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

Zone electrophoresis in open-tubular glass capillaries

SUMMARY Capillary electrophoresis offers the possibility of rapid and effective separations in an instrumental format. The two most serious obstacles for future development are the present insufficient detector sensitivity and residual adsorption of solutes to the capillary surface. Capillaries offer some advantages over traditional gel systems. Efficient heat dissipation from capillaries permits the application of unusually high voltages which results in fast and efficient separations. On-line sample injection and detection along with the reusable nature of capillaries makes this format suitable for automation. This could be a significant advantage in situations where repetitive analyses must be run. Capillaries lend themselves to collection of accurate physico-chemical data such as mobilities from migration times and diffusion coefficients from zone widths. They are also an ideal system in which to investigate non-aqueous separation media (ref. 14). Forming gels in a variety of non-aqueous solvents might be difficult, and capillaries offer an uncomplicated approach to the problem. Non-aqueous solvents, although not traditional in electrophoresis, offer exciting possibilities. The wider range of acid and base strengths possible in solvents other than water, as well as the ability to solvate very hydrophobic solutes, could greatly expand the range of substances amenable to electrophoretic separation and analysis.

https://science.sciencemag.org/content/sci/222/4621/266.full.pdf

Capillary zone electrophoresis

High-resolution separations based on electrophoresis and electroosmosis

The use of extremely high pressures in liquid chromatography can improve the efficiency and reduce analysis time for columns packed with small particles. In this work, fused-silica capillaries with inner diameters of 30 μm are slurry packed with 1.5 μm nonporous octadecylsilane-modified silica particles. These columns are prepared in lengths up to 66 cm with packing pressures as high as 4100 bar (60 000 psi). Near the optimum flow rate, columns generate as many as 300 000 theoretical plates for lightly retained compounds (k‘ < 0.5) and over 200 000 plates for more retained compounds (k‘ ≈ 2). These translate to plate heights (Hmin) as low as 2.1 μm. The pressures required to run at optimum flow rates are on the order of 1400 bar (20 000 psi). Analysis times at these pressures are on the order of 30 min (k‘ ≈ 2) and can be reduced to less than 10 min at higher than optimum flow rates. Capacity factors are observed to increase linearly with applied pressure.

Ultrahigh-pressure reversed-phase liquid chromatography in packed capillary columns.

Fused-silica capillaries with inner diameters of 33 μm and lengths of 25−50 cm are slurry-packed with 1.0-μm nonporous octadecylsilane-modified (C18) silica spheres. These columns are used to perform ultrahigh-pressure reversed-phase liquid chromatographic analyses in both isocratic and gradient elution modes. Mobile-phase pressures as high as 5000 bar (72 000 psi) are applied to column inlets to generate more than 200 000 theoretical plates in 6 min (k‘ ≈ 1) for small, organic analytes. Average capacity factors of analytes are found to increase linearly with applied pressure. An electrically driven constant-flow syringe pump capable of generating mobile-phase pressures as high as 9000 bar (130 000 psi) is described. This pump is used in conjunction with an exponential dilution method for the gradient separation of peptides from a tryptic digest on a 27-cm-long capillary packed with 1.0-μm particles. A peak capacity of 300 is demonstrated for a 30-min analysis.

James W. Jorgenson

Papers

Zone electrophoresis in open-tubular glass capillaries

Capillary zone electrophoresis

High-resolution separations based on electrophoresis and electroosmosis

Ultrahigh-pressure reversed-phase liquid chromatography in packed capillary columns.

Ultrahigh-Pressure Reversed-Phase Capillary Liquid Chromatography: Isocratic and Gradient Elution Using Columns Packed with 1.0-μm Particles