Abstract
We have developed a method for online collection and quantitation of neuropeptides in rat brain microdialysates using on-column dimethylation with capillary liquid chromatography-tandem mass spectrometry (cLC-MS(2)). This method addresses a number of the challenges of quantifying neuropeptides with cLC-MS. It is also a completely automated and robust method for the preparation of stable isotope labeled-peptide internal standards to correct for matrix effects and thus ensure accurate quantitation. Originally developed for tissue-derived proteomics samples ( Raijmakers et al. Mol. Cell. Proteomics 2008 , 7 , 1755 - 1762 ), the efficacy of on-column dimethylation for native peptides in microdialysate has not been demonstrated until now. We have modified the process to make it more amenable to the time scale of microdialysis sampling and to reduce the accumulation of nonvolatile contaminants on the column and, thus, loss of sensitivity. By decreasing labeling time, we have a temporal resolution of 1 h from sample loading to elution and our peptide detection limits are in the low pM range for 5 μL injections of microdialysate. We have demonstrated the effectiveness of this method by quantifying basal and potassium stimulated concentrations of the neuropeptides leu-enkephalin and met-enkephalin in the rat hippocampus. To our knowledge, this is the first report of quantitation of these peptides in the hippocampus using MS.