Introduction

It was recently shown [1] that quenching of N-hydroxy-succinimide (NHS)-ester-coupled dyes in comparative fluorescence gel electrophoresis may require a major excess of quencher in order to avoid unspecific labelling. As much as 2.500-fold excess of reaction partners needs to be present to silence the activated dye molecules sufficiently. This is about 100-fold more quencher than is typically used in differential gel electrophoresis (DIGE) [2], a technique, which has been widely employed for protein expression analysis during the past decade [3]. DIGE is a well-designed experimental set-up which represents the state-of-the-art in gel-based regulation studies. The gel-to-gel variation has been reduced to a minimum applying several samples to one gel. This is possible due to labelling with charge- and mass-matched cyanine fluorescent dyes. An internal standard formed by all samples participating in the experiment is furthermore introduced. Software support for statistical data analysis has also been provided. DIGE experiments take 2-4 weeks, are cost-intensive and consume valuable sample material (biological replicates) so that it is important to critically evaluate the influence of the detected cross-talk.

When the principle of DIGE was first introduced by Ünlü et al in 1997 [4], 1,3-diamino-2-hydroxy-propane was used for quenching and, moreover, active dye molecules were physically removed from the solution containing the labelled proteins by overnight adsorption to SM-2 beads. In later work by other authors [5], lysine was introduced as quencher and the dye-removal step was omitted. Quencher was provided in excess (200 pmol dye, 50 µg protein, 10 nmol lysine) so that its was reasonable to assume complete silencing of active dye. This protocol provided the basis for the commercial DIGE system with the only difference that 400 pmol dye labeled 50 µg of protein. It has been evaluated and applied by many researchers including us for protein expression analysis. Ünlü and coworkers have published another protocol [6] using methylamine-HCl and HEPES for quenching, this time not removing the residual dye from the protein solution. We have not used this method and do therefore not discuss it below.

Unspecific labelling is expected to cause increased spot volumes in DIGE. This is due, first, to continued labelling with the dye assigned to the individual sample, because the labelling time cannot be controlled and, second, to unspecific labelling of the other two samples of the set when all three samples are pooled in preparation for isoelectric focussing (Figure S1). We have shown extensively before [1] that the recommended amount of the quencher lysine is not sufficient in labelling protocols as they are used in DIGE [2]; cross-talk of one proteome (sample 1, e.g. control) in the fluorescent gel image of the second proteome (sample 2, e.g. sample of interest) was routinely detected. Different amounts of dye, protein and quencher were tested. To elucidate the impact of limited quenching on expression data, comparative experiments using Escherichia coli cell lysate were performed in this work.

Results and Discussion

Building on the earlier experiments [1], we first reversed labelling and quenching. To that end, 400 pmol Cy2, Cy3, and Cy5, respectively, were quenched with 10.000 pmol lysine as advised [2] and then 50 µg protein were added (for experimental design and protocols see Text S1, Table S1 and Table S2). At this stage in the protocol the dye should be silenced; if it is not, unspecific labelling during sample pooling would occur in DIGE. As can be seen in Figure 1 for Cy2, the proteome was still visualized on the gel although the image should be blank; this was only achieved when 250 times as much lysine was added. That was the maximum amount of quencher which could be used under these conditions for solubility reasons. Similar results were observed for Cy3 and Cy5 (Figure S2). Intermediate concentrations of lysine were tested before [1]. Instead of increasing the amount of quencher one could, in principle, also use less dye. A tenth of the recommended dye amount was added (40 pmol) for protein labelling, but 10-fold more quencher (Figure S2). However, not unexpectedly, sensitivity was lowered and spots were lost. We did not test for further, intermediate, concentrations, because the recommended labelling protocol was a minimal labelling approach in the first place.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 1. Reversal of labelling and quenching.

Cy2 image of E. coli lysate (pH 3–10, molecular weight 10–150 kDa). Protein was added after quenching. A) Regular protocol [2]; 10 mM lysine. Protein spots are detected on the gel. B) 250 times more quencher silences the active dye (lysine to dye ratio 1∶6250 instead of 1∶25).

https://doi.org/10.1371/journal.pone.0018098.g001

Having found a lysine concentration which silenced the dye, we proceeded to perform first a single and later replicate experiments comparing both quencher concentrations (gel 1/10 mM, gel 2/2.5 M) in order to determine an estimate for the differences in spot volumes (for protocol see Table S1). Detailed results for the first experiment are given in Table S3. Twenty-five well defined random spots were chosen for analysis across gel 1. Thereby, care was taken not to select proteins which spread over a large area or appeared as spot series. Of these 25 spots, 17 found a corresponding spot in gel 2 by automatic matching. Some spots showed no great change in spot volume within the variations typically observed in two-dimensional gel electrophoresis, but the majority of spots exhibited a much higher spot volume in gel 1 than in gel 2. For 8 spots even more than twice the volume was detected in gel 1 compared to gel 2 (Figure 2; spot 2, 3, 4, 9, 11 17, 20, 24).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Comparison of spot volumes.

Experiments were performed according to the recommended DIGE labelling protocol (10 mM lysine) and increasing the concentration of quencher to 2.5 M (complete quenching). Single experiment: gel 1 (10 mM lysine) – gel 2 (2500 mM lysine), Cy2 image (left) and Cy5 image (right). Data analysis using DIA module.

https://doi.org/10.1371/journal.pone.0018098.g002

The goal of the replicate runs was to perform a DIGE-like experiment with three replicate samples, because this is a) the minimum of experiments which should be done for statistical validity and b) the number of experiments often desired by researchers in practice. Three E. coli gels were run following recommendations (10 mM lysine [2]) plus three gels where the higher concentration of quencher was used during sample preparation (2.5 M; Text S2, Text S3). Each gel generated a Cy2, Cy3 and Cy5 image, respectively. However, in contrast to DIGE where the Cy2-labelled sample is formed by pooling all samples and used as internal standard, our experiment had to be designed slightly differently to accommodate the fact that Cy2 labelling results in gels 1–3 were expected to differ from those in gels 4–6 due to different quencher concentration. While all gels were run under exact same experimental conditions for best comparison they could, therefore, not be correlated to an internal standard. Spots were matched for all 6 gels and the corresponding spot volumes were compared. All samples, reagents and buffers were freshly prepared. Care was taken to double-check and readjust the required pH values for optimal labelling. Lysine quality would also influence the outcome of the experiment and it was checked by mass spectrometry (Figure S3). Raw volumes of selected spots as well as average spot volumes were evaluated in several ways comparing only few perfectly shaped spots (Text S2) or a larger number of random spots across the gel (Text S3), respectively, using DeCyder DIA and BVA analysis modules. The experimental error became less evident after mathematical data manipulation was performed (Text S2), but even then the results convincingly demonstrated an overabundance of proteins in the gels run with 10 mM lysine. An example is shown in Figure 3 for the average volumes of 36 spots in 3 gels. This image is representative for the two other dyes demonstrating also that individual proteins respond differently to the labelling procedure due to their amino acid sequence, secondary and tertiary structure and possibly due to parameters such as local pH. The introduction of a mathematical factor to correct for the difference in volumes is therefore not feasible. Experiments should be performed using a protocol which ensures that quenching conditions are suitable. The use of 2.5 M instead of 10 mM lysine for quenching serves this purpose in most cases, but a control experiment is advised. Alternatively, a filtration step to physically remove residual dye would also work, but it might also unspecifically remove proteins.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. Comparison of spot volumes.

Experiments were performed according to the recommended DIGE labelling protocol (10 mM lysine) and increasing the concentration of quencher to 2.5 M (complete quenching). Replicate experiments with 6 gels (gels 1–3 10 mM, gels 4–6 2.5 M. Analysis using BVA module. Shown are the average spot volumes of gels 1–3 versus gels 4–6 for Cy2. Unspecific labelling more than doubles the spot volumes in some cases. See Text S2, Text S3.

https://doi.org/10.1371/journal.pone.0018098.g003

Materials and Methods

Experiments were performed according to the manufacturer's recommendations [2] as described earlier [1] unless otherwise noted. Detailed information is available in Text S1, Table S1, Table S2, Table S4.

Supporting Information

Acknowledgments

The authors thank A. Dreiling, who measured lysine using ion trap mass spectrometry, for technical support.