Anal. Chem. 1986, 58, 1303-1307 (6) Lichtin, D. A,; Datta-Ghosh, S.; Newton, K. R.; Bernstein, R. B. Chem. Phys. Len. 1080, 75, 214. (7) Sack, T. M.; McCrery, D. A.; Gross, M. L. Anal. Chem. 1085, 57, 1290. (8) Carlin. T. J.; Freiser, B. S. Anal. Chem. 1983, 55, 1955. (9) Irion, M. P.; Bowers, W. D.; Hunter, R. L.; Rowland, F. S.; McIver. R. T.. Jr. Chem. Phys. Len. 1082, 93, 375. (10) Boesl, U.; Neusser, H. J.; Schlag, E. W. Chem. Phys. 1981, 55, 193. (11) Rhodes, G.; Opsal, R. B.; Meek, J. T.; Reilly, J. P. Anal. Cbem. 1983, 55, 280. (12) Rettner, C. T.; Brophy, J. H. Cbem. Phys. 1981, 24, 89. (13) Rizzo, T. R.; Park, Y. D.; Levy, D. H. J. Am. Chem. SOC. 1985, 107, 277. (14) Pang, H. M.; Sln, C. H.; Lubman, D. M.; Zorn, J. C. Anal. Chem. 1088, 58, 487. (15) . . Frank. Lenore Randall. Ph.D. Thesis, The University of Utah, Sak Lake City, UT, Dec 1979. (16) Posthumas, M. A.; Kistemaker. P. G.; Meuzelaar, H. L. C.; Ten Noever deBrauw, M. C. Anal. Chem. 1978. 50, 985. (17) Cotter, R. J. Anal. Chem. 1979. 51, 317. (18) Conzemius, R. J.; Capellen, J. M. I n f . J. Mass. Specfrom. Ion Phys. 1980, 34, 197. (19) McCrery, D. A,; Ledford, E. B., Jr.; Gross, M. L. Anal. Chem. 1982, 54. 1437. (20) Zakett, D.; Schoen, A. E.; Cooks, R. G.; Hemberger, P. H. J. Am. Chem. SOC. 1981, 103, 1295. (21) Hercules, D. M.; Day, R. J.; Balasannugam, K.; Dong, T. A.; LI, C P. Anal. Chem. 1982, 54, 280A.
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(22) Vastola, F. J.; Pirone. A. J. Adv. Mass Specfrom. 1070, 4 , 107. (23) Wilkins, C. L.; Weil, D. A.; Yang, C. L.; Ijames, C. F. Anal. Chem. 1085, 57, 520. (24) Sherman, M. G.; Kingsley, J. R.; Hemminger, J. C.; McIver, R. T., Jr. Anal. Chlm. Acta 1985, 178, 79. (25) Lindner, B.; Seydel, U. Anal. Chem. 1985, 57, 895. (26) Boesl, U.; Neusser, H. J.; Schlag, E. W. J . Chem. Phys. 1980, 72, 4327. (27) Lubman, D. M.; Naaman, R.; Zare, R. N. J. Chem. Phys. 1980, 72, 3034. (28) Whetten, R. L.; Fu, K. J.; Tapper, R. S.; Grant, E. R. J. Phys. Chem. 1983, 87, 1484. (29) Anderson, J. B.; Andres, R. P.; Fenn, J. B. Adv. Cbem. Phys. 1968, 10 276 .- , . -. (30) Lubman, D. M.; Rettner, C. T.; Zare, R. N. J. Phys. Chem. 1082, 86, 1129.
RECEIVED for review November 20,1985. Accepted January 27, 1986. We gratefully acknowlege financial support from a Cottrell Research Grant and the donors of the Petroleum Research Fund, administered by the American Chemical Society. We acknowledge support of this work under NSF Grant CHE 83-19383 and partial support from the Army Research Office under Grant DAAG 29-85-K-1005.
Glutathione as a Matrix for Plasma Desorption Mass Spectrometry of Large Peptides Mehrshid Alai, Plamen Demirev, Catherine Fenselau, and Robert J. Cotter* Department of Pharmacology, Middle Atlantic Mass Spectrometry Facility, The Johns Hopkins University, Baltimore, Maryland 21205
The plasma desorptlon mass spectra of large peptides, dlssolved and electrosprayed In solutlons contalnlng glutathione, show Increased molecular Ion signal, reductlon of base-llne nolse and peak wldths, and an Increase In multlply charged Ions. The reduced, rather than the oxldlzed, form of glutaihlone Is responsible for these effects. Some other chemlcally slmllar matrlces show slmllar effects while others do not. Several roles for the matrhc are suggested lncludlng prevlously reported effects on protein refoldlng and aggregatlon In solution, as well as posslbllHles for lowering the sample/substrate blndlng energy durlng desorptlon.
The importance of appropriate chemical/physical matrices for the desorption of intractable compounds in mass spectrometry was underscored by the introduction of glycerol (1, 2),as an integral part of the fast atom bombardment technique. From the beginning it was understood that the liquid matrix provided strong stable secondary ion signals under high-flux bombardment necessary for high-performance double-focusing mass spectrometers capable of high-mass ranges (3). More recently, the possibility for reducing the internal energy of secondary ions by solvent shedding has also been suggested ( 4 , 5 ) . The addition of acid to glycerol or the use of monothioglycerol improves the matrix's role as a proton donor ( 3 , 6 ) . Other matrices, such as tetraglyme for cesium perfluoroalkonate clusters (6),or mixtures of thiols, such as dithiothreitol and dithioerythritol for peptides (7), have been employed. Recently solid saccharide matrices have also been proposed for use in FAB mass spectrometry (8).
The plasma desorption technique (9) uses the time-of-flight, TOF, analyzer. While glycerol has been used with TOF analyzers (lo),liquid matrices are generally less compatible with such instruments because of the high electric fields in the source region and because of the desirability of forming all ions on the same focal and equipotential plane. Nevertheless, the importance of the sample matrix is recognized for this technique as well. The dissolution of solid samples in trifluoroacetic acid, TFA, or glacial acetic acid (11) increases sample solubility, improves the electrospraying process by providing a volatile aqueous medium with high ionic strength, and also acta as a proton donor to improve ion signal (as MH+ ions). Concentration studies by Macfarlane (12)have shown that the sample itself, or chemically similar compounds (13), serves as a matrix, improving the molecular ion yield, compared with sample molecules desorbed directly from a metal foil. In that vein, matrices have been introduced that are designed to alter the surface of the sample foils used in plasma desorption and thereby reduce the binding energy between the sample and the surface. Jordan et al. (14) have reported the use of electrosprayed thin films of Nafion, a perfluorinated cation-exchange polymer. More recently, Sundqvist et al. (15) have bonded nitrocellulose to the sample foil prior to electrospraying the sample followed by removal of excess cations (particularly Na'). Initially we also attempted (with some success) to reduce the sample/substrate binding energy by depositing a layer of lactalbumin on the sample foil prior to electrospraying the sample (16).Recently, however, we reported the use of a 1:l mixture of oxidized/reduced glutathione as a matrix for the analysis of relaxins, 3-dimensional
0003-2700/86/0358-1303$01.50/00 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
Scheme I
a. 0
0
I1
II
CH2-CHz-CKH-CH-CNH-CH,-COO-
I I
H-C--SHS+
CHz
coo-
SH
Glutathione (r-~-Glutam)l-~-cvstein~l~l~cine)
z
GSH H
reduced form
GS-SG
t
ZH+
M W 5733.5 e
t
2e-
1900
CHANNEL NUMBER
2800
oxidized form
homologues of insulin containing A and B chains and three disulfide bridges (16). In this case samples are added to solutions of glutathione prior to electrospraying. Glutathione is reported (17)to assist in the refolding of proteins in solution, and we envisioned that solution of peptides in acetic acid f glutathione would increase the yield of intact molecular ions. In addition to pronounced increases in the molecular ion signal to noise, decreases in peak widths and increases in the intensity of multiply charged ions, effects observed with nitrocellulose surfaces BS well, indicated that we had also reduced the sample/substrate binding energy and the internal energy of desorbed ions. This correlation between a reagent whose effects on peptides in solution are known and spectra with well-defined molecular ion peaks led to this present study of the effects of glutathione on several other peptides and a comparison with other reagents whose effects on protein folding and f or aggregation have been reported.
ieeo
CHANNEL NUMBER
2809
Figure 1. Plasma desorption mass spectra of
bovine insulin (a)without glutathione (4 nslchannel)and (b) with GSH (4 nslchannel).
EXPERIMENTAL SECTION Several peptides in the 4000-14000-amu range were studied. Bovine insulin, equine insulin, egg white lysozyme, and carboxypeptidase inhibitor were purchased from Sigma (St. Louis, MO). Porcine insulin, bovine proinsulin, human proinsulin, and protamine I were courtesy of R. E. Chance and B. H. Frank, Lilly Research Laboratories (Indianapolis, IN). Mass spectra were obtained for all of the peptide samples in glutathione matrix. In this case 50-150 pg of sample wag dissolved in an acetic acid/water solution (1005) containing 25 mM glutathione to give an approximately 1:l molar ratio of sample/ glutathione. For comparison, several samples were dissolved directly in glacial acetic acid only. Also for comparison, bovine insulin was prepared with several matrices: oxidized glutathione, reduced glutathione, a 1:1 mixture of oxidized/reduced glutathione, glutamic acid, CysGlyGly, methionine, oxalic acid, and hydrochloric acid, in a similar molar ratio. Sample solutions were electrosprayed on thin aluminum sample foils. Plasma desorption mass spectra were obtained on a BIO-ION Nordic (Uppsala, Sweden), BIN-1OK time-of-flight mass spectrometer with an accelerating voltage of 20 kV and a flight tube length of 15 cm. The mass spectrometer ion source consists of a 1O-pCi sample of californium-252emitting simultaneouslytwo fission fragmentsat the rate of approximately 1100 events/s. One of the fission fragments from each event is detected to provide a “start” pulse for a time-to-digital converter, while the other is used to desorb secondary ions from the sample. The secondary ions are detected and provide “stop” pulses for the TDC, which has a maximum time resolution of 1ns/channel and can acquire up to 64 ions for each desorption event. Spectra were acquired to a preset count of 3 x lo6 primary ion events. Data were acquired on an Apple IIe microcomputer as 4K channels read from the multistop timer. Channel resolution was generally from 2 to 8 ns, depending upon mass range. Mass assignments were made by determination of the time (channel) centroids of the sample peaks in the spectrum and comparison with centroids for Ht and Nat peaks appearing in the same spectrum. Mass accuracy at 5000 amu was approximatelyA3 amu.
RESULTS AND DISCUSSION Although our initial experiments involved the use of a 1:l mixture of oxidized and reduced glutathione (16))we have
2000
CHANNEL NUMBER
40RQ
0 1
lOR0
CHANNEL NUMBER
2000
Figure 2. Plasma desorption mass spectra of equine insulin (a)wffhout glutathione (2 nskhannel) and (b) with GSH (4 nslchannel).
since determined that the reduced form is more effective in producing those effects in the spectrum (narrow peak widths, multiply charged ions) that are indicative of lower binding energy and internal energies of desorbed ions. Scheme I shows the structure of glutathione, a tripeptide, and the redox half-reaction. Comparative results for bovine, equine, and porcine insulin with and without reduced glutathione, GSH, are presented in Figures 1, 2, and 3, respectively. In these three spectra, there is no base-line subtrgction, in order to illustrate the relationship between molecular ion signal and incoherent signal in the raw spectra as measured by the PDMS-TOF analyzer. (Subsequent spectra utilize constant base-line subtraction, although more elaborate curve-fitting methods have been described ( I I ) . ) In addition to the increase in
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
1305
1222
6127
CARBOXYPEPTIDASE INHIBITOR v)
I-
z 3
PORCINE INSULIN
0 0
MW 5777.8
e 2%
CHANNEL NUMBER
503
1500
CHANNEL NUMBER
e50
Flgure 5. Plasma desorption mass spectrum of carboxypeptMase inhibitor in a 1:l mixture of oxidized and reduced glutathione (4 nsl
channel).
57n
I A"
PROTAMINE
MW 4 2 3 5
e
ieee
CHANNEL NUMBER
2812
Figure 3. Flasma desorption mass spectra of porcine Wln (a)withan GSH (4 nshhannel).
glutathione (16 ns/channel) and (b) with
e50
a.
CHANNEL NUMBER
1500
Flgure 6. Plasma desorption mass spectrum of protamine with GSH (4 nskhannel).
QOVINE PROINSULIN
MW 8 8 8 1
Table I. Matrices Used in the Plasma Desorption Mass Spectra of Bovine Insulin and Their Effects on the Ratio of Doubly/Singly Charged Molecular Ions matrix acetic acid only I
1 one
CHANNEL NUMBER
2239
IP50
I
1+z
%BOOR
CHANNEL NUMBER
Figure 4. Plasma desorption mass spectra of bovine proinsulin (a)
without glutathione (8 ns/channel) and (b) with
elutamic acid
0.1 0.6 0.1 0.38
matrix
MHP/MH+
methionine
0.1 0.37
CysGlyGly HCl oxalic acid
0.1
b.
LlRU0
GSH GSSG
MH?/MH+
GSH (4
ns/channel).
signal/noise of the molecular ion, MH+, doubly charged ions are greatly enhanced by the addition of reduced glutathione. Peaks corresponding to the A and B chains of insulin are not enhanced. The spectrum of the heavier bovine proinsulin (Figure 4a) without GSH shows that it contains doubly charged ions, while the spectrum obtained with GSH (Figure 4b) contains abundant doubly charged ions and triply charged ions as well. Spectra of several other large peptides in the mass range of 4000-14000 amu were obtained by using the glutathione matrix. The mass spectrum of carboxypeptidase inhibitor is shown in Figure 5. The complete sequence was obtained by Biemann et al. (18),who noted that about half of the molecules lacked a glutamine (19). The molecule has three disulfide
bridges. The two components are evident from peaks corresponding to both the singly and doubly charged ions. The 1:1mixture is more evident in the doubly charged ion peak, where there is not interference from the "tail", which is pronounced for the singly charged ion peaks. A reasonable spectrum for this compound could not be obtained when glutathione was omitted. While the chemical similarity between glutathione and cysteinecontaining peptides had originally led us to speculate that only peptides which contained disulfide bonds would be enhanced by this matrix, we have since observed effects on the spectra of peptides without disulfide bridges. Figure 6, the spectrum of protamine I, is such an example. Previous attempts to obtain spectra of protamine samples were unsuccessful, and the spectrum with glutathione shows the characteristic enhancement of the doubly charged ion as well. The larger human proinsulin (Figure 7) shows the presence of both doubly and triply charged ions, as well as remarkably good signal to noise. Multiply charged molecular ions dominate the mass spectrum of egg white lysozyme (Figure 8). While samples were generally prepared as equimolar solutions of sample and glutathione, there appeared to be little sensitivity to the relative concentrations. However, when the molar concentration of glutathione was significantly lower than that of the sample, the spectra began to take on the appearance of spectra without glutathione. One quantitative measure of the effect of concentration is to compare the ratio of doubly to singly charged molecular ions, and Figure 9 shows
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ANALYTICAL CHEMISTRY, VOL. Si23
h
58, NO. 7,JUNE 1988
HUMAN PROINSULIN
a.
M W 9388.7 v)
c
z 2
0 U
i 2308
CHANNEL NUMBER
680
I
7
k 1100
0
0.5
1.0
RELATIVE CONCENTRATION
b.
1.5
2.0
QSH:INSULIN
Ratio of the doubly charged, MH ,'; and singly charged, MH', molecular ions of bovine insulln as a function of the relatlve concentratlons of reduced glutathione and insulin. Figure B.
tl
I
+3
II +i
1
innn
CHANNEL NUMBER
nrlrt
Figure 7. Plasma desorption mass spectra of human proinsulin (a) without glutathione (8nslchannel) and (b) in a 1:l mixture of oxidized and reduced glutathione (4 nshhannel). 16386
LYSOZYME
2REE 65
CHANNEL NUMBER
165
Plasma desorption mass spectrum of egg white lysozyme in GSH (64 nskhannel), with linear background subtraction. Figure 8.
the results of one such experiment for bovine insulin. Several other reagents were used as matrices and are listed in Table I. As mentioned previously our initial experiments employed a mixture of oxidized and reduced forms of glutathione. A logical first step was to determine whether the effects were produced by one or the other of these forms. The reduced glutathiohe produced enhanced molecular ion signal and abundant doubly charged ions, while the oxidized form was much less effective. Since this experiment suggested a role for the sulfhydryl proton of glutathione, the tripeptide CysGlyGly (which contains the same proton on a cysteine) and methionine (in which the proton is replaced by a methyl group) were used. The tripeptide produced both enhancement of molecular ion signal and doubly charged ions, while methionine produced no noticeable effects compared to spectra without any matrix. Glutamic acid also produces enhanced spectra. However, oxalic acid and hydrochloric acid did not lead to any improvements in the spectra. Table I summarizes these results in terms of the ratio of doubly/singly charged molecular ions. While all of the matrices studied are proton donors, the addition of acid does not necessarily produce the desired effects on the spectrum. Addition of HC1 results in rather poor spectra. It is well-known that extreme pH results in the
denaturing of peptides (i.e., unfolding and reduction of disulfide bonds), and it may perhaps be assumed that unfolding reduces the probability that the protein molecules will be desorbed intact. Glacial acetic acid or TFA solutions, used to dissolve and electrospray the samples, may likewise result in denaturing of the proteins. We have found, for example, using high-pressure liquid chromatography, that insulin recovered from thioglycerol (used in FAB) or from foils after electrospraying with acetic acid contain increased amounts of A and B chain (20). In general protein denaturing is a reversible process, thermodynamically driven by noncovalent interactions. For peptides that contain disulfide bonds, thiols such as glutathione have been used to facilitate disulfide bond reformation during renaturing (I 7). Refolding is generally accelerated by thiols in the presence of oxidizing and reducing agents, or with a mixture of reduced and oxidized thiols (17). Saxena and Wetlaufer (21) determined that the optimal conditions for the refolding of reduced and denatured hen egg white lysozyme was a mixture of 5 mM GSH and 0.5 mM GSSG, resulting in approximately50% recovery to the native form in less than 5 min. The predominance of the reduced form results from the need to reduce illicit disulfide bond formation during the renaturing process and therefore correlates well with our observation that the reduced form is more effective in improving the molecular ion signal than the oxidized form. If acetic acid solutions result in protein denaturing, then evaporation of the solvent during the electrospraying process and the resulting sudden increase in concentration may give rise to disulfide bond formation between neighboring molecules, thus reducing the available intact molecules for desorption. Minimization of denatured species in the presence of thiols may thus reduce the possibility for such covalent aggregation. Aggregation of human insulin at high concentrations and the problem that this poses for drug delivery are well-known (22) and can proceed as a result of van der Waals forces and hydrogen bonding as well (23). Bringer et al. (24) have demonstrated a very specific reduction of insulin aggregation using the dicarboxylic amino acids, glutamic acid and aspartic acid, at their isoelectric (acidic) pHs, while lowering of pH has in itself been shown to be insufficient to prevent aggregation (24, 25). Although this effect is not completely understood, we note that the reagents (glutathione, CysGlyGly, glutamic acid) which produce changes in the spectra are all amino acids which contain an additional labile hydrogen. If the improvements in the spectra result from reducing noncovalent aggregation, then it would not be surprising that these effects are produced on peptides without disulfide bridges as well. We note in this respect that the mixture of DTT/DTE used by Cook et al. (7) for FAB mass spectrometry enhances the molecular ion formation of peptides without disulfide bridges. Aggregation
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
of insulin is also reduced in human blood serum, and experiments with serum albumin have indicated that, while this reagent reduces adsorption to some surfaces, it does not prevent aggregation (24). The decrease in adhesion to surfaces, however, motivated our earlier experiments in the analysis of relaxins using a-lactalbumin deposited on a sample foil prior to sample deposition (16).We note that glycerol has been used to prevent aggregation of insulin (26). Glutathione may of course play an important role in the desorption process itself, which would account for the increased ease of desorption and reduced peak widths. Shedding of solvent molecules might then serve to reduce the internal energy in analogy to that proposed for glycerol in the FAB technique (5). Alternatively, the reduced glutathione anion, GS-, or glutamate anion may provide a better counterion than C1- from which protonated species can be separated. Separation might occur at the surface boundary or at some small distance above the surface after desorption of a neutral ion pair. The latter process has been suggested previously by Macfarlane (13)and is based upon the desorption model of Norskov and Lundqvist (27). In this model, clusters of two similar molecules are desorbed and after desorption are separated either as neutrals or with the transfer of an electron from the highest occupied orbital of one molecule to the lowest unoccupied orbital of the other to form a positive and a negative ion. The two dissociation channels differ in energy by AH = EA + IP (where EA = electron affinity and IP = ionization potential). The extension of this model to the protonated, even electron, ions observed in desorption mass spectrometry requires that protons rather than electrons be transferred. Differences in proton affinity are, in general, much lower than the energies involved in electron transfer, so the cluster could dissociate to form the protonated insulin ion provided sufficient internal energy exists to overcome the coulombic attraction between the charged species. This mechanism has the attractive feature that desorption as a neutral will be easier than that of an ion and that species with proton affinities very closely matched with that of the sample will readily produce proton transfer. Strong proton donors may tend to preform sample ions whose desorption will be less favorable than neutrals, which dissociate in the gas phase.
CONCLUSIONS We note the remarkable similarity between the changes in plasma desorption mass spectra through the addition of gluathione (16)and those produced using a nitrocellulose backing (15). These include enhancement of the molecular ion signal, reduction of tailing of peaks and base-line signal due to metastable decomposition, decrease in the peak widths, and increases in multiply charged ions. The nitrocellulose technique was intended to reduce the electrostatic attractions of molecular ions to the metal substrate by promoting nonionic binding to the surface. The changes observed in the spectra indicate that as a result even multiply charged species are more easily desorbed and the ions have lower internal energy. While the mechanisms involved in the use of glutathione are not entirely understood a t this point, it seems clear from the spectra that a similar result is produced. On the other hand,
1307
the role of glutathione in promoting and preserving native forms of peptides or reducing aggregation may be equally important. While not completely understood, the glutathione matrix appears to have a beneficial analytical usefulness for large peptides comparable to that of the glycerol (and related) matrices used in fast atom bombardment.
ACKNOWLEDGMENT We are grateful to R. E. Chance and B. H. Frank, Lilly Research Laboratories, for providing several of the samples used in this study. Registry No. Bovine insulin, 11070-73-8; equine insulin, 11061-70-4; porcine insulin, 12584-58-6; bovine proinsulin, 11062-00-3;human proinsulin, 67422-14-4;lysozyme, 9001-63-2; carboxypeptidase, 9031-98-5; glutathione, 70-18-8.
LITERATURE CITED (1) . . Surman. D. J.; Vickerman, J. C. J . Chem. SOC.,Chem. Commun. 1981,324. (2) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. J . Chem. SOC.,Chem. Commun. 1981, 325. (3) Barber, M.; Bordoli, R. S.; Elliott, G. J.; Sedgwick, R. D.; Tyler, A. N.; Green, B. N. J . Chem. SOC.,Chem. Commun. 1982, 938. (4) Orth, R. G.; Jonkman, H. T.; Michl, J. J . Am. Chem. SOC.1982, 704, 1834. (5) Cooks, R. G.; Busch, K. L. Int. J . Mass Spectrom. Ion Phys. 1983, 53,111. (6) Heller, D. N.; Fenselau, C.; Yergey, J. A.; Cotter, R. J.; Larkln, D. Anal. Chem. 1984. 56. 2274. (7) Whitten, J. L:; Schaffer, M. H.; O'Shea, M.; Cook, J. C.; Hemling, M. E.; Rhinehart, K. L., Jr. Biochem. Biophys. Res. Commun. 1984, 724, 350. (8) Ackerman, 8.; Watson, J.; Holland, J. Anal. Chem. 1985, 57, 2658. (9) Macfarlane, R. D.; Torgerson, D. F. Sclence (Washington, D.C.) 1976, 797,920. (10) Cotter, R. J. Anal. Chem. 1984, 56, 2594. (1I) Sundqvist, 6.; Kamensky, I.; Hakansson, P.; Kjellberg, J.; Salehpour,
M.; Widdiyasekera, S.; Fohlman, J.; Petersen, P. A,; Roepstorff, P. Biomed. Mass Spectrom. 1984, 7 7 , 242. (12) Macfarlane, R . D. Phys. Scr. 1983, T 6 , 110. (13) Macfarlane, R. D. Acc. Chem. Res. W82, 75, 288. (14) Jordan, E. A.; Macfarlane. R. D.; Martin, C. H.; McNeal, C. J. Int. J . Mass Spectrom. Ion Phys. 1983, 53, 345. (15)Jonsson, G. P.; Hedin, A. 6.;Hakansson, P. L.; Sundqvist, B. U.; Save, G. B. S.; Nielsen, P. F.; Roepstorff, P.; Johansson, K.-E.; Kamensky, I.; Lindberg, M. S. L. Anal. Chem. 1988, 58, 1084-1087. (18) Cotter, R. J.; Honovich, J.; Olthoff, J.; Demirev, P.; Alai, M. In Ion Formation for Organlc Sollds; Benninghoven, A., Ed.; Springer-Verlag: Berlln, 1988. (17) Ghelis, C.; Yon, J. Protein Folding; Academlc Press: New York, 1982; pp 225-297. (18) Nau, H.; Biemann, K. Anal. Biochem. 1978, 73, 175. (19) Hass, G. M.; Nau, H.; Biemann, K.; Grahn, D. T.; Ericsson, L. H.; Neurath, H. Blochemistry 1975, 74, 1334. (20) Demirev, P.; Alai, M.; van Breemen, R.; Cotter, R. J.; Fenselau, C. Proceedings of Flfth Internatlonal Conference on Secondary Ion Mass Spectrometry; Washington, DC. In press. (21) Saxena, P.; Wetlaufer, D. B. Biochemlstry 1970, 9 , 5015. (22) Bringer, J.; Heldt, A.; Grodsky, G. M. Diabetes 1981,30, 83. (23) Blundell, T. L.; Dodson, G. G.; Dodson, E. J.; Hodgkin, D. C.; Vijayan, M. frog. Horm. Res. 1971,27, 140. (24) Albisser, A. M.; Lougheed, W.; Perlman, K.; Bahoric, A. Diabetes
1980,29, 241. (25) Lougheed, W. D.; Woulfe-Fionagon, H.; Clement, J. R.; Aibisser, A. M. Diabetologla lB80, 79,1. (26) Blackshear, P. J.; Rhode, T. D.; Palmer, J. L.; Wigness, B. D.; Rupp, W. M.; Buchwald, H. Diabetes Care 1983, 6 , 387. (27) Norskov, J. K.; Lundqvist, B. I. Phys. Rev. B 1979, 79,5861.
RECEIVED for review December 16,1985. Accepted February 1, 1986. This work was supported in part by Grant PCM82-09954 from the National Science Foundation and an Institutional Research Grant (NIH) from The Johns Hopkins University. Work was carried out at the Middle Atlantic Mass Spectrometry Facility, an NSF Shared Instrumentation facility.
