Assessing Detection Methods for Gel-Based Proteomic Analyses Lauren R. Harris, Matthew A. Churchward, R. Hussain Butt, and Jens R. Coorssen* Departments of Physiology & Biophysics, Biochemistry & Molecular Biology, and Cell Biology & Anatomy, Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary AB, T2N 4N1, Canada Received January 15, 2007
Proteomic analyses using two-dimensional gel electrophoresis (2DE) depend heavily upon the quality of protein stains for sensitive detection. Indeed, detection rather than protein resolution is likely a current limiting factor in 2DE. The recent development of fluorescent protein stains has dramatically improved the sensitivity of in-gel protein detection and has enabled more accurate protein quantification. Here, we have evaluated the overall quality and relative cost of five commercially available fluorescent stains, Krypton, Deep Purple, Rubeo, Flamingo, and the most commonly used stain, Sypro Ruby (SR). All stains were found to be statistically comparable with regard to number of protein spots detected, but SR was superior with regard to fluorophore stability (e.g., capacity for repeated use of the stain solution). Notably, colloidal Coomassie Blue was also found to be comparable to SR when detected using an infrared fluorescence imaging system rather than standard densitometry. Thus, depending on available equipment and operating budgets, there are at least two high-sensitivity alternatives to achieve the best currently available in-gel protein detection: Sypro Ruby or Coomassie Blue. Keywords: fluorescent dyes ‚ two-dimensional gel electrophoresis ‚ protein staining ‚ proteomics ‚ comparative study ‚ infrared
Introduction Sensitivity of protein detection is a primary concern in proteomic studies using two-dimensional gel electrophoresis (2DE). In recent years, 2DE has been coupled with mass spectrometry for routine and high-throughput proteomic analyses.1,2 Ongoing refinements of 2DE protocols have enabled the resolution of thousands of proteins, including membrane proteomes, within a single gel,3,4 thus making the detection of proteins a limiting factor. Given that proteomic analyses are only as effective as their ability to account for the underlying complexity of the sample, the sensitivity and selectivity of a stain are key factors in enabling the accurate, quantitative representation of a given proteome. Traditional total protein detection methods include radiolabeling, Coomassie Brilliant Blue (CBB), and silver stain. Over the past decade, the fluorescent family of metal-chelate stains such as ruthenium bathophenanthroline disulfonate and the related Sypro Ruby (SR) have become the new gold standards.5-8 New detection methods continue to be developed, with the goal of increased stain selectivity and sensitivity, while maintaining practicality with regards to cost, ease of use, and compatibility with downstream protein characterization and identification technologies such as mass spectrometry. Radioactive labeling was once the primary method of postelectrophoretic protein detection in polyacrylamide gels.9 Various radioactive isotopes were incorporated into proteins * To whom correspondence should be addressed. Tel.: (403) 220-2422. Fax: (403) 283-7137. E-mail:
[email protected].
1418
Journal of Proteome Research 2007, 6, 1418-1425
Published on Web 03/17/2007
and were visualized directly by autoradiography. However, it was found that the radiolabeling of metabolic cells causes changes including DNA fragmentation and altered expression of tumor suppressor proteins,9-12 compounding inherent difficulties in achieving consistent, equilibrium labeling. These problems make it impossible to acquire a realistic ‘snapshot’ of the proteome in question. Thus, such labeling methods are unsuitable for the bulk of modern proteomic studies that have increasingly focused on human clinical disorders. Along with the safety and health concerns of handling radioactive compounds, particularly in large-scale proteomic studies, this technique is impractical for routine use. Both fluorescent and nonfluorescent staining methods have proven effective in protein detection and quantification. One of the most commonly used total protein stains is the organic dye Coomassie, both Brilliant Blue (CBB) and its variant, colloidal Coomassie Blue (CB).9,13,14 Originally developed in the 1960s, CBB has proven to be cost-effective, simple to use, and compatible with mass spectrometry; CBB thus remains a popular choice for in-gel protein detection.13,15-17 One of the weaknesses of CBB is relatively high-background staining because of nonselective binding of the dye particles to the gel matrix, as well as a low linear dynamic range (limited to 1 order of magnitude).9,14 The colloidal formulation was designed to overcome the problem of high background by incorporating selective stain particles that do not bind to the gel matrix, thus improving the sensitivity of the dye.9 While varying reports of detection limits of Coomassie exist, it is widely accepted that this stain is less sensitive than both silver-staining and fluo10.1021/pr0700246 CCC: $37.00
2007 American Chemical Society
research articles
Assessing Detection Methods for Proteomic Analyses Table 1. A Summary of Six Commercially Available Protein Stainsa
protein stain
excitation maximum [nm]
excitation filter (bandwidth) [nm]
emission maximum [nm]
Sypro Ruby Rubeo Flamingo Krypton Deep Purple Colloidal Coomassie
450 480 512 520 532 550
480 (30) 480 (30) 480 (30) 520 (20) 540 (30) 685 + 785 (laser)
610 605 535 580 610 670
a
emission filter (bandwidth) [nm]
620 (30) 620 (30) 590 (30) 620 (30)
