Excess salt removal with matrix rinsing: Direct peptide profiling of neurons from marine invertebrates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOP-MS) is a viable technique for the examination of biological environments. Clearly, sample preparation plays a pivotal role in the ability to obtain mass spectra from samples as complex as biological cells. The physiological salt concentrations associated with neurons from marine specimens interfere with MALDI analysis. A unique and simple rinsing procedure allows cellular clusters, individual neurons and connective tissues to be directly assayed for peptides with minimal sample handling. Isolated cells and tissues, including egg-laying hormone-releasing cells, from the central nervous systems of the model marine molluscs Aplysia californica and Pleurobranchaea californica are used to demonstrate the salt removal method. In addition to facilitating sample ionization, the MALDI matrix 2,5-dihydroxybenzoic acid serves to (i) aid in microdissections by stabilizing cell membranes, (ii) deactivate endogenous proteolytic enzymes and (iii) reduce high salt concentrations in order to improve spectral quality. Representative MALDI mass spectra are presented which indicate the presence of several neuroactive peptides previously characterized by conventional biochemical methods. More than ten individual peptides can be detected in a single cell. In spite of the chemically complex sample, the mass spectra are surprisingly free of extraneous peaks. Furthermore, both mass resolution and mass accuracy are similar to those encountered with more common MALDI samples and protocols.