of the diffuse spots corresponds with the distance between the t and t,, bands at the moment of quasi-stationary melting.
we thank professo,.,y.L. pioegilf o r helpful suggestions in the coL‘rse of this and Dr’ A ’ V1ugforproviding nlye‘omaparaproteins. Received August 1992
References [ I ] Creighton.T.E., J. Mol. 5 i o l . 1980, 137, 61-80. [2] Ljunggren, H.G., Stam, N.J., Ohlen. C., Neeljes, J.J.. Hoglund, P., Heemels, M.T., Bastin. J.. Schumacher,T.M.N.,Townsend. A , . Kirre. K . and Ploegh, H.L.. Riarure 1990, 346, 476-480. [3] Deres, K,. Schumacher, T, N,M , , Wiesmiiller, K,. Stevanovic, S.. Greiner, G., Jung, G . and Ploegh. H. L.. Eiir. J . Inrmunol. 1992. 22, 1603-1608. [4] Cerundolo, V.,Elliot, I-.,Elvin, J., Bastin. J., Ranmensee, H . and Townsend, A,. Eur. J . Immunol. 1991,21, 2069-207s.
Christoph Eckerskorn’ Kerstin Strupat’ Michael Karas2 Franz Hillenkamp’ Friedrich Lottspeich’
Mass spectrometric analysis of blotted proteins after gel electrophoretic separation by matrix-assisted laser desorptiodionization
‘Max-Planck Institut fur Biochemie, Martinsried ’Institut fur Medizinische Physik und Biophysik, Ziniversitat Miinster
The molecular masses of electroblotted proteins were determined with a time-offlight mass spectrometer by matrix-assisted laser desorption/ionization directly from blot membranes. Therefore standard proteins, separated by polyacrylamide gel electrophoresis, were electroblotted onto polyvinylidene difluoride or polyamide membranes by standard procedures. Pieces of membrane containing the protein of interest were soaked in matrix solution and analyzed in the mass spectrometer.
Determination of the amino acid sequence and structural elucidation of proteins are essential prerequisites to understand protein function at a molecular level. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and high-resolution two-dimensional polyacrylamide electrophoresis (2-DE) are commonly used as an analytical approach to resolve and detect most of the numerous protein species of an organism. Currently, the most rapid and efficient method for isolating small quantities of proteins for protein chemical analysis is the electrotransfer of the electrophoretically separated proteins onto a suitable membrane. The immobilized proteins can then be directly N-terminally sequenced or subjected to amino acid composition analysis [1,2]. Many aspects of protein structural characterization, such as the accurate measurement of the molecular mass of intact proteins and the identification of post-translational modifications (blocked N-terminus, phosphorylation, glycosylation, etc.) are not readily amenable to standard methods of amino acid sequence analysis and amino acid composition analysis. Mass spectrometry is rapidly becoming the method of choice to help solve these structural problems [3]. Recently, two major developments, matrix-assisted laser desorptiodionization (MALDI) and electrospray ion-
Correspondence: Dr. Chrisloph Eckerskorn, Max-Planck Institut fur Biochemie, DW-8033 Martinsried, Germany
Abbreviations: IR. infra red, MALDI, matrix-assisted laser desorplion/ ionization. MS, mass spectrometry; PVDF, polyvinylidene difluoride
0 VCH Vcrlagsgescllschaft m b H , U-6940
Weinheim, 1992
ization have overcome the difficulties in the desorption and ionization oflarge and labile biomolecules such as proteins. Whereas for electrospray mass spectrometry (MS) the proteins have to be solubilized in a liquid mobile phase, for MALDI-MS the proteins were dried before laser desorption in a suitable form onto solid surfaces. Therefore MALDI-MS is the most promising technique for the direct determination of molecular masses of electroblotted proteins immobilized on membranes. I n the following, first spectra of electroblotted proteins obtained by MALDI-MS are presented
In standard preparations for MALDI-MS a dilute protein solution (lO-’-lO-x mol/L) is mixed with a concentrated matrix solution (typically 0.1 mol/L). Small aliquots (typically 0.1-1 pL) are placed onto a metal substrate and dried in a stream of air before being transferred into the vacuum of the mass spectrometer. Either pulsed lasers emitting in the UV range or in the IR range are used to induce desorption and ionization of small volumes of the analyte-matrix mixture. The alleged functions of the matrix, usually a small organic compound, are to absorb the energy from the laser pulse, to embed and isolate the proteins and to assist in their ionization. Best results are obtained if the proteins are homogeneously distributed and fully embedded in an environment of matrix molecules [41. Generally, singleshot spectra exhibit the molecular ion signal; 10 to 30 spectra are usually accumulated to improve the signal-to-noise ratio. All spectra shown are positive ion spectra. Though the laser beam can easily be focused to the area of interest on the membrane, the strong hydrophobic interaction of protein and membrane has to be overcome by suitable pre0173-0535/92/0910~0hhl$3 50+.25/0
Elerlrophoresis
Mass spectrome:try of' blotted proteins by malrix-assisted laser desorptionfionization
1992, 13. 664-665
r
paration techniques to allow for a successful MALDI-measurement. In a first set of experiments standard proteins (low-molecular-mass protein standard mixture, BioRad) were electrophoretically separated and electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Fluorotrans, Pall) as described [5]. Only the left and the right lanes of the PVDF membrane were stained for localization of the proteins with Coomassie Blue (Serva Blue R, Serva). Small areas, containing the unstained proteins, were cut out of the membrane, incubated in a solution of succinic acid as matrix and introduced into the mass spectrometer. The blotted samples were analyzed in a TOF instrument equipped with a first order reflectron. Desorbed ions were accelerated to a potential of 12 kV and detected by a secondary electron multiplier (EM1 9643, Electron Tube LTD.) after post accelation to 10 to 15 keV. A mechanically Q-switched Er-YAG laser 1-2-3 (Schwartz Electro-Optics) with a wavelength of 2.94 pm and 150 ns pulse width was used for these experiments. Figure 1 shows as an example the MALDI mass spectrum of bovine serum albumin. Compared to spectra obtained from sample prepared by standard techniques the peaks are somewhat broadened, which may result in a somewhat lower accuracy of mass determination. The distribution of single and multiple charged ions and oligomers, however, is comparable to that in standard preparations.
20000
50000
100000
w* Figure 1. Infrared-MALDI mass spectrum of bovine serum albumin blotted onto a PVDF membrane.A piece of the membrane was incubated in a solution of succinic acid as matrix. Sum of 10 single spectra. Laserwavelength: 2.94 vm.
M+
75 loo 50 25
665
2M+
.
10000
40000
Figure 3. Infrared-MALDI mass spectrum of bovine carbonic anhydrase blotted onto polyamide membrane after staining with India ink. A piece ofthe membrane was incubated in a solution ofmalic acid as matrix. Sum of 10 single spectra. Laser wavelength: 2.94 pm.
The sensitive staining of proteins should not interfere with determination of molecular masses. For proteins stained with colloidal metals like gold (Aurodye, Janssen) or India Ink [6],relatively sharp peaks with a shoulder to higher masses were obtained. The peak shoulder indicates that a part of the protein molecules absorb staining material. As an example, in Fig. 2 a MALDI-MS spectrum is shown for soy bean trypsin inhibitor electroblotted onto Immobilon PSQ and stained with colloidal gold. Nevertheless, the expected mass deduced from the known sequence is obtained from the sharp component of the signal. Even better results could be obtained with India ink. An example is given in Fig. 3 for carbonic anhydrase electroblotted onto a polyamide membrane (Sartolon, Sartorius). In the case of Coomassie Blue as staining material and lactic acid as matrix, the protein peaks obtained were shifted to higher masses. This mass shift might be due to strong interaction between Coomassie Blue molecules and protein, which are obviously retained during the desorptionlionization process. These results show that it is possible, in principle, to desorb electroblotted proteins from hydrophobic membranes. It still remains to be shown whether incubation in the matrix solution mainly induces a detachment of the proteins from the membrane, overcoming the strong hydrophobic interaction, or if the only partial embedding by the matrix suffices to desorb the still bound intact protein molecules. Currently, the level of mass determination accuracy achieved is still considerably lower than in standard preparation (> 0.25 O/o as compared to 0.01 O/o). Ongoing work focuses on an optimized combination of membrane, matrix and staining material. Received August 11. 1992
References I
I
I
10000
I
I
40000
Wz Figure 2. Infrared-MALDI mass spectrum of soybean trypsin inhibitor blotted onto PVDF membrane after staining with colloidal gold. A piece ofthe membrane was incubated in a solution of malic acid as matrix. Sum of 10 single spectra. Laser wavelength: 2.94 vm.
[I] Eckerskorn, C. and Lottspeich, F., Electrophoresis 1990, 11,554-561. [2] Aebersold, R., Adv. Electrophoresis 199I, 4, 81-168. [3] McCloskey, J.A., Methods Enzymol.: Muss spectometry Academic Press, Orlando, FL 1990, Vol. 193. [4] Hillenkamp, F., Chait, B.T., Beavis, R.C. and Karas, M., Anal. Chem. 1991, 63, 1193A-1203A. [ S ] Eckerskorn, C., Mewes, W., Goretzki,H.W. and Lottspeich, F., Euro. J. Biochem. 1988, 176, 509-519. [6] Hancock, K. and Tsang, V.C., Anal. Biochem. 1983, 133,157-162.
we thank professo,.,y.L. pioegilf o r helpful suggestions in the coL‘rse of this and Dr’ A ’ V1ugforproviding nlye‘omaparaproteins. Received August 1992
References [ I ] Creighton.T.E., J. Mol. 5 i o l . 1980, 137, 61-80. [2] Ljunggren, H.G., Stam, N.J., Ohlen. C., Neeljes, J.J.. Hoglund, P., Heemels, M.T., Bastin. J.. Schumacher,T.M.N.,Townsend. A , . Kirre. K . and Ploegh, H.L.. Riarure 1990, 346, 476-480. [3] Deres, K,. Schumacher, T, N,M , , Wiesmiiller, K,. Stevanovic, S.. Greiner, G., Jung, G . and Ploegh. H. L.. Eiir. J . Inrmunol. 1992. 22, 1603-1608. [4] Cerundolo, V.,Elliot, I-.,Elvin, J., Bastin. J., Ranmensee, H . and Townsend, A,. Eur. J . Immunol. 1991,21, 2069-207s.
Christoph Eckerskorn’ Kerstin Strupat’ Michael Karas2 Franz Hillenkamp’ Friedrich Lottspeich’
Mass spectrometric analysis of blotted proteins after gel electrophoretic separation by matrix-assisted laser desorptiodionization
‘Max-Planck Institut fur Biochemie, Martinsried ’Institut fur Medizinische Physik und Biophysik, Ziniversitat Miinster
The molecular masses of electroblotted proteins were determined with a time-offlight mass spectrometer by matrix-assisted laser desorption/ionization directly from blot membranes. Therefore standard proteins, separated by polyacrylamide gel electrophoresis, were electroblotted onto polyvinylidene difluoride or polyamide membranes by standard procedures. Pieces of membrane containing the protein of interest were soaked in matrix solution and analyzed in the mass spectrometer.
Determination of the amino acid sequence and structural elucidation of proteins are essential prerequisites to understand protein function at a molecular level. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and high-resolution two-dimensional polyacrylamide electrophoresis (2-DE) are commonly used as an analytical approach to resolve and detect most of the numerous protein species of an organism. Currently, the most rapid and efficient method for isolating small quantities of proteins for protein chemical analysis is the electrotransfer of the electrophoretically separated proteins onto a suitable membrane. The immobilized proteins can then be directly N-terminally sequenced or subjected to amino acid composition analysis [1,2]. Many aspects of protein structural characterization, such as the accurate measurement of the molecular mass of intact proteins and the identification of post-translational modifications (blocked N-terminus, phosphorylation, glycosylation, etc.) are not readily amenable to standard methods of amino acid sequence analysis and amino acid composition analysis. Mass spectrometry is rapidly becoming the method of choice to help solve these structural problems [3]. Recently, two major developments, matrix-assisted laser desorptiodionization (MALDI) and electrospray ion-
Correspondence: Dr. Chrisloph Eckerskorn, Max-Planck Institut fur Biochemie, DW-8033 Martinsried, Germany
Abbreviations: IR. infra red, MALDI, matrix-assisted laser desorplion/ ionization. MS, mass spectrometry; PVDF, polyvinylidene difluoride
0 VCH Vcrlagsgescllschaft m b H , U-6940
Weinheim, 1992
ization have overcome the difficulties in the desorption and ionization oflarge and labile biomolecules such as proteins. Whereas for electrospray mass spectrometry (MS) the proteins have to be solubilized in a liquid mobile phase, for MALDI-MS the proteins were dried before laser desorption in a suitable form onto solid surfaces. Therefore MALDI-MS is the most promising technique for the direct determination of molecular masses of electroblotted proteins immobilized on membranes. I n the following, first spectra of electroblotted proteins obtained by MALDI-MS are presented
In standard preparations for MALDI-MS a dilute protein solution (lO-’-lO-x mol/L) is mixed with a concentrated matrix solution (typically 0.1 mol/L). Small aliquots (typically 0.1-1 pL) are placed onto a metal substrate and dried in a stream of air before being transferred into the vacuum of the mass spectrometer. Either pulsed lasers emitting in the UV range or in the IR range are used to induce desorption and ionization of small volumes of the analyte-matrix mixture. The alleged functions of the matrix, usually a small organic compound, are to absorb the energy from the laser pulse, to embed and isolate the proteins and to assist in their ionization. Best results are obtained if the proteins are homogeneously distributed and fully embedded in an environment of matrix molecules [41. Generally, singleshot spectra exhibit the molecular ion signal; 10 to 30 spectra are usually accumulated to improve the signal-to-noise ratio. All spectra shown are positive ion spectra. Though the laser beam can easily be focused to the area of interest on the membrane, the strong hydrophobic interaction of protein and membrane has to be overcome by suitable pre0173-0535/92/0910~0hhl$3 50+.25/0
Elerlrophoresis
Mass spectrome:try of' blotted proteins by malrix-assisted laser desorptionfionization
1992, 13. 664-665
r
paration techniques to allow for a successful MALDI-measurement. In a first set of experiments standard proteins (low-molecular-mass protein standard mixture, BioRad) were electrophoretically separated and electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Fluorotrans, Pall) as described [5]. Only the left and the right lanes of the PVDF membrane were stained for localization of the proteins with Coomassie Blue (Serva Blue R, Serva). Small areas, containing the unstained proteins, were cut out of the membrane, incubated in a solution of succinic acid as matrix and introduced into the mass spectrometer. The blotted samples were analyzed in a TOF instrument equipped with a first order reflectron. Desorbed ions were accelerated to a potential of 12 kV and detected by a secondary electron multiplier (EM1 9643, Electron Tube LTD.) after post accelation to 10 to 15 keV. A mechanically Q-switched Er-YAG laser 1-2-3 (Schwartz Electro-Optics) with a wavelength of 2.94 pm and 150 ns pulse width was used for these experiments. Figure 1 shows as an example the MALDI mass spectrum of bovine serum albumin. Compared to spectra obtained from sample prepared by standard techniques the peaks are somewhat broadened, which may result in a somewhat lower accuracy of mass determination. The distribution of single and multiple charged ions and oligomers, however, is comparable to that in standard preparations.
20000
50000
100000
w* Figure 1. Infrared-MALDI mass spectrum of bovine serum albumin blotted onto a PVDF membrane.A piece of the membrane was incubated in a solution of succinic acid as matrix. Sum of 10 single spectra. Laserwavelength: 2.94 vm.
M+
75 loo 50 25
665
2M+
.
10000
40000
Figure 3. Infrared-MALDI mass spectrum of bovine carbonic anhydrase blotted onto polyamide membrane after staining with India ink. A piece ofthe membrane was incubated in a solution ofmalic acid as matrix. Sum of 10 single spectra. Laser wavelength: 2.94 pm.
The sensitive staining of proteins should not interfere with determination of molecular masses. For proteins stained with colloidal metals like gold (Aurodye, Janssen) or India Ink [6],relatively sharp peaks with a shoulder to higher masses were obtained. The peak shoulder indicates that a part of the protein molecules absorb staining material. As an example, in Fig. 2 a MALDI-MS spectrum is shown for soy bean trypsin inhibitor electroblotted onto Immobilon PSQ and stained with colloidal gold. Nevertheless, the expected mass deduced from the known sequence is obtained from the sharp component of the signal. Even better results could be obtained with India ink. An example is given in Fig. 3 for carbonic anhydrase electroblotted onto a polyamide membrane (Sartolon, Sartorius). In the case of Coomassie Blue as staining material and lactic acid as matrix, the protein peaks obtained were shifted to higher masses. This mass shift might be due to strong interaction between Coomassie Blue molecules and protein, which are obviously retained during the desorptionlionization process. These results show that it is possible, in principle, to desorb electroblotted proteins from hydrophobic membranes. It still remains to be shown whether incubation in the matrix solution mainly induces a detachment of the proteins from the membrane, overcoming the strong hydrophobic interaction, or if the only partial embedding by the matrix suffices to desorb the still bound intact protein molecules. Currently, the level of mass determination accuracy achieved is still considerably lower than in standard preparation (> 0.25 O/o as compared to 0.01 O/o). Ongoing work focuses on an optimized combination of membrane, matrix and staining material. Received August 11. 1992
References I
I
I
10000
I
I
40000
Wz Figure 2. Infrared-MALDI mass spectrum of soybean trypsin inhibitor blotted onto PVDF membrane after staining with colloidal gold. A piece ofthe membrane was incubated in a solution of malic acid as matrix. Sum of 10 single spectra. Laser wavelength: 2.94 vm.
[I] Eckerskorn, C. and Lottspeich, F., Electrophoresis 1990, 11,554-561. [2] Aebersold, R., Adv. Electrophoresis 199I, 4, 81-168. [3] McCloskey, J.A., Methods Enzymol.: Muss spectometry Academic Press, Orlando, FL 1990, Vol. 193. [4] Hillenkamp, F., Chait, B.T., Beavis, R.C. and Karas, M., Anal. Chem. 1991, 63, 1193A-1203A. [ S ] Eckerskorn, C., Mewes, W., Goretzki,H.W. and Lottspeich, F., Euro. J. Biochem. 1988, 176, 509-519. [6] Hancock, K. and Tsang, V.C., Anal. Biochem. 1983, 133,157-162.
