1. Introduction
The marriage of advanced imaging instrumentation and small-molecule detection reagents has been crucial to inventing new capabilities in electrophoretic detection, especially in the realm of nonradioactive posttranslational modification analysis. Fluorescent total-protein stains, such as SYPRO Ruby dye (Molecular Probes), Deep Purple dye (Amersham Life Sciences), and the cyanine dyes of the DIGE analysis system (Amersham Life Sciences, see Article 30, 2-D Difference Gel Electrophoresis – an accurate quantitative method for protein analysis, Volume 5), provide detection limits matching silver staining but offer significantly better dynamic range of quantitation and better compatibility with protein characterization techniques, such as mass spectrometry (Patton, 2002; Mackintosh et al., 2003). Accurate measurement of total protein amounts is fundamental to evaluating posttranslational modification changes, since it provides a denominator term, serving as a baseline for monitoring potential alterations; that is, the total protein measurement ensures that different samples are present in comparable amounts on the gel and aids in distinguishing between changes in the levels of posttranslational modification per unit protein compared with changes in protein expression levels without concomitant changes in the modification level. Typically, total protein staining is performed after detecting specific posttranslational modification.
2. Acquiring images
The introduction of fluorescent dye technology has played a pivotal role in spurring development of advanced imaging instrumentation, such as CCD camera-based multiwavelength imaging platforms, as well as gel scanners with multiple laser excitation sources (reviewed in Patton, 2000a,b). For example, the ProXPRESS 2D Proteomic Imaging System (Perkin-Elmer) is a sensitive imaging instrument that enables the use of a wide range of fluorescent and colored dyes owing to its CCD camera and multiwavelength emission and excitation capabilities. The instrument’s high-pressure xenon arc lamp provides broadband wavelength coverage and requires modest power, allowing visualization of the wide range of dyes commonly encountered in proteomics investigations, including Coomassie Blue, Amido Black, silver, colloidal gold, and the variety of fluorescent dyes now available. While the spatial resolution of conventional fixed CCD camera-imaging systems is typically inferior to laser-based gel scanners and photographic film, this problem is circumvented with the ProXPRESS instrument by mechanically scanning the CCD camera over the gel or blot and collecting multiple images that are subsequently automatically reconstructed into a complete image. Thus, the system readily delivers the same 50-|J.m spatial resolution obtained with high-end laser scanners, such as the FLA-5000 fluorescent imager (Fuji Corporation). By acquiring images in succession, as many as four different fluorescent labels may be viewed from any single gel.
3. Detecting posttranslational modifications
The rapid and comprehensive elucidation of protein posttranslational modifications is certainly one of the more important challenges facing the field of proteomics. Three prominent protein posttranslational modifications, glycosylation, phospho-rylation, and S-nitrosylation are now readily detectable after polyacrylamide gel electrophoresis using commercially available kits and instrumentation. The basic principles of the detection schemes are summarized below.
3.1. Glycosylation
Alterations in glycosylation profiles arising in human cancer are known to contribute in part to the malignant phenotype observed downstream of oncogenic events (see Article 62, Glycosylation, Volume 6). A sensitive green-fluorescent glycoprotein-specific stain, Pro-Q Emerald 300 dye (Molecular Probes), detects glycoproteins directly in polyacrylamide gels or on polyvinylidene difluoride (PVDF) membranes. Pro-Q Emerald 300 dye is conjugated to glycoproteins by a periodic acid Schiffs base (PAS) mechanism using room-temperature reaction conditions. As little as 300 pg of a1-acid glycoprotein (40% carbohydrate) may be detected in gels after staining with the dye, and a 500- to 1000-fold linear dynamic range of detection is obtained. A UV transilluminator-based imaging system is required to detect the stain, but a related stain, Pro-Q Emerald 488 dye, allows the detection of glycoproteins using visible light excitation sources. GlycoProfile III fluorescent glycoprotein detection kit (Sigma-Aldrich) also operates by a PAS-staining mechanism and is suitable for fluorescence-based detection of glycoproteins. The dye can detect roughly 150 ng of a glycoprotein in gels. Additionally, chemiluminescence-based approaches employing PAS labeling with digoxigenin hydrazide followed by immunodetection with alkaline phosphatase-conjugated antidigoxigenin antibody conjugated (DIG Glycan Detection Kit, Roche Molecular Biochemicals), or PAS labeling with biotin hydrazide followed by detection with horseradish peroxidase-conjugated streptavidin (ECL Glycoprotein Detection kit Amersham Life Sciences) detect glycoproteins on electroblots with high sensitivity.
3.2. Phosphorylation
Reversible protein phosphorylation plays a critical regulatory role in the circuitry of biological signal transduction, and disregulation of this process is a hallmark of carcinogenesis (see Article 63, Protein phosphorylation analysis by mass spec-trometry, Volume 6). Pro-Q Diamond phosphoprotein stain (Molecular Probes) detects phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins on SDS-polyacrylamide gels, isoelectric focusing gels, 2-D gels, electroblots, and protein microarrays by a mechanism that combines a chelating fluorophore and a transition metal ion (Schulenberg et al., 2003). The staining is rapid, simple to perform, readily reversible, and fully compatible with modern microchemical analysis procedures, such as MALDI-TOF mass spectrometry. Pro-Q Diamond dye can detect as little as 8 ng of pepsin, a monophosphorylated protein. The linear response of the fluorescent dye allows rigorous quantitation of phosphorylation changes over a 500-1000-fold concentration range. Alternatively, phosphoproteins may be selectively detected in gels through alkaline hydrolysis of serine or threonine phosphate esters, precipitation of the released inorganic phosphate with calcium, formation of an insoluble phosphomolybdate complex, and then visualization of the complex with methyl green dye, as recently commercialized in the GelCode phosphoprotein detection kit (Pierce Chemical Company). Detection by the staining method is not particularly sensitive, however, with microgram to milligram amounts of phospho-protein required to obtain a discernible signal. Phosphotyrosine residues cannot be detected with the stain.
3.3. S-nitrosylation
In an analogous manner as with protein phosphorylation, protein S-nitrosylation is thought to represent a key mechanism for the reversible posttranslational regulation of protein activity and, consequently, cellular function (see Article 68, S-nitrosylation and thiolation, Volume 6). Until relatively recently, the detection of protein S-nitrosylation in cells and tissues has been limited by a lack of appropriate detection techniques that permit identification of this labile posttranslational modification on target proteins. The Biotin Switch method was devised to facilitate the detection of protein S-nitrosylation after SDS-polyacrylamide gel electrophore-sis and electroblotting (reviewed in Patton, 2002). The basic approach has been commercialized as the NitroGlo nitrosylation detection kit (Perkin-Elmer). The NitroGlo kit provides an optimized procedure for detecting protein S-nitrosylation that has been validated using known S-nitrosylated proteins, such as H-ras and creatine phosphokinase. Following chemical blocking of free cysteine residues, S-nitrosylated cysteine residues are reduced to free thiols. These free thiols are then modified with a biotinylating reagent. Thus, only the cysteine residues that had nitroso modifications are tagged with a biotin label. Gel electrophoresis may then be performed to separate the various proteins in the sample. Alternatively, the labeled proteins may be selectively enriched using streptavidin affinity chromatography prior to gel electrophoresis. Western blotting, using chemiluminescence reagents, such as the Western Lightning kit (Perkin-Elmer), and subsequent imaging of the blots, generates a fingerprint pattern of the S-nitrosylated proteins in the specimen.
In conclusion, small-molecule-based detection molecules, in combination with advanced analytical imaging devices, now permit the routine monitoring of common protein posttranslational modifications in polyacrylamide gels and on electroblot membranes, without resorting to the use of hazardous radiolabeling approaches. Though these probes do not provide the same level of detection sensitivity as radiolabeling, they are safer and more convenient, allowing measurement of native posttranslational modification levels, as opposed to changes in levels associated with a particular labeling incubation time period employed in an experiment.
