Sweet silver: A formaldehyde-free silver staining using aldoses as developing agents, with enhanced compatibility with mass spectrometry. Mireille Chevallet, Sylvie Luche, Hélène Diemer, Jean-Marc Strub, Alain van Dorsselaer, Thierry Rabilloud To cite this version: Mireille Chevallet, Sylvie Luche, Hélène Diemer, Jean-Marc Strub, Alain van Dorsselaer, et al.. Sweet silver: A formaldehyde-free silver staining using aldoses as developing agents, with enhanced com- patibility with mass spectrometry.. Proteomics, Wiley-VCH Verlag, 2008, 8 (23-24), pp.4853-61. 10.1002/pmic.200800321. hal-00348699 HAL Id: hal-00348699 https://hal.archives-ouvertes.fr/hal-00348699 Submitted on 20 Dec 2008 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Sweet silver: a formaldehyde-free silver staining using aldoses as developing agents, with enhanced compatibility with mass spectrometry. Mireille Chevallet 1,2, Sylvie Luche 1,2, Hélène Diemer 3, Jean-Marc Strub 3, Alain Van Dorsselaer 3, Thierry Rabilloud 1,2 1 CEA- Laboratoire de Biochimie et Biophysique des systèmes Intégrés, iRTSV/BBSI, CEA- Grenoble, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9, France 2 CNRS UMR 5092, CEA-Grenoble, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9, France 3 Laboratoire de Spectrométrie de Masse Bio-Organique, UMR CNRS 7178, ECPM, 25 rue Becquerel, 67087 STRASBOURG Cedex2, France Correspondence : Thierry Rabilloud, iRTSV/BBSI, UMR CNRS 5092, CEA-Grenoble, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9 Tel (33)-4-38-78-32-12 Fax (33)-4-38-78-44-99 e-mail: Thierry.Rabilloud@ cea.fr Abstract Protein detection methods after electrophoresis have to be sensitive, homogeneous, and not to impair downstream analysis of proteins by mass spectrometry. Speed, low cost and user- friendliness are also favored features. Silver staining combines many of these features, but its compatibility with mass spectrometry is limited. We describe here a new variant of silver staining that is completely formaldehyde-free. Reducing sugars in alkaline borate buffer are used as developers. While keeping the benefits of silver staining, this method is shown to afford a much better performance in terms of compatibility with silver staining, both in peptide mass fingerprinting by MALDI and in LC/ESI/MS/MS. 1. Introduction Protein detection is still a key step for the proteomics analysis of proteins separated on mono- or bidimensional gels. Besides obvious constraints such as sensitivity, homogeneity from one protein to another and linearity throughout a wide dynamic range, suitable features include compatibility with digestion and mass spectrometry, speed, convenience and low. Up to now, no protein detection method matches perfectly these prerequisites, in the three families of methods that are of current use in proteomics. Detection with organic dyes is simple, cheap and rather linear, shows an adequate compatibility with mass spectrometry, but its lacks sensitivity. In its most widely-used version, namely Colloidal Coomassie Blue [1], it requires long staining times for optimal sensitivity. However, faster protocols in this category are available, such as the dye pair staining technique [2], but the sensitivity remains moreless the same. Fluorescent detection methods, on their side, show a convenient linearity and an adequate sensitivity, although the latter varies from the one of colloidal Coomassie, such as sypro orange [3] to the one of silver (sypro ruby, deep purple) [4], [5] . Although superior to the one of silver staining, their compatibility with mass spectrometry does not always equal the one of Coomassie Blue, and this has been unfortunately shown to be the case for Sypro Ruby [6] and Deep Purple [7], i.e. the most sensitive variants. In addition to this drawback, optimal sensitivity requires rather long staining times, and the cost of the commercial reagents can become a concern when large series of gels are to be produced. Within this category, the covalent labeling used in DIGE is slightly different [8], in the sense that the sensitivity in detection is reached by exploiting the very low fluorescent noise, thereby allowing to use the very high signal to noise ratio to achieve sensitive detection through a high- performance hardware. However, the absolute level of signal is very low in this technique, so that protein excision for identification by MS is made on a more heavily loaded gel stained with noncovalent fluorescent probes. Furthermore, spot excision after fluorescent staining is usually carried out on a UV table, with the associated safety problems. The last family of protein detection methods consists of silver staining [9]. This method is sensitive, but labor-intensive, and its linearity is limited. However, in the proteomics frame, the most important problem lies in its limited compatibility with mass spectrometry. Although this feature has been improved by destaining of the spots or bands after silver staining [10], or by the use of silver-ammonia methods [11], the compatibility with mass spectrometry remains far below what can be achieved with fluorescent probes or colloidal Coomassie [12]. This low compatibility has been attributed to the use of formaldehyde [13], which also induces artefactual formylations. [14]. It would be therefore of great interest to have in hands a sensitive silver staining method totally formaldehyde-free. However, most silver reducers used in silver reduction (e.g. hydroquinone) [15] also show an important ability to induce protein crosslinks. To date, the only formaldehyde-free silver staining method uses carbohydrazide as the reducing agent [13]. While this results in improved compatibility with mass spectrometry, the staining performances are inadequate, both in sensitivity and homogeneity. We describe here a new family of silver staining protocols, using reducing sugars in alkaline borate buffer as developing agents. These methods combine the classical sensitivity and staining homogeneity of classical silver staining methods, while showing a much improved compatibility with mass spectrometry. 2. Material and methods 2.1. Samples Molecular weight markers (broad range, Bio-Rad) were diluted down to 10 ng/µl for each band in SDS buffer (Tris-HCl 125mM pH 7.5, containing 2% (w/v) SDS, 5% (v/v) thioglycerol, 20% (v/v) glycerol and 0.005% (w/v) bromophenol blue). The diluted solution was heated in boiling water for 5 minutes. A tenfold dilution in SDS buffer was performed to get a 1ng/µl per protein dilution . J774 cells (mouse macrophage) and WEHI274 cells (mouse monocytes) cells were grown in spinner flasks in DMEM + 5% fetal calf serum up to a density of 1 million cells /ml. The cells were collected by centrifugation (1000g 5 minutes), washed in isotonic wash buffer (10mM Tris HCl pH 7.5, 1mM EDTA and 250mM sucrose) and centrifuged at 100g for 5 minutes . The final pellet was suspended in its volume of isotonic wash buffer, transferred in an ultracentrifuge tube, and 4 volumes of concentrated lysis solution (8.75M urea, 2.5M thiourea, 5% CHAPS, 50mM DTT and 25mM spermine base) were added. After lysis at room temperature for 30 minutes, the viscous lystae was centrifuged at 200,000g for 1 hour at room temperature. The supernatant was collected, the protein concentration was estimated and the solution was made 0.4% (w/v) in carrier ampholytes (Pharmalyte 3-10). The solution was stored frozen at -20°C until use 2.2 Electrophoresis 2.2.1. SDS electrophoresis 10%T gels (160x200x1.5 mm) were used for protein separation. The Tris taurine buffer system was used [16], operated at a ionic strength of 0.1 and a pH of 7.9. The final gel composition is thus Tris 180mM, HCl 100 mM, acrylamide 10% (w/v), bisacrylamide 0.27%. The upper electrode buffer is Tris 50mM, Taurine 200mM, SDS 0.1%. The lower electrode buffer is Tris 50mM, glycine 200mM, SDS 0.1%. For 1D SDS gels, a 4% stacking gel in Tris 125mM, HCl 100mM was used. No stacking gel was used for 2D electrophoresis. The gels were run at 25V for 1hour, then 12.5W per gel until the dye front has reached the bottom of the gel. 2.2.2. IEF Home made 160mm long 4-8 or 3-10.5 linear pH gradient gels were cast according to published procedures [17]. Four mm-wide strips were cut, and rehydrated overnight with the sample, diluted in a final volume of 0.6ml of rehydration solution (7M urea, 2M thiourea, 4% CHAPS and 100mM dithiodiethanol [18], [19]). The strips were then placed in a multiphor plate, and IEF was carried out with the following electrical parameters 100V for 1 hour, then 300V for 3 hours, then 1000V for 1 hour, then 3400 V up to 60-70 kVh. After IEF, the gels were equilibrated for 20 minutes in Tris 125mM, HCl 100mM, SDS 2.5%, glycerol 30% and urea 6M. They were then transferred on top of the SDS gels and sealed in place with 1% agarose dissolved in Tris 125mM, HCl 100mM, SDS 0.4% and 0.005% (w/v) bromophenol blue. Electrophoresis was carried out as described above. 2.3. Detection on gels Colloidal coomassie blue staining was performed according to the published method [1]. Fluorescent staining was carried out with a ruthenium complex [6] with the improved protocol previously described [20]. The classical silver staining methods used were an ultrafast method [21], a silver-ammonia method [11] and a classical silver nitrate staining [22] The new staining methods were based on the fast silver nitrate method, and all the steps up to silver impregnation were kept constant (see table 1).