We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we com- pared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively glob- al protein expression in cells and tissues.

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Quantitative analysis of complex protein mixtures using isotope-coded affinity tags

Economic Control of Quality of Manufactured Product.

We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides. The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions. The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit. By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively.

Direct analysis of protein complexes using mass spectrometry

We describe a modification of two-dimensional (2-D) polyacrylamide gel electrophoresis that requires only a single gel to reproducibly detect differences between two protein samples. This was accomplished by fluorescently tagging the two samples with two different dyes, running them on the same 2-D gel, post-run fluorescence imaging of the gel into two images, and superimposing the images. The amine reactive dyes were designed to insure that proteins common to both samples have the same relative mobility regardless of the dye used to tag them. Thus, this technique, called difference gel electrophoresis (DIGE), circumvents the need to compare several 2-D gels. DIGE is reproducible, sensitive, and can detect an exogenous difference between two Drosophila embryo extracts at nanogram levels. Moreover, an inducible protein from E. coli was detected after 15 min of induction and identified using DIGE preparatively.

Difference gel electrophoresis. A single gel method for detecting changes in protein extracts

Micromachining technology was used to prepare chemical analysis systems on glass chips (1 centimeter by 2 centimeters or larger) that utilize electroosmotic pumping to drive fluid flow and electrophoretic separation to distinguish sample components. Capillaries 1 to 10 centimeters long etched in the glass (cross section, 10 micrometers by 30 micrometers) allow for capillary electrophoresis-based separations of amino acids with up to 75,000 theoretical plates in about 15 seconds, and separations of about 600 plates can be effected within 4 seconds. Sample treatment steps within a manifold of intersecting capillaries were demonstrated for a simple sample dilution process. Manipulation of the applied voltages controlled the directions of fluid flow within the manifold. The principles demonstrated in this study can be used to develop a miniaturized system for sample handling and separation with no moving parts.

https://science.sciencemag.org/content/sci/261/5123/895.full.pdf

Micromachining a Miniaturized Capillary Electrophoresis-Based Chemical Analysis System on a Chip

Modification of lysine residues in proteins to improve their recovery when using derivatizing reagents.

Two-dimensional liquid chromatography-mass spectrometry for lipidomics using off-line coupling of hydrophilic interaction liquid chromatography with 50 cm long reversed phase capillary columns.

Detection of released egg-laying hormones from single cerebral ganglia of the pond snail Lymnaea stagnalis by means of capillary column liquid chromatography-mass spectrometry

James W. Jorgenson

Papers

Modification of lysine residues in proteins to improve their recovery when using derivatizing reagents.

Two-dimensional liquid chromatography-mass spectrometry for lipidomics using off-line coupling of hydrophilic interaction liquid chromatography with 50 cm long reversed phase capillary columns.

Detection of released egg-laying hormones from single cerebral ganglia of the pond snail Lymnaea stagnalis by means of capillary column liquid chromatography-mass spectrometry

Probeninjektionssystem für die Flüssigkeitschromatographie

Extraction and Gas Chromatographic Determination of Ethanol in Beverages