In situ sequencing of peptides from biological tissues and single cells using MALDI-PSD/CID analysis

The ability to directly sequence peptides from biological cells using matri x-assisted laser desorption/ionization time-of-flight mass spectrometry (MA LDI-TOF MS) with postsource decay (PSD) and collision-induced dissociation (CID) fragment ion mass analysis is explored. Three different sample prepar ation methods are described for sequencing peptides in tissue samples and i n single neurons from the invertebrate model Aplysia californica. To charac terize peptides from the atrial gland, MALDI-PSD/CID is applied directly to a tissue blot covered with the matrix alpha-cyano-4-hydroxycinnamic acid ( CHCA). The resulting fragment ions combined with database searching confirm the structure of several novel peptides encoded by egg-laying hormone gene s. Moreover, MS profiling of a single unidentified neuron detects peptides with molecular weights of myomodulins C and E; this assignment is confirmed using MALDI-PSD with the matrix 2,5-dihydroxybenzoic acid (DHB). DHB does not always provide adequate fragmentation for PSD experiments; therefore, a unique dual-matrix sampling method, employing both DHB and CHCA, is develo ped to directly sequence a decapeptide from a single cerebral ganglion B ce ll. Mass accuracy of fragment ions from cellular samples is typical for the instrument employed and is not deleteriously affected by the morphology an d complexity of the samples.