Neuropeptide Localization in Lymnaea stagnalis: From the Central Nervous System to Subcellular Compartments

The human brain is often considered to be the most cognitively capable among mammalian brains and to be much larger than expected for a mammal of our body size. Although the number of neurons is generally assumed to be a determinant of computational power, and despite the widespread quotes that the human brain contains 100 billion neurons and ten times more glial cells, the absolute number of neurons and glial cells in the human brain remains unknown. Here we determine these numbers by using the isotropic fractionator and compare them with the expected values for a human-sized primate. We find that the adult male human brain contains on average 86.1 +/- 8.1 billion NeuN-positive cells ("neurons") and 84.6 +/- 9.8 billion NeuN-negative ("nonneuronal") cells. With only 19% of all neurons located in the cerebral cortex, greater cortical size (representing 82% of total brain mass) in humans compared with other primates does not reflect an increased relative number of cortical neurons. The ratios between glial cells and neurons in the human brain structures are similar to those found in other primates, and their numbers of cells match those expected for a primate of human proportions. These findings challenge the common view that humans stand out from other primates in their brain composition and indicate that, with regard to numbers of neuronal and nonneuronal cells, the human brain is an isometrically scaled-up primate brain. (c) 2009 Wiley-Liss, Inc.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used to generate ion images of samples in one or more mass-to-charge (m/z) values, providing the capability of mapping specific molecules to two-dimensional coordinates of the original sample. The high sensitivity of the technique (low-femtomole to attomole levels for proteins and peptides) allows the study of organized biochemical processes occurring in, for example, mammalian tissue sections. The mass spectrometer is used to determine the molecular weights of the molecular in the surface layers of the tissue. Molecules desorbed from the sample typically are singly protonated, giving an ion at (M + H)+, where M is the molecular mass. The procedure involves coating the tissue section, or a blotted imprint of the section, with a thin layer of energy-absorbing matrix and then analyzing the sample to produce an ordered array of mass spectra, each containing nominal m/z values typically covering a range of over 50,000 Da. Images can be displayed in individual m/z values as a selected ion image, which would localize individual compounds in the tissue, or as summed ion images. MALDI ion images of tissue sections can be obtained directly from tissue slices following preparative steps, and this is demonstrated for the mapping of insulin contained in an islet in a section of rat pancreas, hormone peptides in a small area of a section of rat pituitary, and a small protein bound to the membrane of human mucosa cells. Alternatively, imprints of the tissue can be analyzed by blotting the tissue sections on specially prepared targets containing an adsorbent material, e.g., C-18 coated resin beads. Peptides and small proteins bind to the C-18 and create a positive imprint of the tissue which can then be imaged by the mass spectrometer. This is demonstrated for the MALDI ion image analysis of regions of rat splenic pancreas and for an area of rat pituitary traversing the anterior, intermediate, and posterior regions where localized peptides were mapped. In a single spectrum from the anterior/intermediate lobe of a rat pituitary print, over 50 ions corresponding to the peptides present in this tissue were observed as well as precursors, isoforms, and metabolic fragments.

Peptide natural products exhibit broad biological properties and are commonly produced by orthogonal ribosomal and nonribosomal pathways in prokaryotes and eukaryotes. To harvest this large and diverse resource of bioactive molecules, we introduce Natural Product Peptidogenomics (NPP), a new mass spectrometry-guided genome mining method that connects the chemotypes of peptide natural products to their biosynthetic gene clusters by iteratively matching de novo MSn structures to genomics-based structures following current biosynthetic logic. In this study we demonstrate that NPP enabled the rapid characterization of >10 chemically diverse ribosomal and nonribosomal peptide natural products of novel composition from streptomycete bacteria as a proof of concept to begin automating the genome mining process. We show the identification of lantipeptides, lasso peptides, linardins, formylated peptides and lipopeptides, many of which from well-characterized model streptomycetes, highlighting the power of NPP in the discovery of new peptide natural products from even intensely studied organisms.