples were placed into the centrifuge for 10 minutes at 13 krpm. The supernatant is what was placed into the Speedvac to evaporate off the solvents. I hope this clears things up!
*A total of 50 µL of desalting solution was added to the samples, so the salts were in the last 10 µL. Thank you!
So if the lowest ratio of IPA to ACN produced the best results you got, why wouldn’t you try a lower ratio such as 70% or 65% to see if you could get an even better result?
Hi Linda, thank you for the question! Since this is an extension of a previous project done in the group, I was able to reference their lab notebooks with solutions they tested. From data they collected, lower percentages of IPA:ACN yielded poorer results than what they utilized for their projects, which was 80% IPA:ACN. This is why I decided on 75% IPA:ACN for my work.