Direct Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometric Identification of Proteins on Membrane Detected by Western Blotting and Lectin Blotting Iwao Ohtsu, Tsuyoshi Nakanisi, Masaru Furuta,* Eiji Ando, and Osamu Nishimura Life Science Laboratory, Analytical & Measuring Instruments Division, Shimadzu Corporation, 1, Nishinokyo-Kuwabaracho, Nakagyo-ku Kyoto 604-8511, Japan Received March 22, 2005
We have developed new procedures to identify proteins after they are detected by Western blotting or other interactions such as lectin blotting on membranes. Our method is based on the combination of on-membrane MALDI-TOF mass spectrometry with piezoelectric chemical inkjet technology. Using this method the GroEL, FtsZ, DnaK, and GroES proteins were successfully identified from Escherichia coli after separation on two-dimensional gels, immunostaining, and on-membrane digestion. A glycoprotein detected by lectin blotting with concanavalin A was also identified using this technique. Keywords: protein identification • lectin blotting • mass spectrometry • Western blotting • on-membrane digestion
Introduction Many proteomics approaches are based on the combined use of electrophoresis and mass spectrometric analysis (MS).1-3 After separation, proteins are sometimes transferred by electroblotting onto a membrane for specific detection. One obvious advantage of having the separated proteins on a membrane is that it is then possible to use immunochemical methods. Several previous reports have described systems involving mass spectrometric analysis and identification of proteins detected by immunostaining.4,5 These technologies comprise indirect procedures that require dual separations of the same sample on gels, or a step in which proteolytic peptides are extracted from the membrane used for immunodetection and then subjected to MS analysis.6,7 We have developed a new method that very simply and effectively allows direct mass spectrometric identification of proteins digested after Western blotting or lectin blotting onmembrane. To implement this process, a Chemical Inkjet Printer was used to capture the sample image, and to microdispense reagents onto the visually designated positions.1,8 Trypsin is microdispensed by Chemical Inkjet Printer onto the membrane used for immunodetection, and then the matrix solution is dispensed directly onto the same position prior to MALDI-TOF MS analysis, which is carried out directly from the membrane surface.1,8 The obvious advantage of this approach is that peptide mass fingerprinting (PMF) of the visualized proteins is performed on the same membrane, without the necessity to extract proteolytic peptides after digestion. Here we present a process designated “Western MS”, for identification of proteins detected by Western blotting on a PVDF membrane. This principle for Western MS can also * To whom correspondence should be addressed. Phone: +81-75-8231351. Fax: +81-75-823-1364. E-mail:
[email protected]. 10.1021/pr050073n CCC: $30.25
2005 American Chemical Society
applied to identify proteins that are detected with lectins, or probe proteins such as tubulin; we call this method “Interaction MS.” The Western and Interaction MS processes are shown schematically in Figure 1. They couple a piezoelectric Chemical Inkjet Printer, which is a micro-jet device utilizing drop-ondemand technology for rapid liquid microdispensing in onmembrane digestion procedures, with a MALDI-TOF MS apparatus.8-11 The method allows one to perform Western blotting and other interactions such as lectin blotting analyses followed by direct MS identification of the relevant proteins on the membrane directly. The present paper describes the direct MS identification of Western blotted bovine serum albumin (BSA); the twodimensionally Western blotted GroEL, FtsZ, DnaK, and GroES against a background of total E. coli cell extracts;, and ovalbumin captured by lectin, as applications of Western, and Interaction MS methods, respectively.
Experimental Section Materials. Dithiothreitol (DTT), 2,5-Dihydroxybenzoic acid (DHB), Poly(vinylpyrrolidone) (PVP-40), R-methyl-mannopiranoside (R-MM), Direct Blue 71 (DB71) and BSA were purchased from Sigma (St. Louis, MO). Immobilon-P membrane was purchased from Millipore (Bedford, MA). E. coli W3110 strain was used for analysis. Cells were grown at 30 °C in Lennox broth consisting of 1% Bacto Tryptone (Difco Laboratories, Detroit, MI), 0.5% Bacto Yeast Extract (Difco), 0.5% NaCl and 0.1% glucose. All other solvents and chemicals were of analytical grade. Instruments. Tryptic digests were analyzed using a MALDITOF-type MS instrument, AXIMA-CFRplus, and/or the matrixassisted laser desorption/ionization- quadrupole ion trap timeof-flight mass spectrometric (MALDI-QIT-TOF MS) instrument, Journal of Proteome Research 2005, 4, 1391-1396
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lecular Probes, Leiden, Netherlands). Ovalbumin was detected by Alexa488-conjugated concanavalin A (Molecular Probes, Invitorogen, Eugene). Two-Dimensional Gel Electrophoresis. The sample for 2-DE gel electrophoresis was prepared according to the protocol of SWISS-2DPAGE (http://kr.expasy.org/ch2d/protocols/).14 A 50 mL culture of E. coli W3110 strain was centrifuged for 30 min at 3000 rpm at 4 °C. The pellets were washed three times with 30 mL of washing buffer (3.0 mM KCl, 1.5 mM KH2PO4, 68 mM NaCl, 9.0 mM NaH2PO4), suspended in 2 mL of a buffer containing 10 mM Tris-HCl, pH 8.0, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.1% SDS, and stored at -20 °C. Thirty milliliters of the sample was mixed with 360 mL of sample solution containing 8 M urea, 4% (w/v) CHAPS, 65 mM DTT, 2% Ampholyte (pH 3.0-10, Bio Rad), and a trace of bromophenol blue (BPB). An Immobiline Gelstrip (pH 4.07.0, 11 cm, Bio Rad) was rehydrated overnight with the prepared sample. Proteins were separated by isoelectricfocusing after the strip was equilibrated for 15 min in equilibration buffer containing 6 M Urea, 0.375 M Tris (pH 8.8), 2% SDS, 20% glycerol, 2%(w/v) DTT and 25 mg/L BPB. The strip was then placed on the stacking gel, and SDS-PAGE (12.5% acrylamide gel) was performed. The proteins separated on SDS-PAGE were electroblotted on Immobilon-P.
Figure 1. Scheme for Direct Identification of Proteins by Western MS or Interaction MS.Typically, a sample is separated by 1-DE or 2-DE and blotted to a membrane, and then molecular interaction analysis is performed on the membrane. Next, after stripping the antibody, positive spots are digested on the membrane using a Chemical Inkjet Printer, and they are identified by mass spectrometry. The print position is determined by the Chemical Inkjet Printer. The program MASCOT (14) is used to correlate the mass spectra of fragmented peptides to amino acid sequences using databases.
AXIMA-QIT (Shimadzu Corporation, Kyoto, Japan and Kratos Analytical, Manchester, UK) equipped with a 337 nm nitrogen laser.13 The MALDI-TOF MS analysis on the PVDF membrane was carried out operating in a positive ion mode by using an internal calibration method. In this method, bradykinin and the peptide fragment of the adrenocorticotropic hormone corresponding to 18-39 (ACTH) were used as internal standards. The MALDI-TOF MS/MS analysis on the PVDF membrane was carried out in the same mode by using an external calibration method with a mixture of angiotensin II and the ACTH peptide. For on-membrane digestion, the Chemical Inkjet Printer (CHIP-1000), developed by Shimadzu Corporation (Kyoto, Japan) in collaboration with Proteome Systems Ltd. (Sydney, Australia), was used for microdispensing the reagents onto blotted protein bands.1,8 SDS-PAGE Analysis of BSA and Ovalbumin. Titrated amounts of BSA (20, 10, 5, 2, 1, 0.5 pmol) and ovalbumin (20, 10, 5, 2, 1 pmol) were denatured in the presence of 1% SDS by 5 min heat treatment in a boiling bath. The proteins were electrophoresed on a 0.1% sodium dodecyl sulfate (SDS)-12.5% (wt/ vol) polyacrylamide gel (1-DE). After SDS-PAGE, proteins in the gel were electroblotted onto a PVDF membrane, and BSA was detected immunologically by sequentially adding an anti-BSA rabbit antibody (INC Pharmaceuticals, Inc., OH) and an Alexa488 conjugated anti-rabbit IgG (H+L) goat antibody (Mo1392
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Western Blot Analysis Using A Modified Rapid Immunodetection Method. The rapid immunodetection method (http:// www.Millipore.com/publications.nsf/docs/TN051) reported by Millpore Co. was performed as described with slight modifications.12 After transfer, the blots were soaked in 100% methanol for 10 s to drive out the water. Then the blot was placed on a piece of filter paper for 15 min and in a vacuum chamber for 30 min. At last the blot was incubated at 37 °C for 1 h and at room temperature for 2 h. Primary and secondary antibodies were diluted 1:1000 and 1:2000, respectively, into 0.25% PVP40, in Tris-buffered-saline (TBS, 50 mM Tris-HCl, 0.15 M NaCl, pH 7.6) and added to the blots at a ratio of 0.09 mL/cm2 of membrane surface area. Blots were washed in plastic containers using TBS buffer at a ratio of 0.9 mL/cm2 of membrane surface area. After visualization using fluorescence, the blotted spots were imaged on the FLA-5000 analyzer (Fujifilm, Tokyo, Japan). Stripping of Antibodies and Lectin from Membrane. After Western blot analysis, antibodies were eliminated by washing the membrane in 0.1 M Gly-HCl (pH 2.0) for 20 min, three times. Alexa488-conjugated concanavalin A was stripped by washing with 50 mM Tris-HCl (pH 7.5) containing 0.5 or 1.0 M R-MM for 30 min, twice. After the stripping of antibodies or lectin, the remaining proteins were visualized on the membrane by DB 71 staining for on-membrane digestion using the Chemical Inkjet Printer. On-Membrane Protein Digestion. The Immobilon-P membrane was adhered to the MS target plates using 3M electrically conductive tape 9713 (St. Paul, MN). Seven nl of 0.25% (w/v) PVP solution in 60% methanol was printed to pre-wet the membrane and then 50 nL of trypsin (Promega, Madison, WI) at 200 µg/mL in 25 mM NH4HCO3 containing 10% (v/v) 2-propanol was dispensed to each target position. Digestion was performed for 16 h at 30 °C in a humidified chamber. After digestion, 100 nL of 10 mg/mL of 2,5-DHB in 0.1% trifluoroacetic acid (TFA) containing 25% (v/v) acetonitrile was printed to each position on the membrane.
Identification of Proteins on Membrane
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Figure 2. Identification of BSA by Western MS. Each concentration of BSA was electrophoresed on a 0.1% SDS-12.5% (wt/vol) polyacrylamide gel. (A) Each concentration of BSA was detected immunologically using an anti-BSA antibody (Top panel); subsequently, antibodies were eliminated (under panel). The blot was then adhered to an MS plate using double-sided conductive tape. On-membrane tryptic digestion and subsequent direct MALDI-TOF MS analysis from the membrane were then performed as described in Materials and Methods. (B) MALDI-TOF MS analysis of on-membrane tryptic peptides derived from 1 pmol and 5 pmol of BSA. The pmf analysis was performed before and after the stripping of antibodies, respectively.
Figure 3. Identification of FtsZ from total E. coli extract by Western MS. (A) Staining of the membrane used in Western MS analysis of FtsZ, with anti-FtsZ (left), with DB71 added after stripping off antibodies (right). Tryptic digestion sites are indicated by white spots caused by bleaching of the DB71 (see inset) (3). X and Y coordinates of the digestion sites were integrated across to the mass spectrometer, thereby permitting precise peptide analysis on the membrane. (B) The mass spectrum (m/z range of 790 to 2100 Da) of tryptic peptides derived from FtsZ is shown. The Mascot-PMF search identified the sequence as belonging to E. coli cell division protein ftsZ (CEECZ), and resulted in an identification score of 112 and sequence coverage 27%(see inset). From 11 peptide matches this protein was identified as FtsZ. *: Masses assigned to the FtsZ sequence; T: tryptic autodigest peptides.
Results Blocking reagents such as skim milk and Tween-20, which are often used in conventional Western blot analysis, disturb
analyses using MALDI-TOF MS. A rapid immunodetection method without blocking has been recently developed by Millipore Co. and has been applied to Western blot with great success.12 However, it is still not sufficient for direct identificaJournal of Proteome Research • Vol. 4, No. 4, 2005 1393
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Figure 4. Identification of GroES from total E. coli extract by Western MS. (A) Staining of the membrane used in Western MS analysis of GroES, with anti-GroES (left), with DB71 added after stripping of antibodies (right). (B) The mass spectrum (m/z range of 800 to 1600 Da) of tryptic peptides derived from GroES is shown. This protein was not identified unambiguously as GroES (see right of inset). (C) Database search result after submission of the uninterpreted MS/MS from (B). The search identified the sequence as belonging to E. coli GroES and resulted in an identification score of 46. Subsequently, an ion signal at m/z 1495.50 was matched to the sequence “VGDIVIFNDGYGVK”. *: Peptides from GroES; T: tryptic autodigest peptides.
Figure 5. Identification of Ovalbumin by Interaction MS. Each concentration of ovalbumin (20, 10, 5, 2, 1 pmol) was electrophoresed on a 12.5% SDS-PAGE. (A) Each concentration of ovalbumin was detected using Alexa-488-conjugated concanavalin A (left); subsequently, lectin was eliminated (right) and the sample was re-visualized with DB 71. The blot was then adhered to an MS plate using double-sided conductive tape. On-membrane tryptic digestion and subsequent direct MALDI-TOF MS analysis from the membrane were then performed in the same manner as for BSA. (B) The mass spectrum (m/z range of 780 to 2700 Da) of on-membrane tryptic peptide derived from 5 pmol of ovalbumin is shown. (C) The Mascot-MS/MS ions search of a peptide with ion signals at m/z 2009.11 and 1687.93 identified the sequence as belonging to chicken ovalbumin (OACH) and resulted in identification scores of 20 and 30, respectively. The ion signals at m/z 2009.11 and 1687.93 corresponded to the sequences “GGLEPINFQTAADQAR and EVVGSAEAGVDAASVSEEFR”, respectively. *: Peptides from GroES; F: fragmentation ions.
tion of proteins and peptides by MALDI-TOF MS, because the membrane is treated with detergents such as Tween-20. Our goal was to perform protein identification by MALDI-TOF MS on the same membrane upon which Western blot analysis had been performed, and we improved the rapid immunodetection method to achieve this goal. Direct MALDI-TOF MS of On-Membrane Digested BSA after Western Blotting. BSA was separated by SDS-PAGE, blotted 1394
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to the membrane and detected by the modified rapid immunodetection method (Figure 2A) as described in Materials and Methods. The membrane was stained with DB 71 to re-visualize BSA after Western blotting, and was immobilized to an MS target plate with double-sided conductive tape. To digest the proteins on the Western-blotted membrane, both PVP-40 and trypsin solutions were microdispensed to BSA bands (0.5-20 pmol/lane). The matrix solution was also printed to exactly the
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Identification of Proteins on Membrane
same position as above after incubation for digestion. The digested area shown in Figure 3 occupied approximately 10% of each protein band; therefore, it was estimated that 1/10 (0.05-2 pmol) of the BSA blotted on the membrane was used to analyze directly. For effective detection of peptides resulting from on-membrane digestion after Western blot, it was estimated that 500 fmol (5 pmol/band) of BSA is sufficient. This level of sensitivity should allow for reliable protein identification by pmf analysis (Figure 2B). Improvement of Western MS in Sensitivity. To increase the sensitivity of the Western MS process we attempted to strip the antibody from the membrane. After Western blot analysis, the membrane was soaked with 0.1 M Gly-HCl (pH 2.0), and then the antibody was stripped from it (Figure 2A). By introducing these procedures, even 100 fmol (1 pmol/band) of BSA proved sufficient to generate pmf analysis for reliable protein identification (Figure 2B). We conclude that this Western MS process enables the direct identification of protein bands visualized on the membrane by Western blot. Identifying Proteins from E. coli by Western MS. To confirm the usefulness of the Western MS process developed here, we separated 180 µg of total cellular proteins from E. coli. W3110 by 2-DE (Figure 3A), then blotted them to a membrane. The proteins of interest were detected by Western blot using antiFtsZ, anti-GroES anti-DnaK and anti-GroEL antibodies as described above. After stripping these antibodies from the membrane, we carried out on-membrane digestion of GroEL (60 kDa), FtsZ (40 kDa) and DnaK (20 kDa) using a Chemical Inkjet Printer. The proteins were then identified directly by pmf analysis. Figure 3B shows the result of FtsZ analysis as an example. The small protein GroES (10 kDa) was directly identified by MS/MS analysis of the 1495.52 ion using MALDIQIT-TOF (Figure 4). Application of Western MS. As a further application of Western MS, we attempted MS identification of a glycoprotein (ovalbumin) detected by lectin blotting. Various concentrations of ovalbumin were separated by SDS-PAGE, transferred onto a PVDF membrane and detected by Alexa-488-cojugated concanavalin A (Figure 5). To strip the lectin, the membrane was soaked in R-MM solutions at two different concentrations for 30 min each, as described in Materials and Methods. The Alexa488-conjugated concanavalin A was completely removed from the membrane at 1 mM R-MM (Figure 5). We also confirmed that ovalbumin was detected with Pro-Q Emerald (data not shown). We thus showed that 500 fmol (5 pmol/band) of ovalbumin was successfully identified using MALDI-TOF-MS and MALDI-QIT-TOF MS with both staining methods.
Discussion Proteome analysis has its origins in 2-DE, a technique developed more than twenty years ago that remains an important tool for protein identification in combination with mass spectrometry. Conventional pmf analysis involves identification of individual protein spots from a gel followed by ingel digestion, peptide extraction, desalting, and finally loading of the extracts onto a MALDI-TOF target.15 Alternatively, the gel-separated proteins can be transferred to a membrane support such as nitrocellulose or PVDF by electroblotting followed by on-membrane cleavage and mass spectrometric analysis. Generally, this membrane technique has great advantages in terms of easy manipulation for desalting, and the potential for storage of used membranes. If the manipulating facility has an inkjet printer such as that shown here, then these
advantages are increased. Utilizing chemical printing strategies for microdispensing trypsin and matrix solutions, Sloane et al. recently demonstrated that the targeted protein was sufficient for generating pmf analysis for reliable protein identification, hence bypassing multiple liquid-handling steps.1,8 Furthermore, the ability to dispense small volumes (87 pL∼) of reagents to specific locations easily permits multiple-enzymes analysis of a single spot visualized by 2-DE.1,8 Using a Chemical Inkjet Printer, we demonstrated here the applicability of the MALDITOF MS approach, in conjunction with immunodetection, to on-membrane identification of proteins. The Western MS described here produced particularly high-quality mass spectra. The use of blocking reagents (PVP-40) without detergents such as Tween-20, and the use of stripping reagents without resorting to detergents such as SDS, did not affect the quality of the mass spectra. Recently, we have demonstrated the use of direct MS/MS analysis to identify blotted proteins on membranes at a low picomole level by using the Chemical Inkjet Printer in combination with a MALDI-QIT-TOF MS. We succeeded in conducting a direct MS/MS analysis on membranes, and our result showed highly accurate mass number and a high resolution.1 Using this MS/MS analysis, Western MS can also be used to identify proteins that were not identifiable by pmf analysis (Figure 4). Furthermore, as a broader application of the Western MS process, we successfully identified ovalbumin after detection by Alexa-488-conjugated concanavalin A, which exploited the interaction between the lectin and the sugar chains. We named this process Interaction MS in order to distinguish it from Western MS. When the Western MS or Interaction MS methods developed here are applied to profiling of phospho- and glyco-proteins, they will help to advance the field of proteomics, presenting a new selective detection and identification method for post-translationally modified proteins. This strategy could also be a powerful tool to detecting and identifying specific autoantigens in autoimmune diseases, and to screen for potential biomarkers. For example, it could be used in the early diagnosis of disease. This new approach offers several other advantages such as saving time, ease of use, effectiveness, and excellent reproducibility.
Acknowledgment. We especially thank Dr. Susumu Tsunasawa in this laboratory for helpful discussions. We are also grateful to Professor Masaaki Wachi (Department of Bioengineering, Tokyo Institute of Technology) for providing antibodies. This work has been done with the help of all the members of this laboratory. References (1) Nakanishi, T.; Ohtsu, I.; Furuta, M.; Ando, E.; Nishimura, O. J. Proteome Res. 2005, 4, 743-747. (2) Yates, J. R. III. J. Mass Spectrom. 1998, 33,1-19. (3) McDonald, W. H.; Yates, J. R. III. Traffic 2000, 1, 747-54. (4) Kaufmann, H.; Bailey, J. E.; Fussenegger, M. Proteomics 2001, 1, 194-199. (5) Klarskov, K.; Naylor, S. Rapid Commun. Mass Spectrom. 2002, 16, 35-42. (6) Dufresne-Martin, G..; Lemay, J. F.; Lavigne, P.; Klarskov, K. Proteomics 2004, 15, 55-66. (7) Shin, Y. S.; Lee, E. G.; Shin, G..W.; Kim, Y. R.; Lee, E. Y.; Kim, J. H.; Jang, H.; Gershwin, L. J.; Kim, D. Y.; Kim, Y. H.; Kim, G..S.; Suh, M. D.; Jung, T. S. Proteomics 2004, 4, 3600-3609. (8) Sloane, A. J.; Duff, J. L.; Wilson, N. L.; Gandhi, P. S.; Hill, C. J.; Hopwood, F. G.; Smith, P. E.; Thomas, M. L.; Cole, R. A.; Packer, N. H.; Breen, E. J.; Cooley, P. W.; Wallace, D. B.; Williams, K. L.; Gooley, A. A. Mol. Cell Proteomics 2002, 7, 490-499. (9) Adams, R. L.; Roy, J. J. Appl. Mech. 1984, 53, 193-197. (10) Bogy, D. B.; Talke, F. E. IBM J. Res. Dev. 1984, 29, 314-321.
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research articles (11) Dijksman, J. F. J. Fluid Mech. 1984, 139, 173-191. (12) Mansfiedld, MA. Anal. Biochem. 1995, 229, 140-143. (13) Koy, C.; Mikkat, S.; Raptakis, E.; Sutton, C.; Resch, M.; Tanaka, K.; Glocker, M. O. Proteomics 2003, 3, 851-858. (14) Kaga, N.; Umitsuki, G.; Nagai, K.; Wachi, M. Biosci. Biotechnol. Biochem. 2002, 66, 2216-2220.
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