A subcellular prefractionation protocol for minute amounts of mammalian cell cultures and tissue.

Subcellular localization represents an essential, albeit often neglected, aspect of proteome analysis. Generally, the subcellular location of proteins determines the function of cells and tissues. Here we present a robust and versatile prefractionation protocol for mammalian cells and tissues which is appropriate for minute sample amounts. The protocol yields three fractions: a nuclear, a cytoplasmic, and a combined membrane and organelle fraction. The subcellular specificity and the composition of the fractions were demonstrated by immunoblot analysis of five marker proteins and analysis of 43 proteins by two-dimensional gel electrophoresis and mass spectrometry. To cover all protein species, both conventional two-dimensional and benzyldimethyl-n-hexadecyl ammonium chloride-sodium dodecyl sulfate (16-BAC-SDS) gel electrophoresis were performed. Integral membrane proteins and strongly basic nuclear histones were detected only in the 16-BAC-SDS gel electrophoresis system, confirming its usefulness for proteome analysis. All but one protein complied to the respective subcellular composition of the analyzed fractions. Taken together, the data make our subcellular prefractionation protocol an attractive alternative to other prefractionation methods which are based on less physiological protein properties.

Protein analysis in the rat auditory brainstem by two-dimensional gel electrophoresis and mass spectrometry.

A catalogue of the protein repertoire of processing centres in the central auditory system would greatly foster our knowledge on the anatomical and functional properties of this sensory system. Towards this goal, we report on the first mapping study of the protein content in the superior olivary complex (SOC) and the inferior colliculus (IC) of the rat auditory brainstem. The protein content of these two structures was assessed by means of two-dimensional gel electrophoresis (2-DGE) and mass spectrometry. To do so, proteins were first separated into four fractions by differential centrifugation. For comparison, corresponding cerebellar fractions were also analysed. Immunoblot analysis revealed highly enriched microsomal and cytosolic fractions; the other two fractions were mixtures of various subcellular compartments. Separation of the 800 g pellets (enriched for nuclear and plasma membrane proteins) and the 100,000 g supernatants (enriched for cytosolic proteins) by 2-DGE yielded between 456 and 674 distinct protein spots after silver staining. The overall protein pattern of all three tissues was similar for a given fraction. Fifty prominent protein spots of the SOC cytosolic fraction were identified by mass spectrometry and yielded information on thirty different genes with various cellular functions, e.g. primary metabolism, cytoarchitecture, and signal transduction. Sequencing of eleven corresponding spots from the SOC and IC cytosolic fractions confirmed the great similarity between the two samples. The results of this analysis are part of a novel integrated database of the gene repertoire of the auditory brainstem (ID-GRAB), that is publicly available (http://www.id-grab.de).