Anal. Chem. 2006, 78, 2388-2396
Identification of Trichloroethanol Visualized Proteins from Two-Dimensional Polyacrylamide Gels by Mass Spectrometry Carol L. Ladner,† Robert A. Edwards,† David C. Schriemer,‡ and Raymond J. Turner*,†
Department of Biological Sciences and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada T2N 1N4
Proteins visualized by 2,2,2-trichloroethanol (TCE) on two-dimensional electrophoresis gels are efficiently identified by matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) mass spectrometry (MS) and MS/ MS. In a previous study, a method was developed that placed TCE in the polyacrylamide gel so that protein bands can be visualized without staining in less than 5 min. A visible fluorophore is generated by reaction of TCE with tryptophan that allows for protein visualization. In this study, MALDI-TOF MS and LC-MS/MS are used to identify randomly selected Escherichia coli proteins. The identification of TCE visualized proteins is compared to the identification of Coomassie brilliant blue (CBB) stained proteins from two-dimensional gel electrophoresis of E. coli proteins. This study demonstrated that TCE visualized proteins are compatible with protein identification by MALDI-TOF peptide mass fingerprinting. For 10 randomly selected spots, TCE visualization lead to statistically significant identification of 5 proteins and CBB visualization lead to identification of 6 proteins. TCE visualized proteins are also shown to be well suited for protein identification using LC-MS/MS. In 16 spots selected for MS/MS analysis, TCE samples lead to the identification of 79 peptides; while CBB samples lead to the identification of 65 peptides. TCE samples also supported the identification of more proteins. The low stoichiometry of labeling of tryptophan residues does not require inclusion of this modification for database searches. In addition to being a rapid visualization technique compatible with MS, TCE visualization utilizes rapid washing conditions for sample preparation of proteins spots excised from polyacrylamide gels. Aromatic residues of proteins can undergo UV light-dependent reactions. The UV irradiation of tryptophan slowly forms Nformylkynurenine, kynurenine, and cyclic lactams.1 Tyrosine also reacts under UV irradiation to hydroxylate the phenyl moiety, thus forming Dopa (3-hydroxytyrosine).2 A novel tryptophan chemistry * Corresponding author. E-mail:
[email protected]. Phone: 403-220-4308. Fax: 403-289-9311. † Department of Biological Sciences. ‡ Department of Biochemistry and Molecular Biology. (1) Creed, D. Photochem. Photobiol. 1984, 39, 537-562. (2) Creed, D. Photochem. Photobiol. 1984, 39, 563-575.
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was recently discovered where trichloro compounds, such as chloroform, 2,2,2-trichloroacetate (TCA), or 2,2,2-trichloroethanol (TCE), react with tryptophan under UV light to form a fluorescent product.3 The reaction of tryptophan with TCE is predicted to have a hydroxyethanone group (C(O)CH2OH) on carbon 4, 5, or 6 of the the indole ring based on analogy to the published mechanism of the chloroform reaction with tryptophan.3 The rate of reaction of trichloro compounds with tryptophan under UV irradiation is much greater than the rates of reactions induced by UV irradiation alone.3 This tryptophan photochemistry has been applied to visualizing proteins in polyacrylamide gels. Initially, chloroform or TCA was used to visualize proteins.4 A more efficient technique was subsequently developed by polymerizing the polyacrylamide gel in the presence of TCE and after separation of the proteins irradiating the gel with UV light.5 TCE produces a more intense fluorescent band than TCA and is less volatile than chloroform. This method provides visualization at a detection limit of 20-50 ng of protein in less than 5 min. Furthermore, for TCE visualization, the calibration curve of the fluorescence intensity dependence on the mass of tryptophan is linear from 0.7 to 100 ng of tryptophan.5 In comparison, the detection limit of the standard coomassie brilliant blue (CBB) method is in the 100-ng range. Colloidal CBB staining, which is a significant improvement to the standard CBB method, supports visualization in the range of 8-15 ng.6-8 Matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOF MS) and tandem mass spectroscopy (MS/MS) are commonly used to identify proteins. In MALDITOF, proteins are identified by matching potential peptide masses to a protein based on the expected masses in a protein database. In MS/MS, peptide fragmentation patterns are matched to patterns in databases. Proteomic studies using two-dimensional gel electrophoresis (2-DE) commonly use MALDI-TOF for highthroughput protein identification and follow with MS/MS to (3) Edwards, R. A.; Jickling, G.; Turner, R. J. Photochem. Photobiol. 2002, 75, 362-368. (4) Kazmin, D.; Edwards, R. A.; Turner, R. J.; Larson, E.; Starkey, J. Anal. Biochem. 2002, 301, 91-96. (5) Ladner, C. L.; Yang, J.; Turner, R. J.; Edwards, R. A. Anal. Biochem. 2004, 326, 13-20. (6) Patton, W. F. Electrophoresis 2000, 21, 1123-1144. (7) Diezel, W.; Kopperschlager, G.; Hofmann, E. Anal. Biochem. 1972, 48, 617620. (8) Westermeier, R.; Marouga, R. Biosci. Rep. 2005, 25, 19-32. 10.1021/ac051851y CCC: $33.50
© 2006 American Chemical Society Published on Web 02/18/2006
provide sequence-specific identification.9,10 An advantage of MS/ MS is that it allows the identification of a mixtures of proteins.9 One of the key considerations in protein identification is that the visualization technique needs to be compatible with MS. There are a number of visualization techniques with limits of detection in the 1-ng range that are compatible with MS, such as silver staining, SYPRO Ruby, blue silver, and Deep Purple.6,11-13 Analysis of the compatibility of colloidal CBB, silver staining, and SYPRO stains to MS have been performed by various groups.10,14-16 Many of the highly sensitive stains offer a lower limit of detection than the limit of detection of standard MS techniques. The lower limit of protein required for protein identification for the most sensitive mass spectrometers is in the subnanogram range;17 however, the routine amount of protein required for protein identification by MS is 1 pmol, which corresponds with 30 ng of a 30-kDa protein. CBB staining is the most common visualization technique used in association with mass spectrometry because its limit of detection will provide enough protein for routine MS. However, other staining methods that provide adequate sample for routine mass spectrometry and simplified washing protocols such as described in this study will be advantageous. Labeling tryptophan residues for protein visualization exploits a novel chemistry for a different method of visualization versus dyes. Rather than using dyes to bind to the protein, which will later need to be removed for MS analysis, a fluorescent label is created at tryptophan residues in the protein. There are other techniques that allow prelabeling of proteins before electrophoresis, such as 2-methoxy-2,4-diphenyl-3(2H)-furanone and monobromobimane that label lysine and cysteine residues; these labels change the isoelectric point and the molecular weight of proteins.18,19 The technique of two-dimensional difference gel electrophoresis (2-D DIGE) uses sets of N-hydroxysuccinimide ester cyanine dyes (Cy2, Cy3, Cy5) to prelabel proteins for comparison.20 For protein identification of spots from 2-D DIGE, the gels are stained with CBB and then the desired spot is cut out. Some of the CBB spots cannot be matched up with the cyanine-labeled spot because of the mass or pI change due to the cyanine label. (9) Lin, D.; Tabb, D. L.; Yates, J. R., 3rd. Biochim. Biophys. Acta 2003, 1646, 1-10. (10) Lauber, W. M.; Carroll, J. A.; Dufield, D. R.; Kiesel, J. R.; Radabaugh, M. R.; Malone, J. P. Electrophoresis 2001, 22, 906-918. (11) Berggren, K.; Chernokalskaya, E.; Steinberg, T. H.; Kemper, C.; Lopez, M. F.; Diwu, Z.; Haugland, R. P.; Patton, W. F. Electrophoresis 2000, 21, 25092521. (12) Candiano, G.; Bruschi, M.; Musante, L.; Santucci, L.; Ghiggeri, G. M.; Carnemolla, B.; Orecchia, P.; Zardi, L.; Righetti, P. G. Electrophoresis 2004, 25, 1327-1333. (13) 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, 2273-2288. (14) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Anal. Chem. 1996, 68, 850-858. (15) Berggren, K. N.; Chernokalskaya, E.; Lopez, M. F.; Beechem, J. M.; Patton, W. F. Proteomics 2001, 1, 54-65. (16) Lopez, M. F.; Berggren, K.; Chernokalskaya, E.; Lazarev, A.; Robinson, M.; Patton, W. F. Electrophoresis 2000, 21, 3673-3683. (17) Loo, J. A. In Advances in Protein Chemistry; Smith, R. D., Veenstra, T. D., Eds.; Academic Press: Boston, 2003; Vol. 65. (18) Urwin, V. E.; Jackson, P. Anal. Biochem. 1993, 32, 57-62. (19) Barger, B. O.; White, F. C.; Pace, J. L.; Kemper, D. L.; Ragland, W. L. Anal. Biochem. 1976, 15, 327-335. (20) Tonge, R.; Shaw, J.; Middleton, B.; Rowlinson, R.; Rayner, S.; Young, J.; Pognan, F.; Hawkins, E.; Currie, I.; Davison, M. Proteomics 2001, 1, 377396.
Sample preparation of 2-DE spots for mass spectrometry requires consideration of protein visualization, excision of protein spots, washing of gel pieces, and extraction of digested peptides. Because of the laborious washing protocol required for CBB other visualization methods that provide rapid visualization, rapid washing and uniform washing of gel pieces are advantageous for high-throughput and convenient sample preparation. CBB dyes are often difficult to completely remove from gel pieces, and CBB remaining in MALDI samples causes suppression of ionization.10 Proteins spots with large amounts or proteins or proteins that bind CBB particularly well will require more washing then other proteins. Here we demonstrate that rapid protein visualization using the TCE in gel technique is compatible with trypsin in-gel digestion, MALDI-TOF, and MS/MS. The major advantages are the speed of visualization as well as a uniform gel piece washing protocol. This allows for TCE in gel visualization to be used in proteomic studies requiring protein identification. To demonstrate this approach, we used Escherichia coli extract separated on a 2-DE, visualized in parallel by CBB staining and TCE visualization. Then the proteins were identified by MALDI-TOF and MS/MS. TCE in-gel visualization was compatible with standard sample preparation for MALDI-TOF but is simplified for faster sample preparation. The TCE in-gel visualization method has been shown to be a rapid technique to visualize and provide quality sample for MS. The limit of detection of TCE visualization matches the routine amount of protein required for MS. EXPERIMENTAL SECTION Chemicals. The R-cyano-4-hydroxycinnamic acid (CHCA), DNaseI, iodoacetamide, KWK (lysyltryptophanyllysine), phenylmethanesulfonyl fluoride (PMSF), and 2,2,2-trichloroethanol were purchased from Sigma-Aldrich (Markham, ON, Canada). CHCA was recrystallized from 95% ethanol for MS. Bovine modified trypsin was from Roche (Laval, PQ, Canada). Dithiothreitol and SDS were from BioRad (Hercules, CA). The trifluoroacetic acid was from Pierce distributed by BioLynx (Brockville, ON, Canada). C18 ZipTip were from Millipore (Bedford, MA). Immobiline Drystrips were from Amersham Biosciences (Baie d’Urfe, PQ, Canada). OMIX C18 tip were from Varian, Inc. (Lake Forest, CA). TCE Modification of KWK. A reaction mixture of 0.5 mM KWK and 40 mM TCE in 20 mM NH4CO3, pH 7, was irradiated while stirring in a 1.00)cm cuvette for a total of 13 min 40 s at 280 nm in a Fluorolog-3 (Jobin-Yvon Spex) fluorometer using a 450-W Xe lamp. The excitation slit width was set to 10 nm. Emission spectra before and after were generated by scanning from 320 to 540 nm with the excitation slit width at 10 nm and emission slit at 2 nm. For MALDI-TOF, 25 pmol of KWK was spotted with CHCA matrix in 50% ACN/0.3% TFA by the standard dried droplet method. Spectra were then acquired on a Voyager DE-STR (Applied Biosystems, Foster City, CA) in reflectron mode at the Southern Alberta Mass Spectrometry (SAMS) facility (Calgary, AB, Canada). Two-Dimensional Electrophoresis. For 2-DE, membranefree Escherichia coli cell extract was prepared from a 250-mL Luria-Bertani broth HB101 culture inoculated from a 1% overnight culture. At an optical density of 0.5, the cells were harvested at 2700g and washed once in buffer (25 mM NaHPO4 pH7, 75 mM NaCl). After another 2700g centrifugation, the cells were resusAnalytical Chemistry, Vol. 78, No. 7, April 1, 2006
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pended in 5 mL/g 25 mM NaHPO4. Before lysis, 2 mM PMSF and 150 units/mL DNaseI were added. The cells were then lysed at 16 kpsi with two passes in a minicell French press. Cell debris was removed at 7600g for 10 min followed by 346000g for 30 min to remove the membrane. The protein concentration was determined by a Lowry and aliquots frozen at -80 °C. Isoelectric focusing was done on a 7-cm pH 3-10 nonlinear immobilized pH gradient (IPG) Immobiline Drystrip. At the anodic end, 60 µg of protein was loaded in a sample cup with a MultiphorII system (Amersham Biosciences) for 29.6 kV/h at a maximum of 3500 V. The strip was then equilibrated in standard equilibration solution with 2% dithiothreitol followed by equilibration solution with 2.5% iodoacetamide as previously described.5 This strip was loaded onto a 12% polyacrylamide gel containing 0.5% TCE and run with a Bio-Rad Mini-protean III electrophoresis apparatus. Also, a 7-cm pH 4-7 IPG was focused with 150 µg of protein, loaded at the anodic end, for 41.1 kV/h. This pH 4-7 strip was then equilibrated as above and loaded onto a 15% polyacrylamide gel. Two identical gels were generated as described. One was stained using the TCE in-gel method and the other with CBB as described previously.5 Briefly, for the TCE in-gel method, 0.5% TCE is added before polymerization of the gel and after electrophoresis the gel is irradiated on a transilluminator with 302-nm bulbs (emitting from 275 to 375 nm) for 2 min. After UV irradiation, the TCE visualized gel is stored in 50% methanol containing 5% acetic acid. The gel is imaged with a CCD camera using an ethidium bromide filter. For the CBB method, the gel is stained in a solution of 0.05% Coomassie R250, 0.05% Coomassie G250, 0.1% cupric acetate, 25% 2-propanol, 10% acetic acid and destained in 14.25% ethanol, 10% acetic acid. Washing Conditions and Sample Preparation. For CBB stained proteins, the spots were excised from the gel and cut into 2-mm cubed gel pieces in a filtered hood. The gel pieces are then soaked with agitation for 15 min per wash, with water, acetonitrile (ACN), and 50 mM NH4HCO3 (aq), respectively. Then the gel fragments are washed in 50:50 25 mM NH4HCO3/ACN for 1-2 h. The wash with ACN followed by 50 mM NH4HCO3(aq) is then repeated. In some cases, further washing in 50:50 25 mM NH4HCO3/ACN is required to remove remaining coomassie dye. For TCE visualized proteins, personal safety protection was used to prevent UV irradiation of skin and eyes, during excision. Gloves taped to a lab coat, safety glasses, and a UV body shield were used. Various gel washing methods originally used for other stains were explored and compared. The best method was then selected and used for all E. coli 2-DE protein spots visualized with TCE. In the best method, the gel was soaked in 50% methanol, 5% acetic acid for ∼1 h or overnight. This solution was then replaced with water, and the protein spots were excised while being visualized under UV irradiation. Cubed gel pieces were then dehydrated by shaking in 100 µL of ACN for 5 min, removing the ACN, and drying down the sample in a Speedvac. The gel pieces were then washed twice in 250 µL of 50 mM NH4HCO3 as previously described.21 For both CBB and TCE spots, the washed gel pieces were dehydrated with ACN and dried down. The gel pieces were then
rehydrated with 10 µL of 25 µg/mL modified trypsin in 20 mM NH4HCO3 on ice for 30 min. Excess trypsin solution was removed, and the gel pieces were covered with 10 µL of 20 mM NH4HCO3 and incubated at 37 °C overnight. To extract the peptides, the gel pieces were shaken with 50 µL of 70% ACN followed by 50 µL of water and 50 µL of 70% ACN for 1 h for each wash.10 Extractions were removed and pooled from each washing step and dried. MALDI-TOF. MALDI-TOF spectra were obtained on a Voyager DE-STR (Applied Biosystems, Foster City, CA) in the reflectron mode at the SAMS facility (Calgary, AB). Dried down peptides were dissolved in 50% ACN/water (v/v) and 0.3% TFA (v/v) or 50% ACN/0.1% TFA and analyzed with a standard dried droplet method using a saturated solution of CHCA. Peptides were spotted with internal calibrants (angiotensin I 1296.685 [M + H]+, ACTH clip 1-14 1680.795 [M + H]+, and ACTH clip 18-39 2465.199 [M + H]+). Dried spots that appeared to have high salt content were washed with 0.5 µL of 0.1% TFA, pipetted on top of the spot, and then immediately drawn off using a Kimwipe tissue. In cases where a sample produced a spectra of low quality, the sample was cleaned with an Omix C18 tip as well as the corresponding sample visualized with the other method. Briefly, the peptides were aspirated in 0.1% TFA solution, washed, and eluted in 50% ACN/0.3% TFA. Refinement of the calibration for the acquired spectra was done using the M/Z program (Genomic Solutions Inc.). Mascot was used for protein identification.22 Here the NCBI nr protein sequence database for E. coli was searched. The upper mass limit was set to 10 kDa above the expected mass. The error tolerance was set at 100 ppm, allowing two missed trypsin sites. Partial oxidation of methionine and complete blocking of cysteines with iodoacetamide was included to search the database. Proteins were identified above the statistical significance threshold at a probability less than 0.05 that a database match was a random protein. For the E. coli database, the MOWSE score corresponding to p
