MS Analysis of Proteins Blotted on Membranes by a Matrix

Direct MS/MS Analysis of Proteins Blotted on Membranes by a Matrix-Assisted Laser Desorption/Ionization-Quadrupole Ion Trap-Time-of-Flight Tandem Mass Spectrometer Tsuyoshi Nakanishi, Iwao Ohtsu, 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 November 24, 2004

We constructed a system for the microscale identification of membrane-blotted proteins by proteolytic digestion using an instrument developed with piezoelectric chemical inkjet technology and MS/MS analyses of the resulting peptides with a matrix-assisted laser desorption/ionization-quadrupole ion trap-time-of-flight tandem mass spectrometer (MALDI-QIT-TOF MS). Using this system, bovine serum albumin was clearly identified at levels less than 100 fmol, and proteins from an Escherichia coli extract were also identified by an MS/MS ion search. Keywords: matrix-assisted laser desorption/ionization-quadrupole ion trap-time-of-flight mass spectrometry • electroblotting • piezoelectric ink-jet technology • on-membrane digestion

Introduction Two-dimensional electrophoresis is a widely used technique for separating proteins in proteome analysis.1,2 In general, to identify target proteins, the proteins are separated on gels which are then cut, subjected to in-gel digestion, and identified by peptide mass fingerprinting (PMF) and tandem mass spectrometry (MS/MS) analyses using a mass spectrometer (MS). Especially in current proteome analysis, it has become important to identify the differences in protein expression between healthy and diseased state individuals in tissues and body fluids, not only for the development of diagnostic tools, but also for the development of disease-specific therapies. When disease-specific proteins cannot be identified by PMF analysis, MS/MS analysis with electrospray ionization-quadrupole-time-of-flight (ESI-Q-TOF), matrix-assisted laser desorption/ionization (MALDI)-Q-TOF or MALDI-Q-TOF/TOF tandem mass spectrometers, in which characterization of the amino acid sequences and post-translational modifications of the proteins is carried out, is often used as an effective method for identifying the proteins.3-5 There is an alternative protocol to the in-gel digestion of proteins separated with 2-D PAGE, in which proteins are electroblotted onto a membrane, for example a nitrocellulose, nylon or poly(vinylidene difluoride) (PVDF) membrane, after electrophoresis and on-membrane digestion is conducted. This protocol is more effective for enzymatic digestion than the ingel method.6,7 On-membrane digestion, furthermore, has advantages for long-term storage of blotted membranes and for easy removal of low molecular weight contaminants. However, most experiments that have been reported use the in-gel digestion protocol instead of the on-membrane digestion * To whom correspondence should be addressed, Phone: +81-75-8231351, Fax: +81-75-823-1364. E-mail: [email protected]. 10.1021/pr0497834 CCC: $30.25

 2005 American Chemical Society

protocol, because recovery of peptide fragments from blotted membranes is somewhat low in comparison with in-gel digestion. To keep the advantages of on-membrane digestion and resolve the low recovery issue, some groups have reported the successful analysis of proteins and their protease digests on membranes directly in combination with MALDI-MS.8-10 Furthermore, on-membrane direct PMF analysis has also been conducted for microscale areas of blotted protein spots with a piezoelectric ink-jet device such as the Chemical Inkjet Printer, which allows the microdispensation of reagents at picoliter volumes onto the membranes.11 The piezoelectric drop-ondemand-type ink-jet technology itself has been applied to the fabrication of microarrays of DNA, protein and other bioactive molecules.12,13 The Chemical Inkjet Printer is a novel technology in proteomics, in which the ability of ink-jet printing technology to rapidly dispense a minute amount of solvent also allows high-throughput on-membrane PMF analysis in microscale areas of protein spots. However, MS/MS has not been applied to on-membrane analysis for digested fragments from proteins blotted onto nonconductive membrane despite its utility in proteomics. This is because tandem mass spectra of digests from blotted proteins are hard to acquire due to a charging effect or the roughness of membranes.9,14 The charges created by the MALDI process cannot be dissipated on a nonconductive membrane and accumulate. It is assumed that this charging effect leads to perturbations in the electric field between the sample and the acceleration plate. As a result, a loss of spectral resolution is observed and the mass shift is also observed when the laser energy is increased to ionize the sample. These phenomena mean that direct MS/MS analysis on nonconductive membranes is problematic. To resolve these problems in single MS analysis on membranes, Caprioli et al. have detected peptide/ protein signals using a conductive membrane such as a carbonJournal of Proteome Research 2005, 4, 743-747

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research articles filled polyethylene.15 If proteins blotted onto membranes can be identified with high-throughput on-membrane MS/MS analysis, attractive applications such as direct identification of the target proteins on imaging MS and rapid identification of antigen detected by Western blotting are expected.16-18 Indeed, we have also digested antigen on immunoblot membrane using piezoelectric chemical inkjet technology and rapidly identified antigen with on-membrane MS/MS analysis (in preparation). In the present study, we focused on direct MS/MS analysis to identify blotted proteins on membranes at low picomole levels using the Chemical Inkjet Printer in combination with MALDI-QIT-TOF MS, and succeeded in conducting a direct MS/MS analysis on membranes which gave a highly accurate mass number and a high resolution. The direct profiling of blotted proteins on membranes is very effective for investigating the localization of the objective molecules.16,17 Therefore, the method developed in this study for the direct MS/MS analysis of proteins blotted on a membrane with MALDI-QITTOF MS could prove to be a powerful tool for identifying both the objective molecules blotted on membranes from tissue samples, and antigens detected by Western blotting.

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Figure 1. Microscale dispensing of reagents with the Chemical Inkjet Printer for direct MS analysis on blotted membranes. Each area printed with PVP40, trypsin and matrix solutions (PVP40 solution: 7 nL, trypsin solution: 50 nL and matrix solution: 100 nL), indicated by two arrows, occupies approximately a 10% region of BSA band (20 pmol).

Experimental Section Materials. Escherichia coli K12 lyophilized cells, poly(vinylpyrrolidone) (PVP40) and bovine serum albumin (BSA) were obtained from Sigma (MO). 2,5- Dihydroxy benzoic acid was purchased from Aldrich (WI). Trypsin was obtained from Promega (WI) and the Immobilon-PSQ PVDF membrane was purchased from Millipore (MA). Instruments. Mass spectrometry was performed using a MALDI QIT-TOF-type MS instrument, AXIMA-QIT (Shimadzu Corporation, Kyoto, Japan and Kratos Analytical, Manchester, UK) equipped with a 337 nm nitrogen laser. MALDI-TOF MS/ MS analysis on PVDF membrane was performed in a positive ion mode using an external calibration method with a mixture of angiotensin II and the peptide fragment of the adrenocorticotropic hormone corresponding to amino acid residues 18-39. 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. Preparation of BSA Transferred Membrane. The sample solutions of BSA were prepared at concentrations of 20, 10, 5, 2, 1, and 0.5 p mol/µL and an equal volume of the buffer containing dithiothreitol was added to each BSA sample (10 µL). The solutions were then heated at 95 °C for 3 min, and allowed to cool to room temperature. Two microliters of each BSA sample were electrophoresed in a 12.5% polyacrylamide gel containing 0.1% sodium dodecyl sulfate. This 1-dimensional electrophoresis gel was used for semi-dry type electroblotting to the Immobilon-PSQ membrane. The blotting was performed at 200 mA for 1 h. The blotted membrane was stained with Direct Blue 71 (Sigma, MO) for detection of proteins. On-Membrane Digestion Using Chemical Inkjet Printer. The blotted membrane was adhered to an MS target plate with double-sided conductive adhesive tape. After acquiring the image on the blotted membrane with the scanner connected to the Chemical Inkjet Printer, two printing positions were created on each protein band. Seven nL of 0.25% (w/v) PVP40 solution in 60% (v/v) methanol was printed for pre-wetting the membrane, and trypsin solution at 100 µg/mL in 25 mM NH4HCO3 was then dispensed over 25 iterations onto protein bands 744

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at 2 nL per iteration. Digestion was carried out for 16 h in a humidified chamber at 30 °C, and a matrix solution, 10 mg/ mL 2,5-dihydroxy benzoic acid in 30% acetonitrile/0.1% TFA, was printed on exactly the same positions on the membrane over 25 iterations at 4 nL per iteration (Figure 1). Preparation of 2-DE Blotted Membrane of Escherichia coli Cell Extracts. Escherichia coli lyophilized cells (35 mg) were suspended in 7 mL Chaotropic Membrane Extraction Reagent 3 (ProteoPrep Total Extraction Sample Kit, Sigma). The suspension was ultrasonicated three times on ice for 20 s. The supernatant was recovered and used as cell extracts. After centrifugation (15 000 × g, 30 min, 4 °C) of the suspension, the proteins in the solution were reduced by adding tributylphosphine to a final concentration of 5 mM for 1 h at room temperature, and then alkylated with a final concentration of 15 mM iodoacetamide for 1.5 h at room temperature. The solution was centrifuged at 20 000 × g for 5 min at 4 °C, and the supernatant was subjected to 2-DE as follows. Pharmalyte (pH 5-8, Pharmacia) and bromophenol blue were added at final concentrations of 0.2 and 0.0125% respectively, to 200 µL of the protein sample solution, prepared as described above, and the supernatant was recovered by centrifugation (15 000 × g, 10 min, r.t.). Dry 11 cm IPG strips (Bio-Rad, Hercules, CA) were rehydrated for 8 h with the sample solution and focused on a Protean IEF Cell (Bio-Rad) for 100 kV/h at a maximum of 8 kV. The focused IPG strips were equilibrated for 10 min with equilibration buffer, and SDS-PAGE (10-20%) was then performed on these strips. The protein spots on the 2-DE gel were blotted to the Immobilon-PSQ membrane by the semi-dry electroblotting method. The blotted membrane was then stained with Direct Blue 71, and the target proteins were digested on the membrane using the Chemical Inkjet Printer, as described above. After printing the matrix solution (10 mg/ mL 2,5-DHB in 30% acetonitrile, 0.1% TFA) on the target proteins, the membrane was placed in a mass spectrometer for analysis. Primary sequence database searches were conducted using the MSDB database with the aid of Mascot software (Matrix Science, MA), which was set at a tolerance of 0.3 Da for both the MS and MS/MS analyses, and at one missed cleavage site as fixed parameters.

Direct MS/MS Analysis of Proteins Blotted on Membranes

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Results and Discussion On-Membrane MS/MS Analysis for BSA Using a Chemical Inkjet Printer. Different amounts (20, 10, 5, 2, 1, 0.5 pmol) of bovine serum albumin (BSA) separated by one-dimensional SDS-PAGE were electroblotted onto a PVDF membrane, and the protein bands were detected by staining with Direct Blue 71. The membrane was then immobilized on an MS target plate with double-sided conductive adhesive tape. To digest the protein bands on the blotted membrane, both PVP40 and trypsin solutions were microdispensed onto the BSA bands using the Chemical Inkjet Printer. After incubating the membrane at 30 °C for 16 h, the matrix solutions were then printed onto each digested position. Figure 1 shows the printed area of the microdispensed reagents. The area was approximately a 10% region of the protein band and therefore it was estimated that 2 pmol to 50 fmol amounts of protein were used for the MS analyses, respectively. Figure 2a shows the spectra of MS analyses on the membrane. The peaks for tryptic fragments were observed with sufficient intensity for identification (S/N > 10) in the MS analyses, even at the 500 fmol BSA band (corresponding to approximately 50 fmol in the digested area). Importantly, the loss of resolution and mass shift caused by a charging effect were not observed here but they were when we analyzed proteins blotted onto a PVDF membrane with a single TOF MS instrument (data not shown). In the case of onmembrane MS analysis, it seems to be effective to combine an ion-trap type MALDI-TOF MS with direct analysis of proteins blotted on nonconductive polymeric membranes. An ion signal at m/z 1639.94, which was observed with high intensity in all MS spectra, was focused, and MS/MS analysis was carried out on the membrane (Figure 2b). The MS/MS spectrum obtained was used to search the MSDB database using the Mascot software and the ion at m/z 1639.94 was identified as a peptide corresponding to KVPQVSTPTLVEVSR (residues 437-451 in the amino acid sequence of BSA). The probability-based Mowse scores were 23, 34, and 62 at 0.5, 1, and 5 pmol BSA, respectively. These scores are high enough for identification of proteins and it seems likely that onmembrane MS/MS analysis would be effective for highthroughput protein identification. For the MS/MS analyses on the membrane, the y- and b-type ions were obtained as the predominant fragment ion signals similar to a typical analysis on a stainless steel plate, and signals consistent with the loss of water and ammonia were also found. The spectrum in Figure 2b for on-membrane MS/MS analysis is similar to MS/MS spectra acquired on metal targets. Our results confirmed the fact that the MS/MS analysis on membranes was effective for identification of faint protein spots of less than 500 fmol. A mass shift was not observed in the MS or MS/MS analyses on membrane using the MALDI QIT-TOF MS and the observed resolution of on-membrane MS spectrum was more than 6000 (specification of resolution is >6000 on metal targets in this mass spectrometer). Spectral quality (mass accuracy and resolution) was on the same level as that on metal targets. On-Membrane MS/MS Analysis for a 2-DE Blotted Membrane of Escherichia coli Cell Extracts Using Chemical Inkjet Printer. To prove the effectiveness of the on-membrane MS/ MS analysis for blotted proteins, Escherichia coli extracts were separated by 2-D PAGE. The blotted membrane was stained, immobilized to the MS target plate, and subjected to direct MS/ MS analysis. The protein spots selected arbitrarily for this experiment are shown in Figure 3. Both the PVP40 and trypsin solutions were microdispensed for the indicated protein spots

Figure 2. On-membrane MS and MS/MS analyses of BSA using the Chemical Inkjet Printer. (a) Spectra of the on-membrane direct MS analysis for different amounts of BSA (20, 10, 5, 2, 1, 0.5 pmol). The peaks denoted with asterisks correspond to peptides produced by the tryptic digestion of BSA. (b) Spectra of onmembrane direct MS/MS analysis of BSA (20, 10, 5, 2, 1, 0.5 pmol). The direct MS/MS analyses were carried out for the peaks selected by a solid line in (a) (precursor ion ) 1639.94 Da).

on the blotted membrane, and the membrane was incubated at 30 °C for 16 h for digestion. After printing the matrix solution, single TOF MS analyses were carried out directly on the membrane, without a desalting step. After the on-membrane MS analyses for the digested positions in the protein spots were conducted, high-intensity peaks were selected as precursor ions for the MS/MS analysis. Table 1 shows the selected precursor ions and their MS/MS ion search results on the basis of the spectrum obtained at each protein spot. The differences between the observed and calculated molecular masses [M+H]+ of the precursor ions were less than 0.1 Da for all of the selected ions. These protein spots were identified with high enough scores for identification in the MS/MS ion search, except for spots 1 and 3. The low scores for spots 1 and 3 might be due to a C-terminal lysine residue of the precursor ions selected in the MS/MS mode. It is known that Arg-terminated tryptic Journal of Proteome Research • Vol. 4, No. 3, 2005 745

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Table 1. Precursor Ions Selected for the MALDI MS/MS Spectrum of Each Spot and Proteins Predicted from the MSDB Databasea spot no.

precursor ion (observed)

precursor ion (calculated)

protein

score

sequence

1 2 3 4 5 6 7 8 9 10

1806.94 1877.96 1990.10 1610.85 1598.76 1610.83 1568.91 1708.91 1545.79 1552.75

1806.90 1877.93 1990.06 1610.79 1598.69 1610.79 1568.86 1708.87 1545.75 1552.75

Phosphate - repressible phosphate - binding protein precursor FKBP-type peptidyl - prolyl cis-trans isomerase fkpA Thiosulfate-binding protein cvsP precursor Glycerol-3-phosphate-binding protein precursor ECOPDXA NID Glycerol-3-phosphate-binding protein precursor Succinyl-CoA synthetase alpha chain 2,5-Diketo-D-gluconic acid reductase A Transcription antitermination protein nusG YbdQ protein

14 44 18 45 40 61 67 36 34 39

RADGSGTSFVFSYLAK LSDQEIEQTLQAFFEAR GLGDVLISFESEVNNIRK GNYEQNLSAGIAAFR FSEEAASWMQEQR GNYEQNLSAGIAAFR GGTTHLGLPVFNTVR SIGVCNFQIHHLQR WYVVQAFSGFEGR FEEHLQHEAQER

a

Both the observed and calculated precursor ions are shown as [M+H]+.

Figure 3. Electroblotted membrane of an Escherichia coli extract separated with 2-DE. Protein spots indicated with arrows were subjected to on-membrane MS and MS/MS analyses.

peptides show better positive ion MALDI response than do Lysterminated tryptic peptides. It seems that Arg-terminated peptides also have more intense MALDI signals than those of Lys-terminated peptides in MS analysis on polymeric membranes. Although the Mascot scores for spots 1 and 3 were not high enough for clear protein identification, the proteins predicted from the MS/MS ion search on these two spots corresponded to the results of the PMF analysis (the probability-based Mowse scores of spots 1 and 3 were 57 and 61 in the PMF analysis, respectively; data not shown). As examples, the typical MS/MS spectra of precursor ions at m/z 1610.83 and 1568.91, observed in spot 6 and 7, respectively, are shown in Figure 4. Fragment ion signals corresponding to both b- and y-type ions were observed with enough intensity for identification in the spectrum, as well as when a normal stainless steel plate was used. With the intensity of the b- and y-type ions in the MS/MS spectrum, amino acid sequencing of the proteins blotted on the membrane could be carried out in combination with software for de novo sequencing. Indeed, we identified BSA by a homology search based on the amino acid sequence, which was determined using de novo sequencing software for on-membrane MS/MS spectra (data not shown). Interestingly, some spots that could not be identified with PMF analysis were identified with MS/MS analysis in this study. In general, there are many cases in which the objective proteins cannot be identified by PMF analysis because of low coverage of the peptide fragments from proteins. However, using a few intense ion peaks, it was possible to identify the objective proteins by on-membrane MS/MS analysis. This suggests the effectiveness of direct MS/MS analysis for high-throughput identification of 746

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Figure 4. Spectra of on-membrane direct MS/MS analysis. Spectra of direct MS/MS analyses of precursor ions at m/z 1610.83 (a) and 1568.91 (b) produced from the MS analysis of spots 6 and 7 in Figure 3, respectively were shown.

proteins blotted on membrane. The use of the ion trap type MALDI MS was thus proven to be especially effective for MS and MS/MS analyses on membranes. In conclusion, the identification of electroblotted proteins on membranes has been successfully carried out using chemical inkjet printing technology in combination with a quadrupole ion trap-time-of-flight mass spectrometry. Microscale direct MS/MS analysis on membranes is expected to be a powerful application for the high-throughput identification of antigens in western blotting and of unknown peptides or proteins in MS imaging.

research articles

Direct MS/MS Analysis of Proteins Blotted on Membranes

Acknowledgment. The authors would like to thank Proteome Systems Ltd. for collaborating in the development of the Chemical Inkjet Printer instrument. The authors would also like to thank Dr. Susumu Tsunasawa for his insightful discussions with us on this project. References (1) Laemmli, U. K. Nature 1970, 227, 680-685. (2) Molloy, M. P.; Herbert, B. R.; Walsh, B. J.; Tyler, M. I.; Traini, M.; Sanchez, J. C.; Hochstrasser, D. F.; Williams, K. L.; Gooley, A. A. Electrophoresis 1998, 19, 837-844. (3) Vinh, J.; Langridge, J. I.; Bre, M. H.; Levilliers, N.; Redeker, V.; Loyaux, D.; Rossier, J. Biochem. 1999, 38, 3133-3139. (4) Wattenberg, A.; Organ, A. J.; Schneider. K.; Tyldesley, R.; Bordoli, R.; Bateman, R. H. J. Am. Soc. Mass Spectrom. 2002, 13, 772783. (5) Guo, Z.; Wagner, C. R.; Hanna, P. E. Chem. Res. Toxicol. 2004, 17, 275-286. (6) Burnette, W. N. Anal. Biochem. 1981, 112, 195-203. (7) Klarskov, K.; Naylor, S.Rapid Commun. Mass Spectrom. 2002, 16, 35-42. (8) Blackledge, J. A.; Alexander, A. J. Anal. Chem. 1995, 67, 843-848.

(9) McComb, M. E.; Oleschuk, R. D.; Manley, D. M.; Donald, L.; Chow, A.; O’Neil, J. D.; Ens, W.; Standing, K. G.; Perreault, H. Rapid Commun. Mass Spectrom. 1997, 11, 1716-1722. (10) Schleuder, D.; Hillenkamp, F.; Strupat, K. Anal. Chem. 1999, 71, 3238-3247. (11) 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, 1, 490-499. (12) Cooley, P.; Hinson, D.; Trost, H. J.; Antohe, B.; Wallace, D. Methods Mol. Biol. 2001, 170, 117-129. (13) Roda, A.; Guardigli, M.; Russo, C.; Pasini, P.; Baraldini, M. Biotechniques 2000, 28, 492-496 (14) Knochenmuss, R. Anal. Chem. 2004, 76, 3179-3184 (15) Chaurand, P.; Stoeckli, M.; Caprioli, R. M. Anal. Chem. 1999, 71, 5263-5270. (16) Pierson, J.; Norris, J. L.; Aerni, H. R.; Svenningsson, P.; Caprioli, R. M.; Andren, P. E. J. Proteome Res. 2004, 3, 289-295. (17) Chaurand, P.; Schwartz, S. A.; Caprioli, R. M. Anal. Chem. 2004, 76, 87A-93A. (18) Krause, E.; Wenschuh, H.; Jungblut, P. R. Anal. Chem. 1999, 71, 4160-4165

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