Mary Zhang with Dr. Lingjun Li and Mingming Ma

UW-Madison Department of Pharmacy

Our research objective was to detect and characterize a varied assortment of neuropeptides within the hemolymph of the cancer borealis crab using mass spectrometry. A Fourier transform (FT) mass spectrometer equipped with a matrix-assisted laser desorption/ionization (MALDI) ion source was used to detect and analyze the neuropeptides present in the hemolymph samples. We studied seven distinct methods used to detect neuropeptides that have been both already discovered and yet to be discovered.

One significant complication of employing the mass spectrometry analysis method is the high concentration of salt, which are naturally present in hemolymph samples, and degraded protein fragments found in the samples. In order to eliminate these fragments and optimize neuropeptide readings, we added ammonium citrate to our preparation techniques.

We employed seven different methods on the hemolymph samples. The most successful method mixed acidified methanol and a citrate buffer to the hemolymph sample. After centrifugation, the sample underwent 0.22 um filtration, 10K molecular weight cut-off, and the C-18 spin column for desaltation. We conducted tests on the effects of ammonium citrate on the analysis of neuropeptides because previous studies show that it is useful in eliminating excess salt. When analyzing the ammonium citrate samples, we discovered numerous peptide peaks, which displays the improved peptide coverage that ammonium citrate demonstrates.