Anal. Chem. 1994,66,471-477
Poly(viny1idene difluoride) Membranes as the Interface between Laser Desorption Mass Spectrometry, Gel Electrophoresis, and in Situ Proteolysis Martha M. Vestling' and Catherine Fenselau Chemistry and Biochemistry Department, University of Maryland Baltimore County, Baltimore, Maryland 2 1228
A 337-nm laser has been used to desorb and ionize proteins from poly (vinylidene difluoride) membranes. Proteins separated by electrophoresis and electroblotted onto 0.5 X 7 cm membranes are located by scanning laser desoqtion, and their molecular weights are determined By mass spectrometry. Overlapping proteinbands are easily deconvoluted. Conditions have been developed to carry out trypsin proteolysis of protein bands directly on the membrane. The same membrane is then reintroduced into the mass spectrometer, and peptide products are mapped by matrix-assisted laser desorption. Gel electrophoresis is the technique used most widely to separate proteins and nucleic acids in biotechnology, molecular biology, and biochemistry.' It also provides estimates of the molecular weights of proteins, based on their Once separated, proteins are visualized on gels by chemical staining or by autoradiography. Gels currently in use are too fragile for most manipulations, and it is increasingly common for separated proteins to be transferred to more robust polymer membranes (poly(viny1idene difluoride) (PVDF), nylon, nitrocellulose) by the application of an orthogonal electrical field.3 When electroblotting is carried out correctly, the proteins are transferred quantitatively and the relative position of each is retained. Proteins are transferred free of buffers, sodium dodecyl sulfate (SDS),and other contaminants. Aebersold has pointed out4 that the greatest single advantage of electroblotting is that it allows concurrent preparation of large numbers of proteins. Immunochemical detection methods (Western blots) have been developed to characterize proteins supported on membranes. In addition, particular bands can be cut out of PVDF for sequencing or further characterization. In one approach, an excised protein band is dropped into a solution of proteolytic enzyme, and peptide products are recovered from the Thus the use of membranes is well established for handling very small amounts of proteins and is potentially important in bringing proteins to mass spectrometry.
Mass spectrometers interfaced with separation techniques are among the most powerful analytical instruments available, and for a number of years there has been considerable interest in the use of membranes as an effective way to interface gel electrophoresis to mass spectrometry (GE/MS). Plasma des0rption,7-~UV laser desorption,6J0 and IR laser desorptionll have been shown to desorb peptides and proteins from nitrocellulose or PVDF membranes. Mass spectrometric anatysis should be an outstanding complement to gel electrophoresis, because it can determine molecular weights with accuracies as high as f0.176,independent of tertiary structure, hydrophobicity, glycosylation, and other features that compromise electrophoretic mobility. Among the several ionization techniques currently applicable to biopolymers, laser desorption is most appropriate for analysis of proteins electroblotted onto membranes, because it was designed to desorb samples from planar stages12 and because matrixassisted laser desorptionionization (MALDI) has been shown13 to provide molecular weights above 100 000. Only a few minutes are required for each molecular weight determination by laser desorption. In the present paper we report techniques by which scanning laser desorption can be used to record electrophoretograms from PVDF strips, to determine molecular weights of electroblotted proteins, and to map tryptic peptides produced by proteolytic reactions carried out directly on the membrane.
EXPERI MENTAL SECTION Materials. Horse heart cyclochrome c, lysozyme, bovine trypsin, and electrophoresissupplieswere obtained from Sigma Chemical Co. (St. Louis, MO). Bovine adenosine deaminase was from Boehringer Mannheim (Indianapolis, IN). BNPS-
(7) Jonsson, G. P.; Hedin, A. B.; Hakansson, P. L.; Sundquist, B. U.;Save, B. J.; Nielson, P. F.; Rocpstorff, P.; Johansson, K. E.; Kamensky, I.; Lindbcrg, M. S. Anal. Chem. 1986, 58, 1084-1087. (8) Woods, A. S.; Cotter, R. J.; Yoshioka, M.; Bullesbach, E.; Schwabc, C. In?. J . Mass Spectrom. Ion Processes 1991, 1 1 1 , 77-88. (9) Klarskov, K.; Rocpstorff, P. Mol. Mass Spectrom. 1993, 22, 433440. (10) Karas, M.; Bahr, U.;Ingendoh, A. I.; Nordhoff, E.; Stahl, B.; Strupt, K.; Hillenkamp, F. Anal. Chim. Acta 1990, 241, 175-185. Hefta, S.A.; Stahl, D. C.; Mahrcnholz, A. M.; Martino, P. A.; Rutherfurd, S. M.; Lee, T. D. (1) Hamcs, B. D.; Rickwood, D. Gel Electrophoresis of Proteins: A Practical Proceedings of the 39th Annual Meeting of the American Society for Mass Approach, 2nd ed.;IRL Press: New York, 1990. Rickwood, D., Hames, B. Spectrometry, Nashville, TN, May 19-24. 1991; pp 1416-1417. Chcvrier, D., Eds. Gel Electrophoresis of Nucleic Acids: A Practical Approach, 2nd M. R.; Cotter, R. J. Proceedings of the 39th Annual Meeting of the American 4.; IRL Press: New York, 1990. Society for Mass Spectrometry, Nashville, TN,May 19-24, 1991; pp 933(2) Matsudaira, P. Ed.A Practical Guide to Protein and Peptide PuriJcation for 934. Mock, K. K.; Sutton, C. W.; Cottrell, J. S. Rapid Commun. Mass Microsequencing, 2nd ed.;Academic Prcss: New York, 1993. Spectrom. 1992, 6, 233-238. (3) Acbcrsold, R. M.;Teplow, D. B.; Hood, L. E.; Kent,%8 . J . Biol. Chem. 1986, 261, 42294238. (1 1) Eckcrskorn, C.; Strupat, K.; Karas, M.; Hillenkamp, F.; Lottspcich, F. Electrophoresis 1992, 13, 664-665. (4) Aebersold, R. M.; Lcavitt, J.; Saavedra, R. A.; Hood, L. E.; Kent, S. B. Proc. Natl. Acad. Sei. U.S.A. 1987, 84, 6970-6974. (12) Posthumus, M. A.; Kistemaker,P. G.;Meuzclaar, H. L.; ten Neuverde Brauw, M. C. Anal. Chem. 1978,50,985-991. (5) LeGendre, N . Biotechniques 1990, 9, 788-805. (13) Hillcnkamp, F.; Karas, M.; Btavis, R. C.; Chait, B. T. Anal. Chem. 1991,63, (6) Hcnzcl, W. J.;Billeci,T.M.;Stults,J.T.;Wong,S.C.;Grimley,C.;Watanabe, C. Proc. Natl. Acad. Sei. U.S.A. 1993, 90, 5011-5015. 1193-1203.
0003-270019410386-0471$04.5010 0 1994 American Chemical Society
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skatole was purchased from Pierce Chemical Co. (Rockford, IL), while the matrices a-cyano-4-hydroxycinnamic acid and 2-aza-2-thiothymine were purchased from Aldrich Chemical Co. (Milwaukee, WI). Peptides from calibration were obtained from Sigma and Bachem (Torrance, CA). The PVDF membranes used were Westran PVDF-hydrophobic with 0.45-pm pores (Schleicher and Schuell, Keene, NH) or Immobilon-P with 0.45-pm pores (Millipore, Bedford, MA). Gel Electrophoresis. A Mini-Protean I1 electrophoresis apparatus (Bio Rad Laboratories, Richmond, CA) was used. The solutions and minigels for Tris/Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were prepared according to Schagger and von Jagow14and run at 150 V for 70-90 min. Electroblotting. Electroblotting was carried out with Towbin’s buffer (tris/glycine, pH 8.3)15in a Mini-Trans-Blot electrophoretic transfer cell (Bio Rad Laboratories) for 50 min at 300 mA. The transfer was monitored by the use of bands of prestained standards. Laser Desorption Mass Spectrometry. Matrix-assisted laser desorption mass spectra were obtained using a Kratos Kompact MALDI I11 mass spectrometer (Manchester, UK), which has a 337-nm nitrogen laser. The analyzer was used in the linear mode, at accelerating voltages of 20 kV. The laser can be fired at any spot or fired continuously along the 7-cm length of the sample holder. Laser power was varied through the middle third of the range. Spectra were calibrated by use of a matrix ion and one or more standard proteins applied to the membrane. For example, with the matrix a-cyano-4-hydroxycinnamic acid, the peak at m/z 172.16 was used, and with the matrix 6-aza2-thiothymine, the peakat m/z 144.17 wasused. Neurotensin (MH+ 1673.9), bovineinsulin (MH+ 5732.6), ubiquitin (MH+ 8565.9), cytochrome c (equine) (MH+ 12361), and lysozyme (MH+ 14307) make good standards when dissolved at 20 pM concentrations in 20 mM NH4HCO3. Calibrations were carried out using the Kratos Kompact calibration program or using TOFWARE (ILYS Software, Pittsburgh, PA). To test the mass accuracy of protein desorbed from PVDF, a mixture of cytochrome c and lysozyme was applied to the PVDF and covered with the matrix a-cyano-4-hydroxycinnamic acid. When eight measurements were acquired from eight spots with the same instrument conditions, the data were calibratedwith thetwolyzosymepeaks(m/z 14307 and7154) and the matrix peak at m/z 172, and the value for cytochrome c was recorded. The parallel experiment was carried out with the metal sample holder. In the present study, two matrices wereused. Thea-cyano4-hydroxycinnamic acid matrix16was applied in 1:1 methanol/ toluene solutions (100 mM) at an estimated matrix:sample molar ratio of 1OOO:l and, in some cases, reapplied to the dried or partially dried membrane to bring the matrixxample ratio as high as 50 0OO:l in order to maximize the sample signal. The matrix 6-aza-2-thiothymine was successively applied in methanol or acetone (100 mM) through the same range of matrixsample ratios. Care was taken to avoid the (14) Schaggcr, H.; von Jagon, G . Anal. Biochem. 1987, 166, 368-379. (15) Matsudaira, P. J . B i d . Chem. 1987, 262, 10035-10038. (16) Juhasz, P.; Costello, C. E. J . Am. Soc. Mass Specrrom. 1992, 3, 785-796.
472
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n
Shot Number
,
Figure 1. Bands of cytochrome c on a PVDF strip, profiled by ion currentsproduced by scanning laser desorption(upper)and chemically stained after mass spectrometry (lower). Reprinted with permission from International Scientific Communications, copyright 1993.27
use of acetonitrile/trifluoroacetic acid or other combinations of acid with organic solvents, because they caused protein bands to spread on the membrane. Usually 5-10 scans were averaged to produce a spectrum. Sensitivity was studied using a 60 X 0.7 mm piece of Immobilon-P taped to a Kratos slide. Three different concentrations of cytochrome c in 20 mM NH4HCO3 were prepared (53.8, 5.38, and 0.538 @ andI) put on ice. The application of protein at each spot was accomplished by successively applying 1 pL of methanol to wet the membrane, 1 p L of 20 mM NHdHCO3, and 1 pL of protein solution. After the protein was applied and before the PVDF had dried, the PVDF on the slide was rinsed extensively with water. It was then allowed to dry. The matrix, 100 mM a-cyano-4hydroxycinnamic acid, was dissolved in 5050 methanol/ toluene and 1 p L was applied to each spot. After spectra were recorded, the PVDF membrane was removed from the instrument and another 1 p L of matrix solution was applied to each spot. Treatment with 2 pL of matrix gave the stronger signals.
Scanning Laser Desorption. The sample holder was automatically advanced so that up to 2000 laser desorption spectra could be collected across the 7-cm length. A 0.5 X 7 cm piece of PVDF was taped to the slide, containing one lane of electroblotted bands. Electrophoretograms were constructed by plotting or pooling the ion current from the most abundant peak within a selected range in each scan. The mass range 3000-15 000 was used in this study. Spectra were pooled in groups of 10. In the experiment summarized in Figure 1, 1.0, 5.0, and 10 pL of 80 pM cytochrome c in Towbin’s buffer15 were applied in duplicate on a PVDF strip. A 2-pL sample of a 100 mM solution of a-cyano-4-hydroxycinnamic acid in 1:1 methanol/toluene was applied to each dry protein band.
A 1-nmol sample of bovine adenosine deaminase was reacted with BNPS-skatole,17 and the product mixture was separated by gel electrophoresisand electroblotted onto PVDF. A 60-pL solution of a-cyano-4-hydroxycinnamic acid (100 mM) in 1:l methanol/toluene was applied across the strip. Trypsin Digestion on the Membrane. Cleavage of cytochrome c on a membrane recovered from the mass spectrometer was accomplishedby wetting the protein spot on the membrane with 1 pL of methanol and adding 4 pL of 50 mM NH4HC03, 2 pL of trypsin (44 pM) in 0.01% TFA, and then 2 pL of 50 mM NH4HCO3. The reaction was allowed to proceed -20 min (until the membrane looked dry, changing from grey to white), after which more matrix was applied (1 pL of 100 mM 6-aza-2-thiothymine in methanol) and the sample was reintroduced into the spectrometer. To compare on-membrane digestion with test tube digestion, five cytochrome c spots were digested as above. The trypsin products were removed from the membrane in 40 pL of 0.1% TFA/water and 40 pL of acetonitrile. The extract was then subjected to HPLC analysis. A parallel digestion was carried out in 50 mM NH4HCO3, with a weight-to-weight ratio of 50:l cytochrome cltrypsin and subjected to HPLC analysis. As each HPLC run required only 6 min, the test tube digestion was monitored for 2 h. The products from both cytochrome c digestions were mapped by HPLC using a POROS 11 R / H (2.1 X 30 mm) column (PerSeptive Biosystems, Cambridge, MA). The flow rate was 1 mL/min while the detection was at 215 nm. The gradient was (A = 0.1% TFA in water, B = 0.08% TFA in acetonitrile) 0.5 min 5% B, 4.5 min to 35% B, 0.5 min to 60% B, and 0.5 min 60% B. On-membrane digestion of lysozyme was accomplished by two different methods. One digestion was performed with native lysozymewhile the second was performed on denatured lysozyme. After a mass spectrometric analysis of intact native lysozyme on PVDF using 6-aza-2-thiothymine as the matrix, the membrane was recovered from the mass spectrometer and wet with methanol. A 5-pL aliquot of buffer solution (50 mM sodium phosphate pH 7.8) was placed over the protein, followed by 1 pL of trypsin (44 pM) in 0.1 mM CaC12. After -20 min, the membrane was rinsed with water and 1 pL of 100 mM 6-aza-2-thiothymine in acetone was applied. The second method for on-membrane reduction and alkylation of lysozyme started with a 9 X 60 mm piece of PVDF. The PVDF was placed on a piece of No. 1 Whatman filter paper. The edge of the PVDF was marked with a pencil, so that the reagents could be applied to the same spots as the protein. Each location was then wet with 1 pL of methanol followed immediately by 1 pL of 10 mM 3-(cyclohexy1amino)1-propanesulfonic acid (CAPS) at pH 11 and 10% methanol. A 0.5-pL aliquot on a 1.0-pL aliquot of a 291 pM lysozyme solution (also in 10 mM CAPS buffer) was applied to the wet spot. Next, 1 pL of dithiothreitol(l0 pg/pL of CAPS buffer) was added. After 4 min, 1 pL of iodoacetamide (100 pg/pL of CAPS buffer) was added. When the PVDF had dried, 30 min; it was rinsed extensively with water to remove the reagents. For the tryptic digestion, the PVDF was taped at each end to a Kratos slide. Each spot was set with 2 pL of
methanol, followed immediately by 4 pL of 50 mM NH4HC03, 2 pL of trypsin (44 pM), and 2 pL of 50 mM NH4HC03. After 30 min, the spot of PVDF had dried enough to go from grey to white, and 2 pL of 50 mM 6-aza-2thiothymine in methanol was added to each spot. Trpytic digestion of the electroblotted protein mixture derived from adenosine deamidase was carried out after first rewetting the membrane with methanol and rinsing with water. A 10-pL aliquot of 50 mM NH4HCO3 was applied over the protein bands. Then 1 pL of trypsin in 0.1 mM CaC12 solution was added and the resultant mixture stirred. After the PVDF looked dry, -20 min, the membrane was rinsed with water. A 4-pL aliquot of a-cyano-4-hydroxycinnamic acid was applied, and the strip was scanned by laser desorption. Subsequently another layer of a-cyano-Chydroxycinnamic acid was applied, and the strip was reexamined. The MacBioSpec computer program (Sciex, Toronto, ON) was used to predict tryptic and other peptides.
-
RESULTS AND DISCUSSION In order to evaluate the ability of scanning laser desorption to detect protein bands on a membrane and to record the electropherogram with fidelity, standard solutions of cytochrome c were applied to a 0.5 X 7 cm strip and detected by direct visualization, by scanning laser desorption and by staining with Coomassie blue. Figure 1 shows the ion profile recorded from bands of cytochrome c applied in duplicate in three different amounts. Comparison of the ion profile with the bands visualized by staining (Figure 1) indicates that the resolution of scanning laser desorption is sufficient to detect protein bands and to record electrophoretic mobilities. When the membrane was removed from the mass spectrometer, no laser burn could be detected, indicating that very little of the sample is consumed by the desorption process. It can be seen in Figure 1 that the duplicate applications provide ion profiles with similar areas. However, the different amounts of protein (80, 400, and 800 pmol) applied to the membrane are not quantitatively reflected in the relative areas of the ion profiles. This scanning laser desorption experiment was carried out using a constant amount of matrix and, consequently, variable matrix to sample ratios. Others have reported that desorption efficiency is very sensitive to this ratio.'" In another experiment, scanning laser desorption was applied to a mixture of unknown proteins that had been separated by gel electrophoresis and electroblotted onto a PVDF strip. These proteins were generated by cleavage of 1 nmol of bovine adenosine deaminase at its tryptophan residues as part of a study of the sequence of that enzyme.19 The electropherogram is shown in Figure 2. Here, as in GC/ MS, the information obtained is three dimensional, and scanned mass spectra can be plotted for each protein band detected. Such MALDI spectra are displayed for four protein bands in Figure 2 and provide molecular weights for these new proteins. Masses have been assigned only to those peaks that occur reproducibily in several lanes on the gel. The
(17) Crimmins, D. L.; McCourt, D. W.;Thoma, R. S.;Scott, M. G.; Macke, K.; Schwartz, B. D. Anal. Biochem. 1990, 187, 27-38.
(18) Billcci, T. M.; Stults, J. T. Anal. Chem. 1993. 65, 1709-1716. (19) Kelly, M. A.; Hua, S.;Fcnselau, C. C. Proceedings of the Forty-first Annual Meeting of the American Society for Mass Spectrometry, San Francisco, 1993; pp 355a-35Sb.
Analytical Chemistty, Vol. 60, No. 4, February 15, 1994
473
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spectrum plotted by averaging laser shots 35 1-355 clearly reveals the presence of two proteins, which were found to overlap 80%. The interfaced system provides the advantages of deconvolution, and of more precise mass determinations relative to stand-alone gel electrophoresis. The combined system is also more powerful than laser desorption applied directly to the unseparated digest mixture, because the possibility of suppression that occurs in MALDI analysis of mixtures20 is reduced when the components are separated. The experiment summarized in Figure 2 indicates that analysis of mixtures by interfaced gel electrophoresis mass spectrometry can be carried out comfortably at the 1-nmol level. In the present work, which reports on the development of techniques to permit laser desorption of proteins of PVDF membranes, the limit of detection for desorption by the 337nm laser of cytochrome c from a PVDF membrane with 0.45pm pores was found to be reproduceably below 5 pmol. A typical spectrum is shown in Figure 3, for which a-cyano4-hydroxycinnamic acid was used as matrix at a matrix:sample molar ratio of 40 000. We have found that with membranes, as with metal surfaces, sensitivity is influenced by both the ratiola and the nature of the matrix.*O Membrane pore size and the nature of ionic groups on the surface have also been found to affect sensitivity. The molecular weight assignments for the unknown proteins in the digest map in Figure 2 depend on the accuracy and
Table 1. Molecular Welght of Cytochrome E calcd metal support membrane support
(20) Vestling, M. M. In Time-of-Night Mass Specfrometry; Cotter, R . J., Ed.; American Chemical Society: Washington, DC, in press.
(21) Daddona, P. E.; Shewach, D. S.;Kelley, W. N.; Argos, P.; Markham, A. F.; Orkin, S. H . J . B i d . Chem. 1984, 259, 12101-12106.
-
474
Analyticai Chemistry, Voi. 66, No. 4, February 15, 1994
12 360 a
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precision of the technique. An independent assessment of mass and precision is presented in Table 1, where it can be seen that they are comparable with and without a membrane. Both sets of numbers are within the manufacturer’s specifications for the Kompact I11 used in the linear mode, f0.1% Figure 2 presents a map of the regions in bovine adenosine deaminase that are separated by tryptophan residues.” Our strategy for mapping and sequencing this protein involves comparison with the sequence already reported for human adenosine deaminase.2’ Molecular weights calculated for BNPS-skatole products from the human protein are 13 044, 11 465, 10 417,4869, and 885. The four heaviest masses in Figure 2 are similar enough to suggest that tryptophan residues occur in about the same positions in the two proteins. (We interpret the origin of the peptide of mass 3590 as a nonspecific cleavage.) The map in Figure 2 also reveals that none of the pairs of products has the identical molecular weights that would be required by identical sequences. Finally, this map
a.
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indicates that tryptophan-based cleavage will provide several large proteins that require further cleavage to form proteins suitable for sequencing. Our second objective was to develop approaches whereby an electroblotted protein could have its molecular weight determined, be recovered from the mass spectrometer, be treated enzymatically or chemically, and be reintroduced into the mass spectrometer for product analysis, still on theoriginal membrane. It is anticipated that sample losses will be minimized by eliminating sample handling and transfers. Several laboratories have already demonstrated this approach8*22 with plasma desorption. In the present study, special considerations included the effect of the matrix and the dry condition of the membrane from the initial MALDI analysis on the enzyme reaction and the effect of buffers used in the enzyme reaction on the MALDI product analysis. The details of strategic accommodation will vary according to the matrix and the proteolytic, glycolytic, or other enzyme. We have initially studied proteolysis with trypsin, since that is the most widely used proteolytic enzyme. Figure 4 shows spectra of (a) tryptic peptides produced when solutions of buffer and trypsin were applied to lysozyme on the membrane and (b) peptides produced when disulfide bonds in lysozyme were reduced and alkylated on the membrane and the resulting denatured protein was incubated with trypsin on the membrane (see Experimental Section). After a 20min incubation, the membranes were rinsed with water to (22) Chait, B. T.; Chaudhary, T.; Field, F. H. In Merhods in Protein Sequence Atiulysis; Walsh, K.A., Ed.; Humana Press: Clifton, NJ, 1987; pp 483-492. Roepstorff, P. Merhods Enzymol. 1990, 193, 432-441.
MasdCharge
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Figure 4. (a) Mass spectrum of peptkles produced by incubation of trypsinand lysozyme on the membrane. (b) Mass spectrum of peptkles produced by reduction and alkylatlon of lysozyme on the membrane, followed by tryptic digestion on the membrane. (c) Mass spectrum of peptides produced as in (b) using twice as much lysozyme on the membrane. Numbers in brackets are the sequence positlons of the first and last amino acids in the tryptic peptides.
remove salts. The matrix was added and the spectra in Figure 3 were obtained. Peptides are detected or mapped that represent 21% of the amino acid sequence of lysozyme2' in the spectrum in Figure 4a. Not unexpectedly, proteolysis is more extensive with the denatured sample, and -36% of the amino acid sequence is mapped in the spectrum in Figure 4b. In another experiment, twice as much lysozyme was applied to the membrane and all other conditions were held constant. In this case, 77% of the amino acid sequence was mapped (Figure 4c). In a control reaction, peaks due to contaminants
-
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(23) Canfield, R. E. J . B i d . Chem. 1963, 238, 2698-2707.
Analytical Chemistry, Vol. 66,No. 4, February 15, 1994
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in the commercial sample of bovine trypsin or autolysis peptides did not coincide with any of the peaks annotated as peptides (with the sequence number of the first and last amino acid) in Figures 4, 5 and 7. Tryptic peptides are mapped in Figure 5 from (a) a digestion of cytochrome c carried out in solution and applied to a PVDF membrane strip for analysis and (b) a digestion carried out on the membrane and analyzed directly. Tryptic peptides are assigned in the figure, indicated by the sequence number of the first and last amino acid. In both spectra enough tryptic peptides are mapped to identify the sequence in protein database^;^^^^ however, the two maps are noticeably different. These differences may be ascribed to different accessibilities of cleavage sites in solution and on the membrane. The occurrence of different product mixtures is further supported by a comparison of high-pressure liquid chromatograms of both sets of products, presented in Figure 6 . This strategy for trypic digestion was applied to the unknown electroblotted protein bands from bovine adenosine deaminase detected with scanning laser desorption in Figure 2. Each band was digested and the membrane was reintroduced into the mass spectrometer. The tryptic peptide map produced from the band containing two unresolved proteins (laser shots 351-355) is shown in Figure 7. As in Figures 4 and 5, only a portion of the product mixture is characterized; (24) Mann, M.; Hojrup, P.; Roepstorff, P. B i d . Mass Spectrom. 1993, 22, 338345. Pappin, D. J.; Hojrup, P.; Bleasby, A. J. Curr. B i d . 1993, 3, 327-332.
476
AnalWalChemistry, Vol. 66,No. 4, February 15, 1994
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i
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Flgure 5. (a) Mass spectrum of peptides formed by the reaction of trvnain ",." and rvtnehmma ' P in enhitinn
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Flgure 6. (a) HPLC of peptides formed by the reaction of trypsin and cytochrome c in solution. (b) HPLC of peptides formed by the reaction of trypsin and cytochrome c on a PVDF membrane. I
l l
,
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however, enough peptide molecular weights are provided to search sequence databases. The map confirms that we can prepare peptides small enough for sequencing by these two proteolytic steps, and the molecular weights in the map provide end points and cross-checks for sequencing those peptides. For proteolytic cleavage with trypsin, the presence of a matrix such as a-cyano-4-hydroxycinnamic acid did not interfere with the reaction. It was necessary, however, to maintain the pH in the range 7-9, optimal for enzyme activity. It was important to rewet the membrane recovered from the vacuum system in order to add the enzyme, and the sequence
of methanol and then water was found to be effective. Involatile salts introduced in the enzyme reaction were preferentially removed from the dry membrane with water, so that they would not interfere with laser desorption. The low-picomole range of the protein bands analyzed in the present study is well within the binding capacity of the PVDF membraneZSand is comparable with the 1-pg detection capability reported26~~' for the widely used Coomassie blue stain. The weak link in the strategy proposed may well be the electroblotting process, which is not reproducibly quantitative as presently practiced. It has been pointed out by Roepstorffg that the exquisite sensitivity of mass spectrometry can provide successful analyses, even in cases where as little as 1%of the sample applied to the original gel reaches the mass spectrometer. Matrix selection and matrix:sample ratio will also influence sensitivity when MALDI is used. Although not encompassed by the objectives of the present report, we have desorbed heavier proteins such as trypsin and adenosine (25) The Immobulon-WTransfer MembraneData Sheet, Millipre Corp., Bedford, MA. (26) Mini-Protean I1 Ready Gels Instruction Manual, Bio-Rad Laboratories, Melville, NY. (27) Fensclau, C.; Vestling, M. Am. Lub. 1993, 25 (16). 74.
deaminase from PVDF, as well as proteins detected with chemical staining. This study confirms an earlier report" that membranes such as PVDF can be used to bridge the capabilities of gel electrophoresis and laser desorption mass spectrometry. The three-dimensional capabilities of scanning laser mass spectrometry are shown to provide both the physical locations of protein bands following gel electrophoresis and the molecular weights of proteins in each band. Proteolysis directly on a PVDF gel is combined for the first time with MALDI to provide partial maps of the peptides produced, which appear to be sufficient to identify proteins from sequence databases. Conditions are being developed to support activities of other enzymes on the membrane, and the interfaced GE/MS system is being applied to on-going structural studies.
ACKNOWLEDGMENT This work was supported by the National Science Foundation and Sterling Winthrop Laboratories. Received for review August 18, 1993. 1993." 0
Accepted October 8,
Abstract published in Aduance ACS Abstracts, December I , 1993.
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