Performance validation of an improved Xenon-arc lamp-based CCD camera system for multispectral imaging in proteomics.

Advances in gel-based nonradioactive protein expression and PTM detection using fluorophores has served as the impetus for developing analytical instrumentation with improved imaging capabilities. We describe a CCD camera-based imaging instrument, equipped with both a high-pressure Xenon arc lamp and a UV transilluminator, which provides broad-band wavelength coverage (380-700 nm and UV). With six-position filter wheels, both excitation and emission wavelengths may be selected, providing optimal measurement and quantitation of virtually any dye and allowing excellent spectral resolution among different fluorophores. While spatial resolution of conventional fixed CCD camera imaging systems is typically inferior to laser scanners, this problem is circumvented with the new instrument by mechanically scanning the CCD camera over the sample and collecting multiple images that are subsequently automatically reconstructed into a complete high-resolution image. By acquiring images in succession, as many as four different fluorophores may be evaluated from a gel. The imaging platform is suitable for analysis of the wide range of dyes and tags commonly encountered in proteomics investigations. The instrument is unique in its capabilities of scanning large areas at high resolution and providing accurate selectable illumination over the UV/visible spectral range, thus maximizing the efficiency of dye multiplexing protocols.

An improved formulation of SYPRO Ruby protein gel stain: comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation.

SYPRO Ruby protein gel stain is compatible with a variety of imaging platforms since it absorbs maximally in the ultraviolet (280 nm) and visible (470 nm) regions of the spectrum. Dye localization is achieved by noncovalent, electrostatic and hydrophobic binding to proteins, with signal being detected at 610 nm. Since proteins are not covalently modified by the dye, compatibility with downstream proteomics techniques such as matrix-assisted laser desorption/ionisation-time of flight mass spectrometry is assured. The principal limitation of the original formulation of SYPRO Ruby protein gel stain, is that it was only compatible with a limited number of gel fixation procedures. Too aggressive a fixation protocol led to diminished signal intensity and poor detection sensitivity. This is particularly apparent when post-staining gels subjected to labeling with other fluorophores such as Schiff's base staining of glycoproteins with fluorescent hydrazides. Consequently, we have developed an improved formulation of SYPRO Ruby protein gel stain that is fully compatible with commonly implemented protein fixation procedures and is suitable for post-staining gels after detection of glycoproteins using the green fluorescent Pro-Q Emerald 300 glycoprotein stain or detection of beta-glucuronidase using the green fluorescent ELF 97 beta-D-glucuronide. The new stain formulation is brighter, making it easier to manually excise spots for peptide mass profiling. An additional benefit of the improved formulation is that it permits staining of proteins in isoelectric focusing gels, without the requirement for caustic acids.

Multiwell in-gel protein digestion and microscale sample preparation for protein identification by mass spectrometry.

In-gel peptide digestion has become a widely used technique for characterizing proteins resolved by two-dimensional gel electrophoresis. Peptides generated from gel pieces are frequently contaminated with detergent and salts. Prior to matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis, these contaminants are removed using micro scale C18 sample preparation columns. In this paper, data are presented to demonstrate the application of a solvent resistant MultiScreen 96-well plate with a low peptide binding membrane and ZipTip micropipette based sample preparation. Recoveries of peptides (m/z of 1000 to 5000 Da) derived from standard protein protease digests, were estimated at various stages of the analytical process. An optimized protocol has been established and all the reagents and consumables have been packaged in a ready to use commercial kit. Data will be presented to show the application of this technology package to accelerate the throughput of protein characterization by protease fragmentation.