1084
Anal. Chem. lQ86, 58,1084-1087
Plasma Desorption Mass Spectrometry of Peptides and Proteins Adsorbed on Nitrocellulose Gunnar P. Jonsson, Allan B. Hedin, Per L. Hakansson, Bo U. R. Sundqvist,* and B. Goran S. Save Tandem Accelerator Laboratory, Uppsala University, Box 533, S- 751 21 Uppsala, Sweden Per F. Nielsen and Peter Roepstorff Department of Molecular Biology, Odense University, Denmark Karl-Erik Johansson Mycoplasma Laboratory, National Veterinary Institute, Uppsala, Sweden Ivan Kamensky and Maria S. L. Lindberg Bio-Ion Nordic AB, Uppsala, Sweden
Nitrocellulose membranes and films have been used as backlngs In plasma desorption mass spectrometry (PDMS) studies of adsorbed peptides and proteins. This method allows for effectlve ellmlnatlon of salt contamlnants In the blomolecular film, which Is Important to Improve molecular Ion yields. Furthermore, formation of multiply charged molecular Ions Is enhanced when desorbed from a nltrocellulose backing. The pattern of peaks due to those ions Is helpful for Identification of the molecular Ions. With this new method of sample preparatlon several large molecules, llke human growth hormone and lysozyme, now give useful mass spectra with the PDMS technique.
In secondary ion mass spectrometry of biomolecules, the sample preparation is a very important step in the analytical procedure. This is the case regardless of the primary particle used to bombard the sample surface. In the original experiments by Macfarlane and co-workers ( 1 ) and Benninghoven et al. (2)the sample material was simply deposited as a droplet dissolved in a suitable solvent on top of a metal backing. The solvent was then allowed to evaporate. Macfarlane used fast ions as primaries and time-of-flight mass analysis in his method, called 252Cfplasma desorption mass spectrometry (PDMS) (3). In later studies performed with PDMS, sample application by the electrospray method ( 4 ) has mainly been used. The major drawbacks of this technique are the limited number of solvents useful for spraying and a rather undefined surface consisting of inhomogeneous layers of small sample particles. Recently the spincoating technique (5)was applied in studies where layers of well-defined thickness were needed. This method has the disadvantage that quite large amounts of sample are needed. In studies with low-energy primary particle impact, like those originally performed by Benninghoven, very well-defined surfaces of small volatile biomolecules have been prepared with the molecular beam method (6). With magnetic sector instruments high primary particle fluxes must be used, with the result that radiation damage of solid samples is a major problem. It was therefore an important contribution to the field when Surman and Vickerman (7) introduced the liquid matrix in 1981. Fast primary ions are potentially more interesting than slow ions for large molecules. Fast ions have been shown to be more efficient than slow ions to cause desorption of large intact molecules (8). An important aspect of secondary ion mass spectrometry methods is that they give molecular weight and
structure information on the compounds at the sample surface. Sample preparation techniques in which the molecules are adsorbed on a sample backing should therefore be investigated because with such tools a t hand the ultimate sensitivity of SIMS methods might be reached. Jordan et al. (9,lO) have reported on the use of an ion-containing polymer, Nafion, as backing in PDMS studies. In another study Macfarlane et al. (11)have used Mylar as a backing for Rhodamine G and showed that submonolayer sensitivity can be reached. In the present study nitrocellulose has been used as sample backing for PDMS studies allowing selective adsorption of peptides and proteins from solutions. Both fission fragments from a 252Cfsource (252CfPDMS) and fast ions (90-MeV I T 4 + ) from the Uppsala EN-tandem accelerator were used as primaries. The usefulness of nitrocellulose as sample backing was discovered in attempts to use gel electrophoretic separation of protein mixtures followed by blotting on nitrocellulose membranes and PDMS analysis.
EXPERIMENTAL SECTION Instrumental Setups. Two different instruments were used in this study. The samples prepared on nitrocellulose blotting membranes were studied with a time-of-flight spectrometer coupled to the Uppsala EN-tandem accelerator (12,13).A beam of 90-MeV 12'1'4+from the accelerator was used as primary ions. These experiments will be referred to as 12'1 PDMS. The advantage of this setup is that the backings can be thick because the primary ions hit the sample from the front side as in a lowenergy SIMS setup. In the accelerator spectrometer an acceleration voltage of 20 kV was used. In the other part of the study the Bio-Ion Bin 10K plasma desorption mass spectrometer, installed in Odense, was used. This instrument was operated at an acceleration voltage of 18 kV. The details of the instrument and the spectrum handling procedures used have been described in ref 14. In the discussion these experiments will be labeled 252Cf PDMS. SAMPLE PREPARATION Two slightly different procedures were used. In the first experiments nitrocellulose blotting membranes (Bio-Rad Laboratories; pore size, 0.45 pm) were used. The thickness is such that the fast ions must hit the sample from the front side (sample material side). Therefore this set of initial experiments was performed with the accelerator setup at Uppsala. The molecules studied were dissolved in a PBS solution (PBS consists of 8.00 g of NaC1, 0.20 g of KC1,0.20 g of KH2P0,, 1.44 g of Na2HP0,; 2H20/L of water, adjusted to pH 7.4). Approximately 10 pL of PBS solution containing 30 wg of protein was deposited on a 1 cm2 piece of a blotting membrane and dried. The membrane was then rinsed 3 times
0003-2700/86/0358-1084$01.50/00 1986 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986 L*lU
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for 5 min in a washing solution (acetic acid, methanol, and demineralized water; 1:1:8 v/v/v). Finally, the sample was dried and put in a desiccator until loaded in the spectrometer. In the second set of experiments the method was modified to the standard PDMS technique where the sample molecules
Flgure 2. Background-subtracted positive ion PD spectra of porcine trypsin: electrosprayed sample (upper), nitrocellulose sample (lower); primary ions, 90-MeV 127114+; number of primary ions, 5.7 X lo6 (upper) and 7.5 X lo6 (lower).
are put on a thin foil (200 pg/cm2 Al) that allows the fission fragment to hit the sample from the backside. The aluminum foil was covered with a thin layer of nitrocellulose by distributing 5-10 pL of a 1% nitrocellulose solution in amyl acetate (E. F. Fullam, Inc., NY) on the foil followed by drying in a desiccator. A droplet with the sample molecules dissolved in an appropriate solvent was then applied. Most frequently water or 0.1% trifluoroacetic acid solution was used as the solvent. Immediately after that the droplet was distributed over the nitrocellulose surface, the sample was rinsed with a jet of 1 mL of demineralized water. Finally, the sample was dried with hot air and stored in a desiccator before analysis.
RESULTS In Figure 1 the positive ion 12'1 PD spectra from a sample of phospholipase A2 (MW 13 980) adsorbed on nitrocellulose are shown. The number of primary ions is the same for a sample prior to, and the lower spectra after, the rinsing procedure. Figure 1A shows the low mass part and Figure 1B shows the high mass part of the spectra. As can be seen in Figure 1A sodium is effectively eliminated by the rinsing procedure. In Figure 1B it is shown that the cleaning procedure also improves both the abundance and peak shape of the molecular ions. The broad peaks in the upper part of Figure 1B are due to the decay of molecular ions with very high internal energy. When these molecular ions decay in the field-free region of the spectrometer,internal energy is released in the decay and a broad peak is observed in the spectra (14, 15). In the lower spectrum a broad peak component is also present, but sharper peaks are superimposed. A characteristic of spectra of molecules adsorbed on nitrocellulose is the very intense multiply charged molecular ion species. This point is illustrated in Figure 2, which shows positive ion spectra of porcine trypsin (MW 24 363) from an electrosprayed sample (16)(upper) and a sample prepared with the present method (lower). Gas-phase molecular ions of porcine trypsin are the
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ANALYTICAL CHEMISTRY, VOL. 58, NO.
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largest monomeric biomolecular ions observed so far. The trypsin ions with six positive charges, have an energy of 120 keV. These ions are therefore effectively detected. The pattern of molecular ion peaks due to different charge states is very useful from an analytical point of view. The molecular ion, and thus the molecular weight, can be uniquely identified even if the peaks due to singly charged ions are of very low intensity. The method of sample preparation described has been applied to a number of molecules in the molecular weight range 500-25 000. The general experience is that multiply charged ion formation is enchanced, at least for molecules with molecular weights above 5000. Several proteins that have failed to give useful spectra from electrosprayed samples have been successfully analyzed with the new method. Examples of such molecules are P-2-microglobulin (MW 11800), lysozyme (MW 14000), and HGH, human growth hormone (MW 22 005). The spectrum of positive ions of HGH is shown in Figure 3. In the following, some results using nitrocellulose backings in combination with 252CfPDMS are described. In Figure 4 spectra of positive ions (molecular ion region) from LHRH, luteinizing hormone releasing hormone (MW 1182),are shown. In the upper spectrum, obtained by using the electrospray technique for sample application, strong cationization is observed. The middle spectrum, taken with a rinsed sample of LHRH adsorbed on nitrocellulose, shows no sign of cationization. A minor adduct ion peak is observed 31 mass units above MH+. The origin of this adduct ion, which is frequently observed in the spectra of peptides adsorbed on nitrocellulose, is not known. It is often also observed when Nafion is used as backing. The lower spectrum illustrates that the sample can be applied from extremely diluted solutions. A nitrocellulose-covered A1 foil was dipped in a solution containing 50 pg of LHRH in 200 mL of water for 2 min and then dried before analysis. In Figure 5 the yield, defined as the number of molecular ions desorbed per incident primary ion, of LHRH is plotted as a function of the amount of LHRH in the droplet applied to the nitrocellulose backing. Estimates indicate that the saturation "knee" corresponds to a few monolayers of LHRH. However, the effective nitrocellulose area may be much larger than the foil area, and the yield probably saturates a t monolayer coverage. In order to further test the sample application from diluted solutions, a sample of porcine insulin (100 pg) was applied to a reversed-phase HPLC column and eluted with 0.1% trifluoroacetic acid and a linear gradient of 2-propanol from 0 to 50%. A nitrocellulose backing was dipped in the HPLC fraction (2-3 mL) containing the insulin. The PD spectrum of positive ions is shown in Figure 6.
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eye. Another application-orienkd experiment was based on the original idea of combining gel electrophoresis and PDMS. Approximately 5 pg of phospholipase A2 was subjected to SDS-gel electrophoresis and electroblotted onto a nitro-
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
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cellulose membrane. The electrophoretic band containing the protein was identified by staining of a parallel sample lane, cut out and washed, and subjected to 12’1 PDMS. The P D spectrum of positive ions showed the expected pattern of molecular ion peaks. A careful investigation of the spectra of phospholipase A2 (Figure l),porcine trypsin (Figure 2), and human growth hormone (Figure 3) indicates that the calibration of the mass scale is very rough. The problem is probably connected to the use of thick insulating backings and should be studied in more detail. The mass calibration in the case of LHRH (see Figure 5 ) deposited on a thin backing is correct.
DISCUSSION The method used in this study allows for an effective removal of salt contaminants. This is important because it is known that Na salts quench molecular ion yields. This effect is clearly demonstrated in Figure 1. The removal of Na salts seems to result in a decrease in the average internal energy of the desorbed molecular ions, which may in part explain the enhanced molecular ion yields. This study has also demonstrated that the use of nitrocellulose enhances multiply charged molecular ion formation. This is very useful as the higher energy acquired in the acceleration process makes a multiply charged ion easier to detect ( 1 7 ) . The observation of very high charge states in this study indicates that the detection of large molecular ions may be a less severe problem then earlier anticipated. The enhanced formation of multiply charged ions, when the proteins are desorbed from a nitrocellulose backing, is not understood a t present. The nature of the actual bonding of a protein to nitrocellulose is poorly known and claimed to be nonionic at.pH around 7 (18) but rather dominated by hydrophobic interactions. A nitrocellulose surface is described as negative (18), because the nitrate ester groups are polarized to form dipoles with the
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“negative” oxygen atoms directed toward the outer surface. A possible explanation is that the positvely charged groups in a protein are attracted to the “negative” groups on nitrocellulose and therefore adsorbed and desorbed as preformed ions. From a practical point of view the use of nitrocellulose backings is superior to the electrospray technique, mainly because the nitrocellulose method allows for sample application from water solutions. The sample can be applied from dilute water solutions, and buffers and salts necessary to keep the protein in solution do not interfere because they are removed in the washing procedure. This implies that application of a protein in either its native or denaturated state is possible. Sample application simply by dipping the backing in HPLC fractions and the use of electroblotting directly from a gel onto a nitrocellulose backing have been demonstrated. These results indicate that plasma desorption mass spectrometry may become an extremely useful tool in protein chemistry. Further studies will show whether nitrocellulose also can be used as backing in PDMS studies of other biomolecules like nucleic acids. Registry No. HGH, 12629-01-5; LHRH, 9034-40-6; phospholipase A,, 9001-84-7;trypsin, 9002-07-7;insulin, 9004-10-8; nitrocellulose, 9004-70-0.
LITERATURE CITED (1) Torgerson, D. F.; Skowronski, R. P.; Macfarlane, R. D. Siophys. Res. Commun. 1974, 6 0 , 616. (2) Benninghoven, A.; Jaspers, D.; Sichtermann, W. Appl. Phys. 1978, 1 1 . 35. (3) Macfarlane, R. D.; Torgerson, D. F. Science (Washington, D.C.) 1978, 19 1 , 920. (4) McNeal, C. J.; Macfarlane, R. F.; Thurston, E. L. Anal. Chem. 1979, 51, 2036. (5) Jonsson, U.; Olofsson, G.; Malmquist, M.; Save, G.; Fohlman, J.; Hakansson, P.; Sundqvist, B., submitted for publication. (6) Benninghoven, A.; Lange, W.; Jirikowsky, M.; Holtkamp, D. Surf. Sci. 1982, 123, L721. (7) Surman, D. J.; Vickerman, J. C. J. Chem. SOC.,Chem. Commun. 1981, 170. (6) Kamensky, I.; Hakansson, P.; Sundqvist, 6.; McNeal, C. J.; Macfarlane, R. D. Nucl. Instrum. Methods 1982, 198, 65. (9) Jordan, E. A.; Macfarlane, R. D.; Martin, C. R.; McNeal, C. J. Int. J. Mass Spectrom. Ion Phys. 1983, 5 3 , 345. 10) Jordan, E. A.; Martin, C. R.; McNeal, C. J.; Macfarlane, R. D. Presented at the 33rd Annual Conference on Mass Spectrometry and Allied Topics, San Antonio, TX, 1984. 11) Macfarlane, R. D. J. Trace Microprobe Tech. 1984, 2 , 267. 12) Hakansson, P.; Sundqvist, B. Radiat. Eff. 1982, 6 1 , 179. 13) Sundqvist, 6.; Hedin, A.; Hakansson, P.; Save, G.; Salehpour, M.; Widdiyasekera, S.;Johnson, R. E. Proceedings from SIMS-V, Washington, DC, Oct 1985. (14) Sundqvist, 6.; Hakansson, P.; Kamensky, I.; Kjellberg, J.; Salehpour, M.; Widdiyasekera, S.; Fohlman, J.; Peterson, P.; Roepstorff, P. Biomed. Mass Spectrom. 1984, 1 1 , 242. (15) Chait, B. T.; Field, F. H. Int. J. Mass Spectrom. Ion Phys. 1981, 4 1 , 17. (16) Sundqvist, B.; Roepstorff, P.; Fohlman, J.; Hedin, A.; Hakansson, P.; Kamensky, I.; Lindberg, M.; Salehpour, M.; Save, G. Science (Washington, D . C . ) 1984, 226, 696. (17) Sundqvist, B.; Hedin, A.; Hakansson, P.; Kamensky, I . ; Salehpour, M.; Save, G. Int. J. Mass Spectrom. Ion Processes 1985, 6 5 , 69. (18) Gershoni, J. M.;Palade, G. F. Anal. Biochem. 1983, 131, 1.
RECEIVED for review September 17,1985. Accepted November 21, 1985.
