Ultrasensitive Detection of Unstained Proteins in Acrylamide Gels by Native UV Fluorescence

Abstract

Visualization of proteins inside acrylamide and other gels usually relies on different staining methods. To omit the protein-staining procedure, we visualized unstained proteins inside acrylamide gels by laser excitation with ultraviolet (UV) light (280 nm, 35 mJ/cm 2 ) and directly detected native UV fluorescence. In one-dimensional gels, a detection limit as low as 1 ng for bovine serum albumin and 5 ng for other proteins with a linear dynamic range (2.7 orders of magnitude) comparable to state of the art fluorescent dyes could be achieved. In addition, the application of this method to 20 µg of a whole cell lysate separated in a two-dimensional gel showed more than 600 spots. Since protein labeling always represents a serious obstacle in protein identification technologies, the working efficiency with our procedure can be considered as a significant improvement for protein visualization and reproducibility in proteomics.Currently, two-dimensional gel electrophoresis (2-DE) represents the technology most widely used to separate complex protein mixtures for subsequent differential comparison (proteomics). Proteins are separated according to their isoelectric point (pI) in the first dimension and according to their apparent molecular mass in the second dimension. This method was first introduced by Klose and O'Farrell in 1975. 1,2 Continuous improvements in mass spectrometry (MS) over the last 10 years routinely allow protein spot identification in 2-DE. For proteomics research, 2-DE gels of different states (e.g., healthy vs diseased states) are acquired and compared in order to investigate biochemical processes (e.g., disease-causing mechanisms). The image analysis of several dozens of gels is a bottleneck in proteomics because of limitations in reproducibility of 2-DE and staining processes.Currently, different staining methods for the visualization of proteins in a gel have been established. Staining after separation: Silver staining is the most sensitive standard detection method (1 ng per band), 3 but it is accompanied by problems, such as chemical modifications of the proteins. 4 Furthermore, the staining and destaining procedures often result in a loss of protein and, therefore, in a loss of sensitivity for mass spectrometrical analysis. Additionally, each protein has an individual staining behavior due to its compositional properties. 5 Another drawback is the low dynamic range of this staining method. Staining with Coomassie Brillant Blue G-250 (CBB) is widely used because it does not interfere with further MS analysis, however, at the cost of a lower detection sensitivity (20-60-fold). 3 In contrast, labeling methods with fluorescent dyes are easier to handle and offer an improved dynamic range, but they are cost-intensive. 6 Staining before separation: Labeling of proteins with fluorescent dyes before separation is a critical process, because the isoelectric point and the molecular mass of the proteins can be changed by this method as a result of the covalent modification. Radioactive labeling ( 14 ...

A fluorescent natural product for ultra sensitive detection of proteins in one‐dimensional and two‐dimensional gel electrophoresis

“…8, methylation of both the 1716 Da and 1196 Da peaks was evident in the peak list and spectrum of the CBB G-250 stained apolipoprotein c-III. Recently a stainfree fluorescent technique has been developed for imaging spots in polyacrylamide gels [28]. Unfortunately, the technique was less sensitive than silver with a detection limit of around 5 ng per band for four proteins in 1-D gels.…”

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

“…The LLD of PhosB, CA and glyceraldehyde 3-phosphate dehydrogenase was 5 ng and 1 ng for BSA. The LDR was between 1 and 500 ng of protein and 2D analysis of EA.hy 926 soluble proteins showed that native fluores-cence detected 86% of spots relative to silver staining [194]. This procedure for in-gel protein detection is, however, very lengthy and requires up to two weeks for completion.…”

“…Since the introduction of native protein detection in gels, there have been various attempts to capitalize on and improve this process [185,[191][192][193]. Native fluorescence has demonstrated protein detection comparable to that achieved with silver staining [194]. The LLD of PhosB, CA and glyceraldehyde 3-phosphate dehydrogenase was 5 ng and 1 ng for BSA.…”

“…The maximum signal detectable by this camera was 10 5 electrons while the read noise or minimum signal was 1 electron. The range of detection afforded by this system presented an important development in the routine quantification of proteins and the application of CCD based systems for imaging gels still remains one of the most prevalent technologies in use today [193,194,196,197,236,[247][248][249].…”

The Human Platelet Membrane Proteome Reveals Several New Potential Membrane Proteins

“…Along with thrombospondin they constitute the predominant majority of the total protein amount in platelets, representing the major drawback of complex analyses because all separation and identification techniques are restricted with regard to dynamic range. Protein staining methods have a dynamic range between ϳ50 for Coomassie Brilliant Blue TM , 1,000 for silver staining procedures, and up to 10,000 for fluorescent dyes (13,14). In correlation, chromatographic separation is limited in binding capacity and dynamic range, too.…”