ASMS Annual Conference on Mass Spectrometry and Allied Topics edition:50 location:Orlando, Florida, U.S.A. date:June 2-6, 2002
in situ analysis of a tissue's/cell's peptidome
One of the characteristics of living cells is that they communicate with other cells. Typical extracellular messenger molecules employed by a highly specialized communicating tissue such as the animal nervous system, include (modified) amino acids (e.g., neurotransmitters) and (neuro)peptides. It is evident that the (bio)chemical message originating from a given cell will reflect some aspects of its physiological/pathological condition. Analysis of the cell's peptidome may, therefore, be a valuable addition to conventional 2D PAGE based proteomics studies.
Relevant neurosecretory tissues were carefully dissected form both invertebrate (American cockroach, Periplaneta americana) and vertebrate (Japanese quail, Coturnix coturnix japonica) species. Neurohaemal organs investigated included insect corpora cardiaca and avian median eminence and pituitary. After harvesting, (slices of) the neuronal tissues were deposited on the metal surface of a MALDI target plate to which 1-2 .mu.l of a highly concentrated solution of dihydroxybenzoic acid was added. After air drying the target was loaded into the oMALDI source of a QSTAR Pulsar i (Applied Biosystems), and MS as well as tandem MS spectra were acquired.
From all neuroendocrine glands under investigation nice series of in situ (i.e. direct tissue) MS spectra are easily obtained. Quadrupole-based selection and subsequent collision induced dissociation of several peaks in these MS spectra indicated that these represent various (neuro)peptide species. Since these naturally occurring peptides are often amino- and/or carboxyterminally modified, peptide identification from their oMALDI fragmentation spectra is usually not readily feasible through tryptic digest based search routines. The latter are typically used in conventional proteomic identification procedures from protein sequence databases. Suggestions will be made to optimize peptidomics search strategies, in order to make this novel direct tissue analytical method even more elegant.