Multiplex Strategies for Characterization of the Crustacean Neuropeptidome
I am currently working on studying hypoxia in crustaceans using mass spectrometry. Specifically, I am interested in studying the neuropeptidomic response to hypoxia. Neuropeptides are chemically diverse and have low abundance making their study a challenge. Mass spectrometry is an excellent detection method as it is highly sensitive and selective. My research employs custom isobaric tags (DiLeu) that we have developed in the Li Lab to label neuropeptide samples. DiLeu adds a dimethylated leucine to primary amines of peptides. At the MS1 level, DiLeu adds the same nominal mass, but upon fragmentation at the MS2 level, unique reporter ions are formed depending on the isotopes selected for the labels. This allows us to pool samples from control and experimental conditions and analyze them at the same time to perform relative quantification. This reduces the amount of samples required and the amount of instrument time required. Preliminary results have shown that using DiLeu will require extensive optimization. Because it is an MS2 reporter, the peptides need to be selected for fragmentation, but due to their low abundances, many of the peptides in the sample are not being fragmented and therefore not identified/quantified. My current work is to optimize the analysis and separation means for DiLeu labeled neuropeptides. This includes adjusting data-dependent acquisition settings, LC separation settings, and I am also looking to try alternative separation methods (e.g. electrophoresis).