Anal. Chem. 1086, 58,485-487
of these unstable ions will also lower their abundance, but the dissociation products will contribute to the spectrum, because any ions formed for -5 ms after ionization will be detected; in Figure 1 the Cs(CsI),+ ions of n = 22, 31 (3 X 3 X 7), 37, and 62 are much more abundant than nearby iohs of lower, as well as higher, n values. However, also of appreciable abundance (Figure 1) are unusually labile cluster ions such as CS3&+ (mlz 8966)) of which 32% was found to undego metastable decomposition in the time period 0.1-0.2 ms (27). The (Cs1)J ion data (Figure 1)show a striking resemblance in abundance to the positive ion data. The exchange of Cs for I- has surprisingly little effect on the relative stabilities of the clusters. CONCLUSION It is apparent that the long ion residuence times of FTMS measurements does not preclude the detection of relatively high mass ions. This is consistent with the expectation that the intensity of an image current produced by ions of the same mass moving coherently should depend directly on the number (and charge) of ions, not on their mass. Registry No. CsI, 7789-17-5.
(19) Cody, R. B., Jr.; Burnier, R. C.; Cassady, C. J.; Freiser, B. S. Anal. Chem. 1982, 54, 2225-2228. (20) Bowers, W. D.; Delbert, S. S.;McIver, R. T. American Society of Mass Spectrometlsts Meeting, San Diego, CA, May 1985; paper MPI1.
(21) Castro, M. E.; Russell, D. H. Anal. Chem. 1984, 56, 578. (22) Hunt, D. F.; Shabanowitz, J.; McIver, R. T., Jr.; Hunter, R. L.; Syka. J. E. P. Anal. Chem. 1985, 57, 765-768. (23) Shabanowltz, J.; Hunt, D. F.; McIver, R. T., Jr.; Hunter, R. L. American Society of Mass Spectrometry Meeting, San Diego, CA. May 1985; paper MOC-3. (24) Cody, R. B., Jr.; Amster, I . J.; Mclafferty, F. W. Proc. Nafl. Acad. SCi. U.S.A. 1985, 82. 6367-6370. (25) Ens, W.; Beavls, R.; Standing, K. G. Phys. Rev. Left. 1983, 50, 27. (26) Bariak, T. M.; Wyatt. J. R.; Colton, R. J.; DeCorpo, J. J.; Campana, J. E. J. Am. Chem. SOC. 1982, 104, 1212. (27) Baldwin, M. A,; Proctor, C. J.; Amster, I. J.; McLafferty, F. W. Int. J. Mass Spectrom. I o n Phys. 1983, 54, 97-106. (28) Campana, J. E.; Green, B. N. J. Am. Chem. SOC. 1984, 106, 531. (29) Campana, J. E.; Colton, R. J.; Wyatt, J. R.; Bateman, R. H.; Green, B. N. Appl. Spectrosc. 1984, 38, 430. (30) Morgan, T. G.; Rabrenovic, M.; Harris, F. M.; Beynon, J. H. Org. Mass Spe&om. 1984, 19, 315. (31) Katakuse, I.; Nakabushi, H.; Ichlhara, T.; Sakurai, T.; Matsuo, T.; Matsuda, H. I n t . J. Mass Spectrom. I o n Proc. 1984, 62, 17-23. (32) Castro, M. E.; Mallis, L. M.; Russell, D. H. J. Am. Chem. SOC. 1985, 707. 5652.
(33) Abehh, W ;Straub, K. M.; Burlingame, A. L. Anal. Chem. 1982, 54, 2029. (34) Martin, T. P. I n “Advances in Solid State Physics”; Vieweg: Braunschweig, 1984, Vol. 24, pp 1-24.
I. Jonathan Amster Fred W. McLafferty*
LITERATURE CITED (1) Barber, M.; Bordoli, R. S.;Elliott, 0.J.; Sedgwick, R. D.; Tyler, A. N. Anal. Chem. 1982, 54, 645A. (2) Macfarlane, R. D. Anal. Chem. 1983, 55, 1247A. (3) Benninghoven, A., Ed. “Ion Formatlon from Organic Solids”; SprlngerVerlag: Berlin, 1983. (4) Katakuse, 1.; Nakabushl, H.; Ichihara, T.; Sakurai, T.; Matsuo, T.; Matsuda, H. I n t . J. Mass Spectrom. I o n Proc. 1984, 57, 239-242. (5) Sundqvlst, B.; Roepstorff, P.; Fohlman, J.; Hedln, A,; Hakansson, T.; Kamenskv, I.: Lindbera, M.; SalehDour, M.; Sawe, G. Science 1984, 226, 6961698. (6) Heller, D. N.; Fenselau. C.; Yergey, J. A.; Cotter, R. J. Anal. Chem. 1984, 56 2274-2277. (7) Cottrell, J. S.; Frank, B. H. Biochem. Blophys. Res. Commun. 1985, 127. 1032-1038. ..- ... (8) filkins, C. L.; Weil, D. A.; Yang, C. L. C.; Ijames, C. F. Anal. Chem. 1985. 57. 520. (9) Amster, I . J.; Baldwin. M. A.; Cheng. M. T.; Proctor, C. J.; McLafferty, F. W. J. Am. Chem. SOC. 1983, 105, 1654-1655. (IO) McLafferty, F. W., Ed. “Tandem Mass Spectrometry”; Wiley: New York, 1983. (1 1) Chait. B. T.; Field, F. H. I n f . J. Mass Spectrom. I o n R o c . 1985, 85, 169-160. (12) Haddon. W. F.; Mclafferty, F. W. Anal. Chem. 1989, 4 1 , 31. (13) Comlsarow, M. 8.; Marshall, A. G. Chem. Phys. Left. 1974, 2 5 , 282. (14) McIver, R. T., Jr.; Bowers, W. D. I n ref IO, Chapter 14. (15) Johlman, C. L.; White, R. L.; Wilkins, C. L. Mass Spectrom. Rev. 1983, 2 , 389. (16) Gross, M. L.; Rempel, D. L. Science 1984, 226, 261. (17) Marshall, A. G. I n “International Symposium on Mass Spectrometry in Health and Life Sciences”; Burlingame, A. L., Ed.; Elsevier: Amsterdam, 1985. (18) Cody, R. B., Jr. Anal. Chem., in press. I
485
Department of Chemistry Cornel1 University Ithaca, New York 14853
Mauro E. Castro David H. Russell* Department of Chemistry Texas A&M University College Station, Texas 77843
Robert B. Cody, Jr. Sahba Ghaderi* Nicolet Analytical Instruments 5225 Verona Road Madison, Wisconsin 53711 RECEIVED for review March 14,1985. Resubmitted June 17, 1985. Accepted August 26,1985. The Texas A&M research was supported by the National Institutes of Health (Grant GM-33780)) U.S. Department of Energy (DE-AS0582ER13023)) and Texas Agriculture Experiment Station. Cornel1 research was supported by the National Institutes of Health (GM-16609) and Army Research Office (DAAG2982-K-0179).
Mass Spectrometric Determination of Dipeptides after Formation of a Surface Active Derivative Sir: Barber et al. (1) have described a mass spectrometry method that is related to secondary ion mass spectrometry (SIMS) but involves sputtering from a liquid rather than a solid surface. This method is particularly applicable to relatively polar substances that carry a negative or positive charge in solution. It has been demonstrated, however; that such a charge will not by itself ensure high sensitivity and that for materials with similar structures, sensitivity differences are determined largely by differences in surface activity (2, 3). A reviewer of this paper has emphasized that hydrophobicity is an important aspect of the more general concept of surface activity. 0003-2700/86/0358-0485$0 1.50/0
It has been shown that surfactants may be used to control the composition of the liquid surface and that surfactants can function in a manner analogous to anion exchange resinsbinding species of interest to the surface (4). Even in the case of small anions such as nitrate and sulfate, which in glycerol solution are entirely lacking in surface activity, high sensitivities can be obtained by using surface active reagents to bind these species to the liquid surface where they are readily sampled by the primary ion beam (5, 6). Great success has been reported in the analysis of peptides by sputtering from liquids such as glycerol (7), but sensitivities vary widely. In particular small relatively hydrophilic peptides 0 1986 American Chemical Soclety
486
ANALYTICAL CHEMISTRY, VOL.
58, NO. 2, FEBRUARY 1988
Table I. Comparative Results for Derivatized and Underivatized Dipeptides dipeptide glycylglycine alanylalanine methionylmethionine valylvaline leucylleucine phenylalanylalanine
intensity ratio" solution concn, M underivatized derivatized 0.007
O.OOb
0.75
0.006
O.OO*
0.0025 0.0035
0.11 1.60 1.8 0.1
0.62 0.74 3.90 1.3 0.81
0.0029
0.003
"Molecular ion intensity divided by the sum of the intensities found for glycerol ( m / z 91) and glycerol dimer ( m / z 183). Three scans were averaged for each point. A value of zero indicates that the material could not be detected. such as those derived largely from glycine and alanine give very poor sensitivity. When considered from the standpoint of relative surface activity, this sensitivity behavior is entirely predictable. These peptide species lack a long hydrocarbon tail, which could provide the hydrophobic character necessary for surface activity in glycerol. In this correspondence, I report a new derivatization procedure that confers enhanced surface activity to molecules of this type. Table I summarizes the negative secondary ion mass spectra obtained for a variety of underivatized dipeptides dissolved in glycerol at similar concentrations. The exact concentrations are given in the table. The glycerol used contained tetramethylammonium hydroxide a t the 0.02 M level. It is interesting that for purposes of generating anions in glycerol, we have found tetramethylammonium hydroxide to provide generally improved spectra compared with the more common sodium or potassium hydroxides. Samples of the dipeptides were obtained from Sigma Chemical Co., St. Louis, MO, and tetramethylammonium hydroxide was obtained from Alfa Products, Danvers, MA. The peptide samples and the organic base were used "as received. The mass spectrometer used was a Finnigan-MAT, Model 731. The operating parameters for SIMS experiments have been described previously (2). The primary particle gun was operated with xenon gas and produced a beam consisting of both ions and neutrals in the ratio of about 3:l (6). The signals observed correspond to the molecular species less a proton and are reported as a ratio to the glycerol matrix ions (mlz 91, 183), which always occur. Note that the dipeptides glycylglycine and alanylalanine are not detected at all at this concentration whereas other dipeptides, which bear hydrocarbon chains on the a carbon, can be detected. The differences in detectability are hypothesized to arise from differences in surface activity. In this context the term "surface activity" is intended to include such related phenomena as hydrophobicity, solubility, and solute-induced variations in surface tension. The concentrations provided for the peptide solutions assume that, as received, the materials were of reasonable purity. The SIMS spectra did not reveal any significant impurities in any of the samples; however the purity of the peptide samples specifically has not been established using an alternate method such as liquid chromatography. Accordingly the reader should not attempt to draw conclusions based on small differences in response among the various peptides (e.g., val-val vs. leu-leu). Such differences may arise both from minor differences in purity and from other presently unknown factors that may influence the ion yield for molecules of varying structure. Table I reports the results for these same dipeptides after they have been treated with dodecanal. These analytical solutions were prepared by treating an aqueous solution (30 FL, about 0.1 M) of each dipeptide with an equal volume of
300
350
400 MASS (DALTONS)
450
Figure 1. Negative secondary ion mass spectrum obtained for the following mixture of dipeptides after treatment with dodecanal: (A) glycylglycine, 0.0016 M; (6) alanylalanine, 0.0019M; (C) valylvaline, 0.00095 M; (D) phenylalanylalanine, 0.0008M; (E) leucylleucine, 0.0008 M; (F) methionylmethlonlne, 0.0012 M.
a 0.1 M solution of dodecanal (Aldrich Chemical Co.) dissolved in methanol. The resulting solution was warmed to the boiling point of methanol. Under these conditions, dodecanal forms a Schiff base or imine with peptides having a free primary amine function, thereby attaching a long hydrocarbon chain to the molecule. After warming, the reaction mixture was mixed directly with 1 g of glycerol that contained tetramethylammonium hydroxide at 0.02 M. A 2-hL aliquot of this glycerol solution was loaded onto the target of the mass spectrometer. If desired, excess dodecanal reagent can be used since any unreacted aldehyde is readily removed by the fore-vacuum system of the mass spectrometer before analysis. It can be seen from the table that sensitivities for all of the more polar peptides are improved by derivatization, but most notably glycylglycine and alanylalanine are now readily detected. It may be noted that the signal for leucylleucine is slightly reduced. It can also be seen that the relative sensitivities for the various peptides are now much more similar. Figure 1shows the negative secondary ion mass spectrum obtained after treating a mixture of this same group of peptides with dodecanal prior to dissolving them in glycerol. This solution was prepared by using exactly the same method as described above. This glycerol solution is about 4 times less concentrated in each peptide than are the solutions described in Table I. Exact concentrations are reported in the figure caption. Note that, even at these relatively low concentrations (250-600 ppm), an excellent mass spectrum with very low matrix interference is obtained. Traces of underivatized dipeptides are not observed at lower mass. In this experiment, it is important to ensure that the aldehyde used is of high purity. The analysis is greatly complicated if the aldehyde contains homologues. The analysis may fail entirely if the aldehyde has been partially oxidized to the corresponding acid, which can by itself entirely dominate the SIMS spectrum. In this case, the absence of homologues was established by gas chromatography and the SIMS spectrum showed no evidence of dodecanoic acid. Experience suggests that longer chain aldehydes might provide even greater enhancements in sensitivity (2, 4 ) . Unfortunately, difficulties have been encountered in finding suitable reaction solvents that will produce homogeneous solutions of such longer chain aldehydes and dipeptides. AS a result, such experiments have been largely unproductive. It has been noted, however, that attachment of hydrocarbon tails greater than C18 can in certain cases reduce glycerol solubility to an impractical level, thereby establishing an upper
Anal. Chem. i986,58,487-490
limit for the effectiveness of this general strategy. Based on these considerations, the 12-carbon aldehyde is currently considered to represent the optimum reagent for this procedure. It is also important to note that, when primary ion currents exceed about 2 X 10l2 particles cm-2 s-l, the critical first monolayer may be eroded faster than it is renewed. Very high primary ion currents result in an increase in the sampling of bulk solution with the result that advantages which accrue from enhanced surface activities are diminished (2). The observations of McEwen and Hass (8) regarding the use of high intensity guns are very much in agreement with this interpretation. It may be concluded, therefore, that this method provides a simple and rapid strategy for improving the secondary ion yield (sensitivity)of relatively hydrophilic species that contain a primary amine and a negative charge but lack structural components that can confer surface activity. As noted in the case of leucylleucine, relatively hydrophobic peptides (and probably most large peptides) may not benefit from this derivatization. This work also suggests that surface activity should be included among the many factors to be considered
487
in the preparation of samples for this technique. Registry No. Dodecanal, 112-54-9; glycylglycine, 556-50-3; alanylalanine, 1948-31-8; valylvaline, 3918-94-3; phenylalanylalanine, 3918-87-4;leucylleucine, 3303-31-9;methionylmethionine, 7349-78-2. LITERATURE CITED (1) Barber, M.; Bordoli, R. S.; Elliot, G. J.; Sedgwlck, R. D.; Tyler, A. N. Anal. Chem. lQ82, 5 4 , 645A. (2) Ligon, W. V.; Dorn, S. B. Int. J . Mass Spectrom. Ion Proc. 1984, 5 7 , 75-90. (3) DePauw, E.; Pelzer. 0.; Viet, D. D.; Marlen, J. Biochem. Biophys. Res. Commun. lQ84, 123, 27. (4) Ligon, W. V.; Dorn, S. B. Int. J . Mass Spectrom. Ion Proc. lQ84, 6 1 , 113. (5) Llgon, W. V.; Dorn, S. B. Int. J . Mass Spectrom. Ion Proc. 1985, 63,315. (6) Ligon, W. V.; Dorn, S. 6. Anal, Chem. 1985, 5 7 , 1993-1995. (7) Rlnehart, K. L. Sclence lQ82, 278, 254. (8) McEwen, C. E.; Hass, J. N. Anal. Chem. lQ85, 5 7 , 890-892.
Woodfin V. Ligon, Jr. General Electric Company Corporate Research and Development Schenectady, New York 12301 RECEIVEDfor review July 15,1985. Accepted October 7,1985.
Supercritical Carbon Dioxide Injection in Supersonic Beam Mass Spectrometry Sir: The ultracold spectroscopy afforded by supersonic expansions in combination with multiphoton ionization has provided a means of selective ionization for mass spectrometry (1-7). The wavelength selectivity of this method is particularly useful for problems that may be difficult to solve with conventional techniques alone; i.e., one needs to discriminate a small concentration of impurity in a large bath of a similar molecule. Such problems may arise in pharmaceutical synthesis where even trace amounts (
