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SDS-PAGE of Proteins Using a Chameleon-Type of Fluorescent Prestain Robert J. Meier, Mark-Steven Steiner, Axel Duerkop, and Otto S. Wolfbeis* Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany A new prestaining method for protein SDS-PAGE was developed using the fluorogenic amino-reactive label Py1. This resulted in one of the fastest, most sensitive, and environmentally friendly protocols available. It is mainly due to the unique optical properties of Py-1, which is blue and virtually nonfluorescent but turns to red and becomes much more strongly fluorescent once it is conjugated to the amino group of a protein. Staining times of 30 min are adequate to visualize subnanogram quantities of proteins because pre-electrophoretic labeling Py-1 does not require the time-consuming steps of washing or fixation of gels. LODs as low as 16 pg of protein are found which is better than the best (commercial) poststains and comparable to the best (commercial) prestains. In addition, prestaining requires marginal amounts of staining solution. The change in electrophoretic mobility and band broadening is at a low level because Py-1 causes a mass shift of 288 Da per bound molecule only. By virtue of the small mass shift it causes, this stain is compatible with mass spectrometric protein analysis even though it acts as a covalent label. The variety of electrophoretic separation methods has enormously increased ever since Tiselius’ pioneering work on electrophoresis1 for which he was awarded the Nobel Prize back in 1984. Among them, polyacrylamide gel electrophoresis (PAGE) has become particularly important. There has been an additional thrust in PAGE when proteomics began to expand at a rapid pace in the 1990s.2 However, the visualization and quantification of very low levels of the concentration and quantity of proteins remains to be a major challenge in PAGE at present. There are two general approaches for staining proteins in PAGE: (a) the well-established technique of poststaining and (b) the method of prestaining, which is less common. Overall, poststaining relates to a process in which proteins are stained after the electropherogram has been developed. This is used in combination with all noncovalent and most covalent stains. Poststaining requires several additional steps after the separation and is time-consuming. In the prestaining method, proteins * To whom correspondence should be addressed. E-mail: otto.wolfbeis@ chemie.uni-r.de. Web: www.wolfbeis.de. (1) Westermeier, R. Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations; Wiley-VCH: Weinheim, Germany, 2004. (2) Wilkins, M. R.; Appel, R. D.; Van Eyk, J. E.; Chung, M. C. M.; Go¨rg, A.; Hecker, M.; Huber, L. A.; Langen, H.; Link, A. J.; Paik, Y.-K.; Patterson, S. D.; Pennington, S. R.; Rabilloud, T.; Simpson, R. J.; Weiss, W.; Dunn, M. J. Proteomics 2006, 6, 4–8.
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are stained prior to electrophoresis. The proteins are labeled after denaturation and then directly loaded onto the gel where separation takes place. No further steps are required, and the gels can be analyzed instantaneously. Inevitably, covalent labeling alters the molecular mass of proteins, thus a change in the migration rate has to be taken into account. Numerous stains are commercially available that enable colorimetric, radiometric, or fluorescent detection of proteins in SDS-PAGE gels. Colorimetric stains were initially used for protein detection due to their optical visibility and thus the ease of use. Coomassie Brilliant Blue (CBB) stains are quite popular. They were introduced around 40 years ago and still do exist in a variety of protocols.3–7 The silver stains8–11 also are in widespread use. These noncovalent stains offer a relatively simple but multistep staining protocol. CBB suffers from a lack of sensitivity, with limits of detection (LODs) of ∼10 ng per band and high background staining with a poor signal-to-noise ratio. Silver staining, with LODs as low as 1 ng, is known for its limited reproducibility. Further disadvantages include high background staining and often incompatibilities with mass spectrometry due to fixation with glutardialdehyde or formaldehyde. Radioactive labeling is most commonly performed via incorporating 3H, 14C, 35S, 32P, 33P, or 125I into proteins12,13 and allows measurement virtually free of background and results in very low LODs. Limitations result from limited operational lifetime due to radioactive decomposition. Furthermore, handling and disposal of radioactive material requires a high degree of safety monitoring and may cause additional costs. The most popular fluorescent stains are the SYPRO stains.14–17 SYPRO Red, for example, binds noncovalently and unspecifically (3) Groth, S. F. d. S.; Webster, R. G.; Datyner, A. Biochim. Biophys. Acta 1963, 71, 377–391. (4) Weber, K.; Osborn, M. J. Biol. Chem. 1969, 244, 4406–4412. (5) Echan, L. A., Speicher, D. W. Current Protocols in Protein Sciences; Wiley: Weinheim, Germany, 2002; Chapter 10. (6) Neuhoff, V.; Stamm, R.; Pardowitz, I.; Arold, N. Electrophoresis 1990, 11, 101–117. (7) Neuhoff, V.; Arold, N.; Taube, D.; Ehrhardt, W. Electrophoresis 1988, 9, 255–262. (8) Chevallet, M.; Luche, S.; Rabilloud, T. Nat. Protoc. 2006, 1, 1852–1858. (9) Switzer, R. C., 3rd; Merril, C. R.; Shifrin, S. Anal. Biochem. 1979, 98, 231– 237. (10) Merril, C. R.; Goldman, D.; Sedman, S. A.; Ebert, M. H. Science 1981, 211, 1437–1438. (11) Merril, C. R. Acta Histochim. Cytochim. 1966, 19, 655–667. (12) Hames, B., Rickwood, D. Gel Electrophoresis of Proteins: A Practical Approach; Oxford University Press: Oxford, U.K., 1990. (13) Link, A. Methods Mol. Biol. 1999, 112, 285–290. (14) Steinberg, T. H.; Jones, L. J.; Haugland, R. P; Singer, V. L. Anal. Biochem. 1996, 239, 223–237. 10.1021/ac800581v CCC: $40.75 2008 American Chemical Society Published on Web 07/11/2008
to the SDS-coat surrounding the proteins. The dye can be excited by UV light, so that the proteins can be visualized and photographed, respectively. The SYPRO Red method requires the labeled proteins to be fixed with diluted acetic acid, similar to Coomassie. The SYPRO labels have LODs as low as 2-4 ng of protein and a linear range of about 3 orders of magnitude; compared to silver staining, the SYPRO dyes are more sensitive, have a wider linear range, and require a shorter staining procedure. Additionally, there is no risk of overstaining, and stained proteins are detectable in protein mass spectrometry. The SYPRO dyes are all poststains with high molecular mass and noncovalent binding. There are more fluorescent poststains available than prestains, probably because the latter is known much longer. The first attempts in fluorescent prestaining of proteins were reported in the 1970s.18,19 They benefit from being sensitive and enable the monitoring of the progress of the gel-separation online via illumination with UV light. Barger et al.20 have introduced 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) as an amino reactive prestain with LODs of 1 ng. The so-called CyDyes are cyaninebased labels that covalently bind to lysine residues of proteins in a charge-matched mode with LODs of around 0.1 ng and linear ranges of over 3 orders of magnitude.21,22 CyDyes are frequently used in so-called difference gel electrophoresis (DIGE),23 where different samples are prestained with different CyDyes and then are separated via 2D-gel electrophoresis.24 Up to three different samples are detectable and thus comparable in one gel run. Although many fluorescent protein stains are currently available, most of them have spectral properties that are identical (within a few nanometers) in the free and the protein-bound form. Further, they mostly possess small Stokes’ shifts. These two facts require that excess label has to be carefully washed out of the gel after staining to minimize fluorescent background, even in the case of prestaining. The Py dyes reported before25 represent a new class of labels for proteins in displaying large Stokes’ shifts and large differences in emission wavelengths of the bound and free form. They possess an amino reactive pyrylium group that reacts under very mild conditions with primary amino groups by the exchange of the ring oxygen and the nitrogen as shown in Figure 1. Py-1 is one of the smallest blue and fluorescent chromophores known. In its unconjugated form, it is dark blue and virtually nonfluorescent with a quantum yield (QY) of 5 (saturation labeling)
GP 314 109 3000 16 000-30 000 1000 4000
ref
25 14, 14, 14, 14,
18 19 18, 19 18, 19
nd ) not determined.
Table 3. Results from ExPASy and MASCOT of a MALDI-TOF Analysis of Py-1 Prestained and Cbb Poststained BSA ExPASy and MASCOT results Py-1 prestained BSA (1 µg) CBB poststained BSA (1 µg)
SCORE value
sequence coverage rate (%)
329 398
50 48
were determined by five SDS-PAGE gels, each with identically stained 2-fold dilution series of Anamed Protemix protein standard. In the case of BSA, the dilution series ranged from 100 ng (lane 1) to 200 pg (lane 10). Figure 3 shows that intensities of the bands decrease as the amount of protein decreases. Excellent linear responses with correlation coefficients of r > 0.9994 were achieved. Linearity ranges were from 0.8 to 100 ng for BSA. Relative standard deviations (RSD) in the range from 1.25 to 5.85% demonstrate the excellent reproducibility of the Py-1 prestain (Table 1). The LODs somewhat depend on the staining conditions. If staining is performed for 30 min at 50 °C, the LODs range from 314 (GP) to 65 pg (GDH) per band. Labeling overnight at 50 °C and applying the amount of label twice results in an LOD of 16 pg (GDH) per band. Band broadening and higher background fluorescence occurs in the case of overnight labeling. Comparison of LODs of the Py-1 prestains with data from literature on other post- or prestains are given in Table 2. The higher amount of labeled BSA, GDH, and CA is dependent on the number of lysine residues per kilodalton. The ratios were 0.89 (BSA), 0.56 (GDH), and 0.45 (CA) lysine residues per kilodalton, respectively. MALDI-TOF and Western Blot Compatibility. Proteins prestained with Py-1 were tested for their general compatibility with matrix-assisted laser desorption ionization mass spectrometry along with time-of-flight detection (MALDI-TOF). Prestained 6278
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proteins (see the previous Prestaining Protocol with Py-1) and unlabeled proteins were separated simultaneously on a 10% SDSPAGE gel in adjacent lanes. The unlabeled proteins were stained with CBB following electrophoretic separation. Tryptic digestion and the mass profiling were accomplished according to the protocol described in the section on Digestion and Mass Fingerprint Analysis of Proteins. BSA was tested as a reference protein. A mass spectrum obtained with a 1 µg sample is depicted in Figure S3 in the Supporting Information. As can be seen, most of the characteristic peptide fragments are still available for protein identification because the recommended protocol for labeling results in a mean of only one to three covalently bound Py-1 labels per protein. The results of the fingerprint analysis are shown in Table 3. The sequence coverage rate of prestained BSA was even 2% higher than in the case of nonstained BSA. The SCORE value of the unstained BSA was calculated to be slightly higher. This results from a superior rate of detection of completely fragmented and higher rated peptides in MALDI-TOF analysis. Since the probability based Mowse Score was considerably higher than the desired value of 67, which gives a significant result,34 and the sequence was covered by half, the proteins were defined as BSA by ExPASy and MASCOT. The results obtained show a compatibility of the Py-1 prestain with MS analysis. The compatibility of prestained proteins to Western Blotting was investigated according to the procedure given in the section Western Blotting. Evaluation of the gel and the blotting membrane (data not shown) revealed that almost all stained proteins were transferred from the gel onto the blotting membrane. Analysis of the residual fluorescent bands in the gel via Quantity One software showed less than 2% of the initial intensity after the 2 h of blotting. (34) Pappin, D.; Hojrup, P.; Bleasby, A. Curr. Biol. 1993, 3, 327–332.
Figure 4. Gel with a 2-fold dilution series of protein standard. Dilution ranges from 100 ng in lane 1 to 200 pg in lane 10 in the case of BSA. The fluorescent spot in lane 3 is an artifact. The gel was visualized using the PCO camera.
DISCUSSION A highly sensitive and time-saving fluorescent staining approach in gel electrophoresis is introduced that is based on the amino reactive protein label Py-1. LODs as low as 16 pg were achieved for GDH and generally are lower than those obtained with CBB, silver stain, or the SYPRO stain but are comparable, if not distinctly better, than those of the Cy dyes. The prestaining technique offers several advantages over postelectrophoretic labeling in that (a) gels can be analyzed subsequent to separation without additional staining, washing, or workup steps; (b) separation can be monitored online; (c) the method is environmentally friendly; (d) only smallest quantities of dye and solvents are necessary (note that postelectrophoretic stains necessitate up to 10 000-fold the volume of staining solution per gel); (e) sensitivity is distinctly better. On the other side, both the linear range of the Py-1 prestain and its molar absorbance is smaller than that of many CyDyes and SYPRO stains. On the other side, the Py-1 prestaining procedure is more rapid. The entire staining process takes 30 min, which is clearly advantageous over the 4 h using SYPRO Red,17 > 3 h with CBB, and ∼40 min for the CyDyes.35 Protein labeling with Py-1 occurs at weakly basic pH values and at moderate temperatures. The Py-1 prestain is compatible with common sample and running buffer systems used in SDSPAGE. The change in electrophoretic mobility and band broadening is a general problem when using covalent labels in prestaining. Py-1 is one of the smallest fluorescent labels known. With a mass shift of 288 Da per bound label, migration rates are nearly identical to those of nonstained or poststained protein bands. Band broadening, caused by statistical variation of the DPR within a protein band, is acceptable and can be ignored in the case of separating proteins with a mass higher than ∼30 kDa (as can bee (35) Alban, A.; David, S. O.; Bjorkesten, L.; Andersson, C.; Sloge, E.; Lewis, S.; Currie, I. Proteomics 2003, 3, 36–44.
seen in Figure 4). Smaller proteins are slightly more affected by these phenomena, and the degree of separation in this range is weaker as a result. The investigation of two protein extracts shows that prestaining with Py-1 still offers a high degree of separation as it can be seen in Figure S4 in the Supporting Information. In comparison to the popular CyDyes prestains, Py-1 offers the following features: (a) It displays a unique chameleon behavior in that labeling is accompanied by a transition from a blue and virtually nonfluorescent label to a red and fluorescently labeled protein. Obviously, excess label does not interfere in fluorescence detection because the free label is nonfluorescent in practice, and because fluorescence of the conjugate is excited at a wavelength where the free (blue) label does not absorb; moreover, background staining is low. (b) The stability and storability of the label is excellent because staining is not based on the use of a rather reactive (and thus unstable) NHS ester; in fact, solutions of Py-1 in methanol are stable for at least 6 months at 4 °C. (c) The labeled proteins display a rather large Stokes’ shift (>100 nm). This strongly facilitates the separation of scattered excitation light from fluorescence and thus enables easier instrumentation. (d) Staining of proteins causes a smaller mass shift. CyDyes cause an increase in mass by approximately 0.58 kDa per label18 and the CyDye DIGE Fluor dyes an increase of even 0.7 kDa. (e) Both the conventional CyDyes and Py-1 enable charge-matched labeling, but in the case of Py-1 the positive charge of the amino group labeled is replaced by a positively charged pyridinium group; no countercharge is introduced at another site of the protein. (f) Py-1 is available at lower costs. Furthermore prestained proteins in gels that were fixed with methanol-TEAA buffer mixture gels can be stored for several months without any crucial decrease in fluorescence intensity. The Py-1 prestain was found not to be compatible with glutardialdehyde fixation and fixation with a 5:4:1 (v/v) mixture of methanol, water, and acetic acid. Given this and the fact that Py-1 prestained proteins are still compatible with mass spectrometry protein analysis and fingerprint protein mapping, the Py-1 prestaining method is deemed to represent a most perfect alternative to the commonly used staining methods in SDS-PAGE. Conceivable, it is also a possible candidate for prestaining in 2D-DIGE, because labeling occurs charge matched like in the use of the CyDyes and thus does not interfere with isoelectric focusing (IEF). SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
Received for review March 21, 2008. Accepted June 10, 2008. AC800581V
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