Influence of the Protein Staining in the Fast ... - ACS Publications

Apr 15, 2008 - The influence of four staining methods (Coomassie brilliant blue, silver nitrate, Sypro Red, and Sypro Orange) on the ultrafast ultraso...
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Influence of the Protein Staining in the Fast Ultrasonic Sample Treatment for Protein Identification through Peptide Mass Fingerprint and Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry M. Galesio, D. V. Vieira, R. Rial-Otero, C. Lodeiro, I. Moura, and J. L. Capelo* REQUIMTE, Departamento de Quı´mica, Faculdade de Cieˆncias e Tecnologı´a, Universidade Nova de Lisboa, 2829-516 Monte de Caparica, Portugal Received December 14, 2007

The influence of the protein staining used to visualize protein bands, after in-gel protein separation, for the correct identification of proteins by peptide mass fingerprint (PMF) after application of the ultrasonic in-gel protein protocol was studied. Coomassie brilliant blue and silver nitrate, both visible stains, and the fluorescent dyes Sypro Red and Sypro Orange were evaluated. Results obtained after comparison with the overnight in-gel protocol showed that good results, in terms of protein sequence coverage and number of peptides matched, can be obtained with anyone of the four stains studied. Two minutes of enzymatic digestion time was enough for proteins stained with coomassie blue, while 4 min was necessary when silver or Sypro stainings were employed in order to reach equivalent results to those obtained for the overnigh in-gel protein protocol. For the silver nitrate stain, the concentration of silver present in the staining solution must be 0.09% (w/v) to minimize background in the MALDI mass spectra. Keywords: Coomassie blue • silver nitrate • Sypro Red • Sypro Orange • ultrasonic in-gel protein digestion • MALDI-TOF-MS

1. Introduction Mass spectrometry is a rapidly growing field of protein analysis, useful in the identification of proteins separated by 1-D or 2-D gel electrophoresis. The most common mass spectrometry protein identification technique is called peptide mass fingerprinting, PMF.1 PMF involves the generation of peptides from proteins using residue-specific enzymes, the determination of peptide masses by spectrometric techniques, and the matching of these masses against theoretical peptide libraries generated from protein sequence databases to create a list of likely protein identifications. The overnight in-gel protein protocol for protein identification by PMF is a complex and time-consuming procedure with many steps as showed in Figure 1. In the last years, protocols for in-gel protein treatment have been drastically changed after the introduction of ultrasound to enhance enzymatic activity.2–4 The use of different ultrasonic devices such as ultrasonic probe or sonoreactor has allowed enzymatic digestion of proteins to occur in seconds (60-120 s), while former approaches needed from 4 to 12 h to complete the enzymatic process. In addition, it has been demonstrated recently that ultrasonic energy can also be used to accelerate other individual steps of the in-gel protein protocol for protein identification through peptide mass fingerprint, as showed in Figure 1. Thus, Cordeiro et al.5 verified that gel washing, protein reduction and protein alkylation steps * Corresponding author. J. L. Capelo. E-mail: [email protected]; tel., +351 21 294 9649; fax, +351 21 294 8550. 10.1021/pr700850w CCC: $40.75

 2008 American Chemical Society

can be acelerated by using an ultrasonic bath at 35 kHz. As a result, a reduction of about 85% of the total time required for the overnight in-gel protein procedure was obtained. In addition, the sample handling was drastically simplified without compromising the protein sequence coverage or the number of peptides matched. This is a technology developed recently at present in phase of internationalization.6 In proteomics, the selection of the appropriate protein staining to visualize the proteins separated in the gel is an issue of great concern, because it is directly linked to the quality of the results obtained.7 An ideal staining method should accomplish the following requisites: (i) to have a detection limit as low as possible with an optimal signal-to-noise ratio (ii) to have a wide dynamic range (iii) to have a wide linear relationship between the quantity of protein and the staining intensity (iv) to be nontoxic (v) the staining procedure should be easy and fast to perform, environmentally friendly, mass spectrometrycompatible, and not too expensive. To date there is no staining method accomplishing all the items described above.8 Different works have evaluated the MALDI-TOF mass spectrometry compatibility of visible stains and fluorescent dyes such as coomassie brilliant blue and silver nitrate, or Sypro Red, Sypro Orange, Sypro Ruby, Sypro Tangerine, and Deep Purple, respectively.7–11 For a detailed explanation about how the staining process occurs as a function of the stain selected and Journal of Proteome Research 2008, 7, 2097–2106 2097 Published on Web 04/15/2008

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Figure 1. Comprehensive scheme of the in-gel protein protocols studied in this work.

how to choose the best one for a specific proteomic application, the review of Miller and co-workers is highly recommended.12 Coomassie brilliant blue staining (CBB) was introduced for protein detection in 1963 and is still the most frequently used method for protein detection employed nowadays in polyacrylamide gel electrophoresis. In the presence of an acidic medium, this disulfonated triphenylmethane dye sticks to the amino groups of the proteins by electrostatic and hydrophobic interactions.8,12 The proteins have a higher affinity for the dye 2098

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than for the gel matrix; therefore, destaining can be carried out until the background is low and protein bands are clearly visible. CBB staining is easy to use, inexpensive, linear over at least 1 order of magnitude, and MS-compatible, and it has a detection limit of 10-100 ng.12 The disadvantages are long staining times, a relatively low sensitivity, and a narrow dynamic range.8 Silver staining procedures are based on the saturation of the gel with silver ions and the subsequent reduction of the protein-

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Protein Staining Influence in Fast Ultrasonic Sample Treatment 12

bound metal ions to form metallic silver. Today there are more than 100 different variants of silver-staining protocols that can be divided in two categories: alkaline and acidic silver stains, depending on the conditions used for silver impregnation. Silverstaining protocols are multistep procedures that include the following five main steps, apart from numerous washing steps: (i) fixation, to insolubilize the proteins and to remove interferences (ii) sensitization, to increase the subsequent image formation (iii) silver impregnation (iv) image development (v) stop the image formation and preservation of the gel. Silver nitrate is 100 times more sensitive than CBB with detections limits down into the picogram level.8,10 Unfortunately, silver stain shows only a narrow dynamic range and is not reliable for quantification. Moreover, highly abundant proteins produce spots with a yellow center which is a drawback for image analysis, either for qualitative and or quantitative analysis.8 Several fluorescent dyes, such as Sypro Red and Sypro Orange, have been developed during the last years with better sensitivities and properties than CCB or silver staining. Sypro Red and Sypro Orange do not bind the protein or the peptides directly but bind to the detergent coat surrounding proteins in SDS denaturing gels; thus, staining in such gels is not strongly selective for particular polypeptides. Staining is done in a one-step procedure after electrophoresis. After staining, protein bands can be visualized by excitation at the UV, ca. 300 nm, or at visible light wavelengths.12 Fluorescent dyes show very wide linear range, over 4 orders of magnitude, and staining is highly reproducible. Sypro Red and Sypro Orange have sensitivity similar to CBB. Thus, depending on running conditions and dye, 1-10 ng may be detected. In addition, and contrary to CBB, staining is reversible.8 Other advantages of Sypro Red and Sypro Orange are the following: proteins are not chemically modified during staining and none of the Sypro dyes contain superfluous chemicals such as formaldehyde, glutaraldehyde and Tween-20 that frequently interfere with peptide identification in MS.12 However, these dyes are very expensive and require the use of a fluorescence scanner to visualize the protein spots and to acquire the image for image analysis.8 As it was stated above, the introduction of ultrasound in the proteomic area represented an important improvement in terms of sample handling. However, to date, the influence of the protein staining, used to visualize the protein bands after gel electrophoresis, in the correct identification of proteins by PMF after the application of the ultrasonic in-gel protein protocol remains unknown. In the present work, we report, to the best of our knowledge for first time, the influence of four different staining procedures on the application of the ultrasonic assisted sample handling for protein identification by PMF and MS-based techniques. Two visible stains (coomassie brilliant blue and silver nitrate) and two fluorescent dyes (Sypro Red and Sypro Orange) were evaluated using six different proteins as follows: glycogen phosphorylase b (97 kDa), BSA (66 kDa), ovalbumin, (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and R-lactalbumin (14.4 kDa). For comparative purposes, the overnight procedure for in-gel protein identification was used.

2. Materials and Methods 2.1. Standards and Reagents. Standard protein mixture of glycogen phosphorylase b (97 kDa), BSA (66 kDa), ovalbumin, (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and R-lactalbumin (14.4 kDa) was purchased from Amersham Biosciences (Buckinghamshire, U.K., part number 170446-01). R-Lactalbumin from bovine milk (g85%), BSA (>97%) and trypsin enzyme, sequencing grade, were purchased from Sigma (Steinheim, Germany). R-Cyano-4-hydroxycinnamic acid (R-CHCA) puriss for MALDI-MS from Fluka (Buchs, Switzerland) was used as MALDI matrix. All materials were used without further purification. ProteoMass Peptide MALDI-MS Calibration Kit (MSCAL2) from Sigma was used as mass calibration standard for MALDI-TOF-MS. Sypro Red, Sypro Orange and silver nitrate for protein staining were purchased from Sigma, and Coomasie blue R-250 was from Merck (Darmstadt, Germany). Other reagents used in this work were methanol, acetonitrile, iodoacetamide (IAA), DL-dithiothreitol (DTT) (99% w/w), sodium thiosulfate, formaldehyde and glutaraldehyde, purchased from Sigma; formic acid, acetic acid (>99.5% v/v) and ammonium bicarbonate (>99.5% w/w) from Fluka; bromophenol blue, glycine, glycerol, trifluoroacetic acid (TFA, 99% v/v), sodium acetate and Na2EDTA from Riedel-de-Hae¨n (Seelze, Germany); β-mercaptoethanol (>99% v/v), sodium dodecyl sulfate (SDS) and sodium carbonate from Merck; and R,R,R-Tris-(hydroxymethyl)methylamine + Tris(hydroxymethyl) aminomethane, ultrapure grade from Aldrich (Steinheim, Germany). 2.2. Apparatus. Gel electrophoresis was performed with a gel electrophoresis system from Bio-Rad (Hercules, CA) model PowerPac Basic following the manufacturer’s instructions. The image of the gel after staining was acquired in a Gel Doc 2000 from Bio-Rad. Protein digestion was realized in safe-lock tubes of 0.5 mL from Eppendorf (Hamburg, Germany). A vacuum concentrator centrifuge from UniEquip (Martinsried, Germany) model UNIVAPO 100H with a refrigerated aspirator vacuum pump model Unijet II was used for (i) sample drying and (ii) sample preconcentration. A minicentrifuge, model Spectrafugemini, from Labnet (Madrid, Spain), and a minicentrifuge-vortex, model Sky Line, from ELMI (Riga, Latvia) were used throughout the sample treatment, when necessary. A Simplicity 185 from Millipore (Milan, Italy) was used to obtain Milli-Q water throughout the experiments. An ultrasonic bath, model Transsonic TI-H-5, from Elma (Singen, Germany) was used to speed up the gel washing, the protein reduction and the protein alkylation steps, and a sonoreactor model UTR200, from Dr. Hielsher (Teltow, Switzerland), was used to accelerated the enzymatic digestion step. 2.3. Gel Electrophoresis. Amounts of protein ranging from 0.1 to 1.9 µg were dissolved in 5 µL of water plus 5 µL of sample buffer (5 mL of Tris-Base 0.5 M, 8 mL of SDS 10% w/v, 1 mL of β-mercaptoethanol, 2 mL of glycerol, and 4 mg of bromophenol blue in a final volume of 20 mL in water) and then boiled for 5 min to denature the proteins for sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The denatured proteins were loaded in 12.5% polyacrylamide gels with 0.5 mm thickness. For coomassie blue and silver nitrate stains, the SDS concentration in the gel and in the running buffer was 0.1% (w/v), while SDS concentration was reduced to 0.05% (w/v) when Sypro stains were used. Proteins were separated at 120 V and 400 mA for 65 min. After gel electrophoresis, the gel was Journal of Proteome Research • Vol. 7, No. 5, 2008 2099

research articles stained and destained to visualize the protein bands, according with one of the protocols described in the next section. 2.4. Stain and Image Analysis. 2.4.1. Coomassie Blue R-250 Stain. The stain solution was prepared dissolving 1 g of coomassie blue R-250 in 200 mL of a mixture of 45% (v/v) methanol, 7.5% (v/v) glacial acetic acid and 47.5% (v/v) water. After gel electrophoresis, gels were rinsed with Milli-Q water and then incubated for 1 h in the staining solution at room temperature. After incubation, the staining solution was removed, and the gel was rinsed with Milli-Q water and then incubated for 1.5 h at room temperature in the destaining solution (45% v/v methanol, 7.5% v/v glacial acetic acid, and 47.5% v/v water). 2.4.2. Silver Nitrate Stain. After gel electrophoresis, the gel was submitted to a fixation procedure by incubation for 30 min at room temperature in a mixture solution of 40% (v/v) ethanol, 10% (v/v) acetic acid, and 50% (v/v) water. Afterward, the gel was incubated for another 30 min in a sensitizing solution prepared dissolving 0.2 g of sodium thiosulfate and 6.8 g of sodium acetate in 100 mL of a solution of 30% (v/v) ethanol, 0.5% (v/v) of a commercial solution of glutaraldehyde (25% v/v) and 69.5% (v/v) water. Then the gel was washed three times with 50 mL of water for 5 min and incubated for 20 min at room temperature in the staining solution (0.25 g of silver nitrate and 40 µL of 37% v/v formaldehyde solution in 100 mL of water). Afterward, the gel was washed two times with 50 mL of water for 1 min and incubated twice for 5 min with the developing solution (2.5 g of sodium carbonate and 20 µL of 37% v/v formaldehyde solution in 100 mL of water). When the protein bands were visible, the gel was incubated for 10 min in a stopping solution (1.46 g of EDTA-Na · 2H2O in 100 mL of water) and finally, rinsed with 50 mL of Milli-Q water three times for 5 min. 2.4.3. Sypro Red and Sypro Orange Stains. The staining solutions were prepared diluting the stock solutions of Sypro Red and Sypro Orange 1:5000 in acetic acid 7.5% (v/v). Prior to protein staining, gels were rinsed with a solution of acetic acid 1% (v/v) and then the gels were incubated with the staining solution for 1 h in the darkness at room temperature. After incubation, the staining solution was removed. Destaining procedure was not necessary. Protein bands were observed in an electronic UV transilluminator radiating at 300 nm from Ultra.Lum (Claremont, CA). 2.5. Accelerated Procedure for In-Gel Protein Treatment. Ultrasonic in-gel enzymatic digestion was done according to the ultrafast proteolytic digestion protocol previously developed in our laboratory.5 and schematized in Figure 1. Protein bands were manually excised from the gel and placed in safe-lock tubes of 0.5 mL. Protein bands were washed, first with water (100 µL) and then with acetonitrile (100 µL), in an ultrasonic bath operating at 35 kHz (100% amplitude) for 5 min for each step. Then, the piece of gel was dried in a vacuum concentrator centrifuge for 5 min. Protein reduction and alkylation steps were included in the protocol to facilitate the enzymatic action and to increase the protein sequence coverage (%).1 To do so, protein cystine residues were reduced with DTT in an ultrasonic bath operating at 35 kHz (100% amplitude) for 5 min at room temperature, and then, the resulting cysteines were blocked with IAA in an ultrasonic bath operating at 35 kHz (100% amplitude) for 5 min at room temperature. After reduction and alkylation steps, the gel was submitted again to the washing procedure in the same way as described above, followed by another dry step of 30 min. Afterward, the dried 2100

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Galesio et al. protein bands were incubated with trypsin (375 ng in 25 µL) in an ice bath for 60 min to rehydrate the gel and to allow enzyme penetration into it. Subsequently, in-gel protein digestion was performed in a sonoreactor operating at 50% amplitude for 2 or 4 min. Next, trypsin activity was stopped by the addition of 3 µL of formic acid 50% (v/v). This sample solution was then evaporated to dryness and resuspended with 10 µL of formic acid 0.3% (v/v). Prior to MALDI-TOF-MS analysis, the sample was mixed with the matrix solution. R-CHCA matrix was used throughout this work and was prepared as follows: 10 mg of R-CHCA was dissolved in 1 mL of Milli-Q water/acetonitrile/TFA (1 mL/1 mL/2 µL). Then, 10 µL of the aforementioned matrix solution was mixed with 10 µL of sample and the mixture was shaken in a vortex for 30 s. One microliter of each sample was handspotted on a well of a MALDI-TOF-MS sample plate and was allowed to dry. 2.6. MALDI-TOF-MS Analysis. A MALDI-TOF-MS system model Voyager DE-PRO Biospectrometry Workstation equipped with a nitrogen laser radiating at 337 nm from Applied Biosystems (Foster City, CA) was used to acquire the PMFs. Measurements were done in the reflector positive ion mode, with a 20 kV accelerating voltage, 75.1% grid voltage, 0.002% guide wire and a delay time of 100 ns. Two close external calibrations were performed with the monoisotopic peaks of the bradykinin, angiotensin II, P14R, and ACTH peptide fragments (m/z: 757.3997, 1046.5423, 1533.8582, and 2465.1989, respectively). Monoisotopic peaks were manually selected from each of the spectra obtained. Mass spectral analysis for each sample was based on the average of 300 laser shots. Peptide mass fingerprints were searched with the MASCOT [http:// www.matrixscience.com/search_form_select.html] and PROTEIN PROSPECTOR [http://prospector.ucsf.edu/] search engines with the following parameters: (i) Swiss-Prot 2007 database; (ii) molecular weight (MW) of protein, all; (iii) one missed cleavage; (iv) fixed modifications, carbamidomethylation (C); (v) variable modifications, oxidation (M); (vi) peptide tolerance up to 150 ppm. A match was considered successful when the protein identification score is located out of the random region and the protein analyzed scores first.

3. Results and Discussion 3.1. Sypro Orange and Sypro Red. 3.1.1. Optimization of the Sypro Orange and Sypro Red Staining Process. The first attempt was to optimize the staining and destaining conditions. Different staining protocols can be found in the literature regarding this item. For instance, Lauber et al.10 stained the gel in three steps, as follows: (i) First step: protein fixation with a solution of 40% (v/v) ethanol, 2% (v/v) acetic acid, and 0.0005% (w/v) SDS. In this step, proteins are insolubilized and fixed to the gel (ii) Second step: the gel was washed three times with a solution containing 2% (v/v) acetic acid and 0.0005% (w/v) SDS. In this step, the excess of SDS is removed form the gel but not from the proteins (iii) Third step: the gel was stained with a Sypro solution diluted 1:5000 in a solution containing 2% (v/v) acetic acid and 0.0005% (w/v) SDS. In this step, the dye is fixed to the SDS in contact with the proteins. This is a very time-consuming protocol that takes almost 10 h. On the other hand, Valdes and co-workers9 proposed to rinse the gel for 10 min with 7.5% (v/v) acetic acid after

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Protein Staining Influence in Fast Ultrasonic Sample Treatment

Table 1. Visualization of BSA and R-Lactalbumin Proteins Bands for Different Staining Protocols Tested for (0.5 and 0.1 µg) Separated by SDS-PAGE with Sypro Stainsa protein band visualization protocol

rinsing solvent

A B C D E

Water Water Water HAc 1% (v/v) HAc 1% (v/v)

a

staining solvent

HAc HAc HAc HAc HAc

7.5% (v/v) 7.5% (v/v) 1% (v/v) 1% (v/v) 7.5% (v/v)

destaining solvent

HAc HAc HAc HAc -

7.5% (v/v) 7.5% (v/v) 1% (v/v) 1% (v/v)

SDS concentration

0.5 µg

0.1 µg

0.1% (w/v) 0.05% (w/v) 0.05% (w/v) 0.05% (w/v) 0.05% (w/v)

Difficult Good Good Difficult Good

Not possible Only BSA bands were visualized Good Not visible Good

In all the cases, the Sypro stain was diluted 1:5000 in the corresponding staining solution. HAc, acetic acid; –, destaining protocol was omitted.

electrophoresis, then to stain the gel for 30-90 min in a sypro solution diluted 1:5000 with 7.5% (v/v) acetic acid, and finally to rinse the gel for 1-5 min with 7.5% (v/v) acetic acid to remove the excess of dye. The manufacturer of Sypro Red and Sypro Orange recommend to stain the gel for 30-60 min with a Sypro solution diluted 1:5000 with acetic acid 7.5% (v/v), without previous fixing or washing steps, and then to rinse the gel for 1 min with acetic acid 7.5% (v/v) to remove the excess of dye13,14 [disclaimer: specific company, product, and equipment names are given to provide useful information; their mention does not imply recommendation or endorsement by the authors]. Moreover, SDS concentration in the running buffer was 0.05% (w/v) instead of the usual concentration recommended by the manufacturer of 0.1% (w/v) in order to allow faster protein staining. To optimize the best procedure, the staining process was done (n ) 4) maintaining the staining time in 60 min, but the gel cleaning previous and following to staining were varied in order to obtain the most sensitive conditions. BSA and R-lactalbumin proteins (0.5 and 0.1 µg) were separated by SDS-PAGE and then stained as follows: (A) rinse the gel with water, stain for 60 min with a sypro solution diluted 1:5000 in 7.5% (v/v) acetic acid and destain for 30 min with a solution of 7.5% (v/v) acetic acid. Use a running buffer containing 0.1% (w/v) SDS (B) rinse the gel with water, stain for 60 min with a sypro solution diluted 1:5000 in 7.5% (v/v) acetic acid and destain for 30 min with a solution of 7.5% (v/v) acetic acid. Use a running buffer containing 0.05% (w/v) SDS (C) rinse the gel with water, stain for 60 min with a sypro solution diluted 1:5000 in 1% (v/v) acetic acid and destain for 30 min with a solution of 1% (v/v) acetic acid. Use a running buffer containing 0.05% (w/v) SDS (D) rinse the gel with 1% (v/v) acetic acid, stain for 60 min with a sypro solution diluted 1:5000 in 1% (v/v) acetic acid and destain for 30 min with a solution of 1% (v/v) acetic acid. Use a running buffer containing 0.05% (v/v) SDS (E) rinse the gel with 1% (v/v) acetic acid and stain for 60 min with a sypro solution diluted 1:5000 in 7.5% (v/v) acetic acid. The destain procedure was not done. Use a running buffer containing 0.05% (w/v) SDS. Results obtained and summarized in Table 1 and in Figure II of Supporting Information, show that, when the SDS concentration in the running buffer was reduced from 0.1% to 0.05% (w/v), keeping constant the other conditions, as described above in A and B, the proteins bands were visualized best. Thus, when SDS concentration in the running buffer was 0.1% (w/v) (protocol A), only the protein bands more concentrated (0.5 µg) were partially visualized. However, when the SDS concentration was reduced to 0.05% (w/v) (protocol B), BSA

bands of 0.1 µg were visible. This is in agreement with results reported by other authors that claim that proteins separated by gel using a running buffer containing 0.1% (v/v) SDS show the same sensitivity for protein staining as those run in SDS 0.05% (v/v), but at SDS 0.1% (v/v) more staining time is needed in order to visualize the proteins better.9 Improved results were obtained with the third protocol described above, protocol C, where the Sypro solution was diluted in acetic acid 1% (v/v), instead of 7.5% (v/v). In this case, protein bands of 0.1 µg, for both proteins tested, were correctly visualized. Interestingly, when the prestaining washing step with water was replaced by a washing step with acetic acid 1% (v/v) (protocol D), the sensitivity of the method decreased significantly again, obtaining similar results to those achieved with the protocol A. Therefore, when the prewashing step is done with acetic acid, then the staining solution should be prepared with higher percentage of acetic acid (7.5% v/v) (protocol E), and in this case, the destaining procedure could be removed. Taken into account that protocol E was faster than the other protocols assayed, and good sensitivity was achieved, it was used in further experiments as explained below. 3.1.2. Optimization of the Ultrasonic Sample Treatment for Protein Identification for Sypro Orange and Sypro Red Stains. BSA and R-lactalbumin proteins (0.5 µg) were separated in a SDS-PAGE gel, and after staining the gel with Sypro Orange, according to the protocol E described above, protein bands were excised and submitted to the ultrasonic protocol schematized in Figure 1. In addition, the overnight protocol was done for comparative purposes. BSA and R-lactalbumin proteins were correctly identified with the overnight protocol, with protein sequence coverages of about 70% for BSA and 45% for R-lactalbumin. On the contrary, when the ultrasonic treatment was applied, lower sequence coverages were found (60% for BSA and 40% for R-lactalbumin). Therefore, optimization of the ultrasonic in-gel protein treatment was considered mandatory. The results obtained with the ultrasonic sample treatment could be linked with an inefficient removal of the colorant during the washing step (step 2, Figure 1). Acetic acid 7.5% (v/v) is used in some protocols to destain the gel or remove the excess of colorant after the staining process; therefore, water in washing steps 2 and 7 of the in-gel protocol, see Figure 1, was replaced for acetic acid 7.5% (v/v). Results obtained with both solvents were equivalents (data not shown). For simplicity, water was chosen as optimum. Lauber et al.10 reported good results for Sypro Red and Sypro Orange after application of one variant of the overnight in-gel protocol, where the reduction and alkylation steps were omitted. However, when we tested this procedure, worse results were obtained either for the overnight or the ultrasonic treatments. For example, protein sequence coverage of about Journal of Proteome Research • Vol. 7, No. 5, 2008 2101

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Figure 2. Protein sequence coverage (% ( SD) and number of peptides matched ((SD) obtained for the Sypro Orange and Sypro Red stains with the overnight and the ultrasonic in-gel protein methods (n ) 2). Amounts of proteins ranged between 0.8 and 1.8 µg.

70% were obtained for the BSA protein with the overnight protocol, while, when the reduction and alkylation steps were omitted, the protein sequence coverage drastically decreased to 33%. Inconsistent results were also obtained for the R-lactalbumin protein, as its correct identification was not possible after removing the reduction and alkylation steps from the sample treatment. These results are in agreement with previous work from our group.3 As a result, reduction and alkylation steps were always included in the subsequent experiments. Different authors have suggested that some peptides are retained in the gel, after the protein digestion has been done. Therefore, to increase the sensitivity of the method, the peptides retained in the gel are further extracted using a mixture of acetonitrile and trifluoroacetic acid (TFA).9–11 To test this, after digestion, trypsin activity was stopped by the addition of 3 µL of formic acid 50% (v/v), and the sample was mixed in a vortex. Then, the digestion solution was removed and peptides were extracted twice from the gel with 25 µL of the extraction solution (50% v/v of acetonitrile, 0.1% v/v of TFA, and 49.9% v/v of water) in a sonoreactor operating at 50% of amplitude for 2 min. Afterward, the digestion solution and the two extraction solutions were combined and evaporated to dryness, and then peptides were resuspended with 10 µL of formic acid 0.3% (v/v). With this additional step ,an increment of about 8-10% in the protein sequence coverage was observed in both protocols. However, values achieved with the ultrasonic treatment were lower than those obtained for the overnight protocol, ca. 10% (v/v) lower. As a final attempt to improve results, the enzymatic digestion time was increased from 2 to 4 min. Reduction, alkylation and peptide extraction steps were included in the protocol. In the case of BSA, no significant variations were observed either with 2 or 4 min. However, in the case of R-lactalbumin, improved results were obtained using 4 min. Thus, increments of about 8% (v/v) in the protein sequence coverage for this protein were observed. Therefore, 4 min of digestion was selected as optimum time of sonication for further experiments. 2102

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Finally, a standard protein mixture of six proteins (glycogen phosphorylase b, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor and R-lactalbumin) was submitted to SDS-PAGE, and then, protein bands were visualized with the Sypro Orange stain prior to perform the overnight and the ultrasonic protocols. Results are shown in Figure 2. As it can be seen, equivalent results in terms of protein sequence coverage and number of peptides matched were obtained with both protocols tested. All proteins were identified positively for a total amount of protein in-gel comprised between 0.8 and 1.8 µg. Therefore, compatibility of the ultrasonic sample treatment with the Sypro Orange stain was demonstrated. On the basis of the similar characteristics of the Sypro Orange and Sypro Red stains, the ultrasonic protocol optimized for the Sypro Orange stain was also applied to the Sypro Red stain. The standard protein mixture of six proteins (glycogen phosphorylase b, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor and R-lactalbumin), with amounts of proteins ranging between 0.8 and 1.8 µg, was submitted to SDS-PAGE, and then, protein bands were visualized with the Sypro Red stain according with the protocol E described. Protein bands were excised and submitted to the overnight and the ultrasonic protein protocols. Results obtained are also shown in Figure 2. Similar results were obtained with both protocols in terms of protein sequence coverage and number of peptides matched. Only differences were observed for R-lactalbumin, for which slightly better results were obtained with the ultrasonic protocol. Therefore, the ultrasonic sample treatment can also be applied to the identification of proteins stained with Sypro Red. 3.2. Silver Staining. BSA and R-lactalbumin proteins (0.5 µg) were separated in a SDS-PAGE gel, and then, protein bands were visualized with a solution of 0.25% (w/v) silver nitrate, according to the protocol described in section 2.4. Afterward, protein bands were treated following the overnight protocol and the ultrasonic protein protocol (in the latter case, employing enzymatic digestion times of 2 and 4 min (step 11, Figure 1). The same peptide extraction step using acetonitrile and

Protein Staining Influence in Fast Ultrasonic Sample Treatment

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Figure 3. MALDI mass spectra of BSA and R-lactalbumin (0.5 µg, each one) obtained after the application of the ultrasonic protocol to gels stained with Sypro Orange and Sypro Red. The ultrasonic enzymatic digestion time was 4 min.

trifluoroacetic acid, as described above, was included in both protocols in order to improve results. MALDI mass spectra obtained in these conditions for the overnight and the ultrasonic sample treatments showed a higher background than those obtained previously with the Sypro stains, as it can be seen in Figures 3 and 4A,C, for the BSA protein. Only peaks with a high signal-to-noise ratio were selected and introduced in the search engine for protein identification. Results obtained are listed in Table 2. Regarding R-lactalbumin protein, only protein bands treated according to the overnight protocol were correctly identified, achieving sequence coverage of about 50%. In the case of BSA, correct identification was possible with the overnight method and the ultrasonic sample treatment with 4 min of digestion time; however, protein sequence coverages lower than those previously obtained with the Sypro stains were achieved. These results are in agreement with data reported for other authors. Thus, Shevchenko et al.15 observed that the overnight treatment of gels stained with silver using formaldehyde in the developer gives a much lower sequence coverage than the less sensitive CBB, even when a silver-destaining protocol with ferricyanide or hydrogen peroxide was used prior to MS. Interference by formaldehyde increases with time between silver staining and gel processing, even upon storage in water, probably by cross-linking between reactive amino acid side chains (lysine, cysteine, to a lesser extent also serine and threonine).16 For this reason, in order to make the procedure compatible to MS applications, glutaraldehyde in the sensitizing solution and formaldehyde in the silver nitrate solution should be avoided.12,15 We hypothesize that better MALDI mass spectra could be obtained in presence of a lower amount of silver. To prove this,

a new SDS-PAGE gel was stained according with the silver nitrate protocol where the amount of silver was reduced from 0.25% (w/v) to 0.09% (w/v), whereas other conditions were keept constant. Then, the stained protein bands were excised and submitted to the overnight and the ultrasonic protocol (4 min of digestion time). Results obtained showed that the background of the MALDI mass spectra was appreciably reduced, as it can be seen in Figures 4B,D, for the BSA protein. In addition, the protein sequence coverage and the number of peptides matched increased considerably for the ultrasonic sample treatment, as showed in Table 2, allowing positive identification of R-lactalbumin and achieving equivalent results to the overnight treatment. As a final attempt for this stain, protein bands of a standard protein mixture of six proteins, stained according with the new silver nitrate protocol, were submitted to the overnight and the ultrasonic protein protocols. Results are shown in Figure 5. As it can be seen in this figure, in general, equivalent results were obtained with both protocols used. In addition, better results were obtained for phosphorilase b and trypsin inhibitor, showing a slight increment in the protein sequence coverage and in the number of peptides matched after the application of the ultrasonic sample treatment. Therefore, the accelerated protocol using ultrasound to speed up several steps of the in-gel process (washing, reduction, alkylation and digestion) is compatible with the silver stain protocol. However, to increase the protein sequence coverage and the number of peptides matched, silver concentration of the staining solution should be decreased to 0.09% (w/v) and the ultrasonic enzymatic protein digestion time should be increased to 4 min. It is important to remark that lower Journal of Proteome Research • Vol. 7, No. 5, 2008 2103

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Figure 4. MALDI mass spectra of BSA protein (0.5 µg) obtained under the following conditions: (A) stain with 0.25% (w/v) silver and treated according to the overnight protocol, (B) stain with 0.09% (w/v) silver and treated according to the overnight protocol, (C) stain with 0.25% (w/v) silver and treated according to the ultrasonic protocol, and (D) stain with 0.09% (w/v) silver and treated according to the ultrasonic protocol. Table 2. Effect of the Concentration of Silver into the Staining Solution in the Protein Sequence Coverage (%) and Number of Peptides Matched Obtained for BSA and R-Lactalbumin (0.5 µg) after Overnight and Ultrasonic In-Gel Sample Treatment (Figure 1) with 4 min of Enzymatic Digestion Time for the Ultrasonic Treatment (n) 2)a 0.25% (w/v) of silver overnight treatment

0.09% (w/v) of silver

ultrasonic treatment

overnight treatment

ultrasonic treatment

protein

coverage (% ( SD)

no. peptides (( SD)

coverage (% ( SD)

no. peptides (( SD)

coverage (% ( SD)

no. peptides (( SD)

coverage (% ( SD)

no. peptides (( SD)

BSA r-lactalbumin

65.5 ( 2.1 49 ( 0.1

47.0 ( 2.8 12 ( 0.1

54.5 ( 6.4 -

43.0 ( 2.8 -

71.5 ( 2.1 52.5 ( 0.7

47.5 ( 2.1 14.5 ( 2.1

74.5 ( 6.4 51.0 ( 0.1

51.5 ( 0.7 12.0 ( 0.1

a

–: no positive results were obtained.

sensitivity for the silver stain method could be observed as a consequence of the reduction of the silver concentration. 3.3. Coomassie Blue. Results obtained for Sypro Red, Sypro Orange and silver nitrate stains revealed that improved results, with high sequence coverages (%) and more peptides matched, can be obtained with the ultrasonic sample treatment employing 4 min of digestion time instead of 2 min. At this point, we decided to test if also enhanced results could be obtained for the coomassie blue stain increasing the digestion time from 2 to 4 min. To test this, the standard protein mixture of six proteins was separated by SDS-PAGE, the gel was stained with coomassie blue as described in section 2.4, and then, the protein bands were treated according to the overnight and the ultrasonic protocol described in the Figure 1. As it can be seen in Figure 6, no differences were found among the values obtained for the overnight and the ultrasonic protocol in terms 2104

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of protein coverage or peptides matched, for either 2 or 4 min of digestion time. Therefore, a digestion time of 2 min is enough for achieve good results with this stain.

4. Conclusions The ultrasonic protocol developed previously in our laboratory, where ultrasonic energy is used to speed up several steps of the in-gel protein process (washing, reduction, alkylation and digestion), is compatible with different protein stains such as coomassie blue, silver nitrate and also the fluorescent dyes Sypro Red and Sypro Orange. For the case of silver nitrate and the fluorescent dyes, the ultrasonic enzymatic digestion time must be incremented from 2 to 4 min. In general, equivalent results were obtained for the four stains tested in this work, as it can be seen in Figure I of the Supporting Information.

Protein Staining Influence in Fast Ultrasonic Sample Treatment

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R-lactalbumin. To overcome this problem, the concentration of silver must be reduced in the staining solution from 0.25% (w/v) to 0.09% (w/v). In these conditions, comparable results using silver to other stains were obtained either for the overnight or the ultrasonic protocol. Protein reduction and protein alkylation steps must be included in the overnight and in the ultrasonic protocols regardless of the staining method studied, to achieve good results, in terms of protein sequence coverage and number of peptides matched. Moreover, sensitivity of the method can be improved by increasing the number of peptides extracted, doing several extractions from the gel with a mixture of acetonitrile/TFA/water, once the enzymatic digestion has been completed. With respect to the Sypro Red and Sypro Orange staining, it was confirmed that the stain protocol more recommendable is the following: after digestion, the gel is rinsed with acetic acid 1% (v/v), and then it is stained for 60 min with a Sypro solution diluted 1:5000 in acetic acid 7.5% (v/v). This method is faster than other protocols assayed in this work and also destaining is unnecessary without compromising the sensitivity of the method. Figure 5. Protein sequence coverage (% ( SD) and number of peptides matched ((SD) obtained for the silver stain with the overnight and the ultrasonic in-gel protein methods (n ) 2). The amount of silver used in the staining solution was 0.09% (w/v). Amounts of proteins ranged between 0.8 and 1.8 µg. The ultrasonic enzymatic digestion time was 4 min.

Acknowledgment. R. Rial-Otero and M. Galesio acknowledge the Fundac¸a˜o para a Cieˆncia e a Tecnologı´a (FCT, Portugal) for their postdoctoral grant SFRH/BPD/23072/2005 and the doctoral grant SFRH/BD/31652/2007, respectively. Dr. J. L. Capelo acknowledges the MALDI-TOF-MS service of the Chemistry Department of the New University of Lisbon (http://www.dq.fct.unl.pt/maldi) for their helpful assistance and valuable suggestions. The research findings here reported are protected by international laws under patent pending PCT/ IB2006/052314 and PT 103 303. Supporting Information Available: Figure I, comparative of the results of protein sequence coverage (% ( SD) and number of peptides matched ((SD) obtained with the ultrasonic treatment for all the protein stainings used in this work (n ) 2). Digestion times of 4 min were used for the Sypro Orange, Sypro Red and silver nitrate stains, while 2 min was used for the coomassie blue in the step 11 of the ultrasonic treatment (Figure 1). Amounts of proteins ranged between 0.8 and 1.8 µg. Figure II, photos of the sample treatments A, B, C, D and E concerning optimization of the Sypro Orange and Sypro Red staining process as described in section 3.1.1. This material is available free of charge via the Internet at http:// pubs.acs.org. References

Figure 6. Protein sequence coverage (% ( SD) and number of peptides matched ((SD) obtained for coomassie blue stain with the overnight and the ultrasonic in-gel protocols. Ultrasonic enzymatic digestion time (step 11, Figure 1) of 2 or 4 min (n ) 2). Amounts of proteins ranged between 0.8 and 1.8 µg.

Major interferences were found for silver stain with the overnight and the ultrasonic protocols, including more background in the MALDI mass spectra and lower percentages of protein sequence coverage compromising the positive identification of some proteins with low molecular weight, such as

(1) Lo´pez-Ferrer, D.; Can ˜ as, B.; Va´zquez, J.; Lodeiro, C.; Rial-Otero, R.; Moura, I.; Capelo, J. L. Trends Anal. Chem. 2006, 6, 909. (2) Lopez-Ferrer, D.; Capelo, J. L.; Vazquez, J. J. Proteome Res. 2005, 4, 1569. (3) Carreira, R. J.; Cordeiro, F. M.; Moro, A. J.; Rivas, M. G.; Rial-Otero, R.; Gaspar, E. M.; Moura, I.; Capelo, J. L. J. Chromatogr., A 2007, 1153, 291. (4) Rial-Otero, R.; Carreira, R. J.; Cordeiro, F. M.; Moro, A. J.; Fernandes, L.; Moura, I.; Capelo, J. L. J. Proteome Res. 2007, 6, 909. (5) Cordeiro, F. M.; Carreira, R. J.; Rial-Otero, R.; Rivas, M. G.; Moura, I.; Capelo, J. L. Rapid Commun. Mass Spectrom. 2007, 21, 3269– 3278. (6) Patent Pendings PCT/IB 2006/052314 and PT 103 303. (7) Chevalier, F.; Centeno, D.; Rofidal, V.; Tauzin, M.; Martin, O.; Sommerer, N.; Rossignol, M. J. Proteome Res. 2006, 5, 512. (8) Westermeier, R.; Marouga, R. Biosci. Rep. 2005, 25, 19. (9) Valdes, I.; Pitarch, A.; Gil, C.; Bermu ´dez, A.; Llorente, M.; Nombela, C.; Me´ndez, E. J. Mass Spectrom. 2000, 35, 672.

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research articles (10) Lauber, W. M.; Carroll, J. A.; Dufield, D. R.; Kiesel, J. R.; Radabaugh, M. R.; Malone, J. P. Electrophoresis 2001, 22, 906. (11) Mackintosh, J. A.; Choi, H. Y.; Bae, S. H.; Veal, D. A.; Bell, P. J.; Ferrari, B. C.; Van Dyk, D. D.; Verrills, N. M.; Paik, Y. K.; Karuso, P. Proteomics 2003, 3, 2273. (12) Miller, I.; Crawford, J.; Gianazza, E. Proteomics 2006, 6, 5385. (13) Product information of Sypro Orange protein gel stain. http:// www.sigmaaldrich.com/sigma/datasheet/s5692dat.pdf. (14) Product information of Sypro Red protein gel stain. http:// www.sigmaaldrich.com/sigma/datasheet/S5817dat.pdf.

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Galesio et al. (15) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Anal. Chem. 1996, 68, 850. (16) Richert, S.; Luche, S.; Chevallet, M.; Van Dorsselaer, A.; et al. Proteomics 2004, 4, 909.

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