Amino acid and tripeptide mixture analysis by laser desorption Fourier

Many molecules of biochemical interest are thermally labile ... Detection limits of 10~u mol of the .... LD/FTMS, we decided to investigate the mechan...
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Anal. Chem. 1989, 6 1 , 1895-1900

Amino Acid and Tripeptide Mixture Analysis by Laser Desorption Fourier Transform Mass Spectrometry M. Paul Chiarelli and Michael L. Gross* Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588

Laser desorptlon (LD) at 1064 nm and Fourler transform mass spectrometry (FTMS) were lnvestlgated as a means of analyzing mixtures of amino acids and trlpeptldes. LD mass spectra were obtalned of a 15-component amlno acM mlxture consisting of 3 ng of each in admixture. The mlxture was desorbed from a copper probe at a power density of 2 X 10’ W/cm2. Ail components yleld samplespectfic ions of the (M H 2Na)’ type, but the relative abundances are not proportlonal to concentratlon. The most Important factors Infiuenclng the dlsproportlonate ion abundances are the dlfferences In the Individual amlno acld subllmatlon enthalpies. A five-component mlxture of trlpeptides wRh widely varylng solubllltles was also analyzed successfully. Fast atom bombardment mass spectrometry is a less successful approach than LD/FTMS for analysis of amlno acid and tripeptlde mixtures.

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INTRODUCTION Many molecules of biochemical interest are thermally labile and do not volatilize without decomposing. Mass spectrometric analysis of these molecules must be preceded by a sputtering or a desorption ionization process that preserves the structural integrity of the analyte molecule. Such “soft ionization” techniques have permitted the mass spectrometric analysis of many important biomolecules, particularly polynucleotides, polypeptides, and small proteins. One of the next major challenges in mass spectrometry is the analysis of complex mixtures, such as those that might result from the digest of a large protein. The most common type of desorption ionization employed for digest analysis has been fast atom bombardment (FAB) (1). FAB coupled with tandem mass spectrometry (MS/MS) has also proven to be a powerful tool for the sequencing of proteins (2). Collisional activation of peptide constituents of the digest and analysis by MS/MS can be used to determine the amino acid sequence. The utility of FAB is limited, however, because a liquid matrix is necessary for analyte solvation, and the nature of the matrix can have a profound effect on analyte response (3). Furthermore, the surface activities of the peptides in a given mixture also influence the relative responses (4). The more surface-active components of the peptide mixture preferentially desorb with respect to those peptides having greater matrix solubility, and the latter can be suppresed to the point where they are not observed in a mass spectrum at all (5). The discrimination may be minimized somewhat by the addition of other reagents to the matrix, as was shown for liquid secondary ion mass spectrometry (SIMS) (6). The development of continuous flow FAB is promising because the on-line coupling of a FAB probe to a high-performance liquid chromatograph (HPLC) appears to minimize the effects of competitive ionization (7). Some of these issues are discussed in a recent comprehensive review of peptide analysis by FAB mass spectrometry (8). Other desorption ionization techniques for peptide analysis appear to be less plagued by the discrimination problems of 0003-2700/89/036 1-1895$0 1.50/0

FAB. Field desorption (FD) was successful in identifying 28 amino acids in the digest of a 29 amino acid peptide (9). Plasma desorption (PDMS) is also a powerful means of analyzing peptides. PDMS was employed to determine the molecular weight of human interleukin-2. Moreover, digestion of this protein with CNBr gave five peptide fragments, all of which showed sample-specific ions after dithiothreitol reduction on the sample substrate (10). A comprehensive review of PDMS was also published recently (11). Solid secondary ion mass spectrometry (SIMS) is also an effective approach for analyzing peptides. Detection limits of mol of the peptide bradykinin were established by using SIMS and time-of-flight mass analysis (12). The purpose of this study is to investigate the utility of laser desorption a t 1064 nm and Fourier transform mass spectrometry (FTMS) for analyzing mixtures of amino acids and small peptides. The first portion of this investigation is focused on the mixture analysis of single amino acids because their chemical and physical properties are better known than those of peptides. The second part of the paper deals with the analysis of tripeptide mixtures. To date, most laser desorption (LD) studies of amino acids and small peptides have been directed a t understanding the nature of the desorption process itself. The desorption thresholds of amino acids and dipeptides resonant a t a desorbing wavelength of 266 nm were shown to be proportional to their absorptivities (13). Selectively deuterated amino acids were studied to distinguish functional-group-specific protonation reactions from “random” protonation reactions (14). Amino acids and small peptides were employed to show that UV LD analysis can be carried out on a few monolayers of sample (15) and that matrix assisted laser desorption can also be utilized (16). The latter research has produced particularly spectacular results for desorption of very high molecular weight proteins. Peptides were also used to study the desorption time profiles of ions and neutral molecules under IR desorption conditions (17). Infrared LD/FTMS was demonstrated to be successful for determining the peptide bradykinin (18). No peptide mixture analysis, however, has been undertaken by using LD mass spectrometry.

EXPERIMENTAL SECTION Laser desorption spectra were obtained by using a Quanta-Ray DCR-2 Nd:YAG laser and a FT mass spectrometer constructed in this laboratory (19) and interfaced to a Nicolet 1000 data system. The sample probe was interfaced to the analyzer cell through a 6.35-mm hole at the intersection of the excitation and receiver plates (20). The cell was a cubic design of 5.08-cm dimensions. The laser beam entered the cell along the opposing diagonal, striking the probe normal to the surface. The magnetic field was 1.2 T. A specially designed vespel cone was inserted into the opening of the FTMS cell to ensure reproducible probe placement. The laser probes were made of oxygen-free copper and were prepared by facing off in a mechanical lathe and subsequent sonication in methanol and dichloromethane for 15 min in each solvent. All the amino acids and tripeptide5 employed in this study were combined with an equal weight of NaCl prior to LD/FTMS analysis. No NaCl was used in the FAB determinations. Except for the amino acid detection limit determination, 0 1989 American Chemical Society

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ANALYTICAL CHEMISTRY, VOL. 61, NO. 17, SEPTEMBER 1, 1989

ASN

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1

do

1

io

140

160 MASS

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IN A . M . U . Figure 1. LD/FTMS spectrum of a 15-component amino acid mixture combined with an equal total weight of NaCl acquired at a wavelength of 1064 nm and a power density of 2 X lo* W/cm2. The (M - H + 2Na)' ions are labeled by the three-letter codes of the corresponding amino acids. a total of 10 pg of sample was applied to the laser probe. Singlet-shotLD spectra were obtained at a wavelength of 1064 cm2. The pulse nm. The area of the laser spot was 1.6 X width of the laser was 140 ps. The power of the laser was measured immediately prior to the experiment by using a Scientech Model 362 meter and measuring the average powder output when the laser was operated at 10 pulses/s. The FAB MS experiments were conducted with a Kratos MS-50 triple analyzer equipped with a standard Kratos FAB source (21). The primary beam was 6-8-keV argon atoms at a total current of 2 mA at the cathode of the gun. All amino acids and tripeptides used in this investigation were obtained from Sigma Corp. and were used without further purification.

RESULTS AND DISCUSSION Amino Acid Mixture Analysis. It is necessary to evaluate the generality and detection limits of 1064-nm LD/FTMS for amino acid mixture analysis. Figure 1 shows a 15-component amino acid mixture with 10 ng of each applied to the laser probe. The best sensitivity was found under more nearly "thermal" desorption conditions (22) at a power density of 2 X lo6 W/cm2; 3 ng of each amino acid gave a detectable signal. All the amino acids give sample-specific ions of the (M - H + 2Na)+ type. Some of the larger amino acids also desorb as (M + Na)+ ions (e.g., tryptophan and phenylalanine). FAB mass spectra were obtained for comparison. The best results were found with FAB by using a glycerol matrix; 10 of the 15 amino acids gave (M + H)+ions when 350 ng of each was present in the matrix (Figure 2). In these experiments, LD/FTMS is superior to FAB/MS in sensitivity and relative response for the analysis of amino acid mixtures. The relative ion abundances observed in the LD/FTMS spectra indicate that mixture component discrimination is operative. Because the amino acids were applied in equal weights, one would expect the glycine ion molar response or abundance to be 2.7 times that of tryptophan if their LD characteristics are the same. The larger amino acids, however, are desorbed more abundantly in general (e.g., compare the abundances of glycine and alanine to those of histidine and

Table I. Ion Abundance Ratios from the Laser Desorption of Glycine and Other Alkyl Amino Acids in Binary Mixtures in an NaCl Matrix at a Wavelength of 1064 nm and a Power Density of 2 X lob W/cm2

amino acid

R group

ratio on probe

ion abundance ratio"

Ala Val Leu

CH3 CH(CH& CHCH2(CHJ2

1.2 1.5 1.6

0.37 f 0.07 0.24 f 0.07 0.29 f 0.05

a Ratio of abundance of Gly to the abundance of the other amino acid.

tryptophan in Figure 1). In order to improve the utility of LD/FTMS, we decided to investigate the mechanism(s) responsible for the discrimination. Binary mixtures of amino acids of differing chemical and physical properties were desorbed, and their relative sample-specific ion abundances were compared to obtain insight into the mechanisms responsible for the discrimination. Role of Amino Acid Mass. In the first set of experients, binary mixtures of glycine and other alkyl amino acids were codesorbed under the same conditions as for the 15-component mixture to test if molecular mass affects the desorption efficiency. These amino acids possess similar side chains (Table I) and pK,'s. All the ions were of the (M - H 2Na)+ type. The glycine/alkyl amino acid ion abundance ratios tend to decrease as the size of the alkyl side chain on the amino acid increases (Table I). The results are consistent with the apparent trend seen for the 15-component mixture. The implications are discussed later in the text. Role of Acid/Base Characteristics. In the next set of experiments, binary mixtures of amino acids of different pK,'s were desorbed. One might expect that tendencies for the individual amino acids to form (M - H + 2Na)+ ions are dictated at least in part by the relative carboxyl group acidities; that is, the smaller the pKa the larger the (M - H + 2Na)+ abundance. Binary mixtures of cysteine, valine, and threonine were chosen for these experiments because the pKal's are quite

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M

LEI

M (~114) M(~114

M ( x 114

4d

ME Ti

I20

ao

100

140

PHE

I

I60

M/Z Figure 2. FAB MS spectrum of a 15-component amino acid mixture in a glycerol matrix. The (M are labeled by the three-letter codes. Ions from the matrix are labeled as "M". Table 11 A. pK, Values for the Amino Acids Cys, Val, and Thr

amino acid

PKd

pKa2

pK side chain

cysteine valine threonine

1.71 2.32 2.71

8.18 9.62 9.62

10.28

B. Ion Abundance Ratios of (M - H + 2Na)+Ions Desorbed from Binary Mixtures of Cys, Val, and Thr in an NaCl Matrix amino acids

ions ratioed

abundance ratios"

Val/Thr Cys/Thr

(m/z 162)/(m/z 164) (m/z 166)/(m/z 164) ( m / z 166)/(m/z 162)

0.24 f 0.06 0.21 + 0.02 0.77 f 0.05

Cys/Val

"N

= 8 determinations.

different for the amino acids, and their masses are approximately equal (Table 11). The conditions employed and the types of ions desorbed are the same as discussed above. On the basis of the trend in pK,,, cysteine should yield the largest ion abundance and threonine the smallest, but just the opposite trend is observed. This is good evidence that the acid/base characteristics of the amino acids have little influence on their tendency to form (M - H + 2Na)+ ions under thermal laser desorption conditions. The relative ion abundances, however, do suggest that the desorption characteristics of the amino acids are governed by intrinsic properties rather than by intermolecular interactions. Because the Cys/Thr and the Val/Thr ion abundance ratios are similar (Table 111, the Cys/Val ratio is expected to be close to unity, and it is. Because the desorption characteristics of the three amino acids are not governed principally by their pK,'s, some other physical property must be responsible for the observed results. Some "thermal" decompositions may be competing, as was

200

220

240

4- H)' ions of the 10 amino acids detected

found for sulfur-containing amino acids (methionine and cysteine) in Tian-Calvet calorimetry studies (23). For example, cysteine does not sublime at ca. 400 K because it decomposes. Role of Sublimation Enthalpy. A t this point it seemed logical to consider the role of sublimation enthalpy in amino acid desorption. The temperature dependence of the sublimation enthalpies of glycine, alanine, and proline was previously investigated with Tian-Calvet calorimetry (24, 25). To obtain an estimate of the sublimation enthalpies of these amino acids under the conditions employed for laser desorption, the temperature of the irradiated copper probe must be known. The temperature rise on the copper surface may be estimated by the expression in eq 1

T(0,t)= [2(1 - R)F0/K][Kt/T]'/2

(1)

(26)where R is the reflectivity of the copper at 1064 nm, Fo the power density of the laser, K the thermal diffusivitty, K the thermal conductivity, and t the length of the laser pulse in seconds. The values (27) of these physical parameters for copper are R = 0.92, K = 3.74 W/(deg cm), and K = 1 cm2/s. The calculation yields a maximum surface temperature at 800 K. This value is undoubtedly higher than the actual desorption temperature for several reasons. The expression assumes the laser beam is of infinite radial extent so it does not account for the diffusion of energy into the nonirradiated area around the laser spot or for diffusion of energy into the bulk copper during the time of irradiation. The desorption of ions and neutrals may also proceed before all the energy is absorbed; time-resolved studies under conditions similar to those used here (22) show that ion desorption proceeds on a microsecond timescale, and the laser pulse length used here was 140 ws. There is a relation between the relative ion abundance ratios obtained from laser desorption of binary mixtures and the

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Table 111 A. Enthalpies of Sublimation Estimated for Gly, Ala, and Pro at 800 K amino acids

enthalpy, kJ/mol

ref

GlY Ala

108.9 91.0 22.1

24 24 25

Pro

B. (M - H + 2Na)+ Ion Abundance Ratios of Gly, Ala, and Pro Desorbed from Binary Mixtures in an NaCl Matrix amino acids

ion abundance ratios

NO

GlyJAla Gly/Pro AlaiPro

0.37 f 0.07 0.08 f 0.01 0.45 f 0.09

8 12 10

Table IV. Enthalpies of Sublimation for Alkyl Amino Acids Determined at 455 K by the Knudsen Cell Effusion Met hodn

(I

amino acid

enthalpy, kJ/ mol

GlY Ala Val Leu

136.5 138.1 162.8 150.7

Reference 28.

5

I\

0.9 ' , O h

" N = number of determinations.

difference in sublimation enthalpies of the amino acid constituents of the mixtures (see Table 111). Estimates of the sublimation enthalpies at 800 K were made from literature trends (Table IIIA), and the relative ion abundances were obtained under the laser desorption conditions described earlier in the paper (Table IIIB). Because proline has the lowest sublimation enthalpy, the ion abundance ratios of Gly/Pro and Ala/Pro should be less than unity, and the Gly/Pro ratio should be smaller than Ala/Pro. These predictions are in accord with the experimental data. We also expect that the Gly/Ala abundance ratio should be less than unity; a value of 0.37 is obtained. On a quantitative basis, however, the Gly/Ala abundance ratio should be nearly equal to unity, and, on the basis of the Gly/Pro abundance ratio, the Ala/Pro is expected to be smaller than 0.45 and nearly equal to that of Gly/Pro. We conclude that the model gives semiquantitative predictive capability. A possible weakness in the correlation is that the sublimation enthalpy measurements may include some contribution from decomposition of the amino acids. The extent of decomposition, however, cannot be assessed on the basis of the literature studies of sublimation. Furthermore, there is not a necessary parallel between the thermodynamic quantity (sublimation enthalpy) and the kinetics of desorption. The results of the sublimation study of cysteine (23) mentioned above suggest that no (M - H 2Na)+ should desorb under these conditions if LD were governed only by thermodynamic factors. The low-abundance (M - H + 2Na)+ ions that are observed probably arise by means of nonequilibrium, rapid desorption. The desorption may be evidence for a kinetically driven, nonequilibrium component of the overall desorption. This component is minor for most of the amino acids (except cysteine), but it becomes more important a t higher laser powers. If sublimation enthalpies have a major influence on desorption characteristics of amino acids, then the desorption behavior a t other temperatures should be predictable on the basis of trends of sublimation enthalpies as a function of temperature. Knudsen cell calorimetry was previously employed to determine the sublimation enthalpies of many of the amino acids at 455 K (see Table IVA for values for the alkyl amino acids (28)). At this temperature, glycine has the smallest sublimation enthalpy. The Tian-Calvet calorimetry studies discussed above were conducted for the purpose of extrapolating the sublimation enthalpies of proline, alanine, and glycine to 298 K under standard conditions; the resulting enthalpies are 149 f 4.0, 144 f 4.2, and 138.2 f 4.6 kJ/mol, respectively. On the basis of the Tian-Calvet and the Knudsen cell data, the sample-specific ion abundance of glycine is expected to

+

0,21 0 .I 0

VAL

100

200

300 400 L A S E R ENERGY (mJ)

500

Figure 3. Plot of the ratios of the total samplespecific ion abundances (those of (M - H 2Na)+,(M Na)', and (M + H)') of glycine to alanine, valine, and leucine against laser energy at a wavelength of 1064 nm. For simplicity, points and error bars are shown only for alanine to illustrate typical data.

+

+

increase with respect to the ion abundances of the other amino acids as the laser power is decreased. The alkyl amino acids were chosen for these determinations because their pKa)s are similar, and therefore the energies of formation for each type of sample-specific ion should also be similar. The trends in ion abundance ratios shown in Figure 3 are consistent with the variation of sublimation energies of the three alkyl amino (M + Na)+, and (M acids. Three types of ions, (M + H)+, H 2Na)+, were observed over this power density range; the first two show increasing abundances a t lower laser energies, consistent with the results of UV laser desorption on a time-of-flight mass spectrometer (13). Tripeptide Mixture Determinations. The success obtained with the amino acid mixture analyses prompted the application of 1064-nm LD and FTMS to tripeptide mixtures. First, two binary mixtures of tripeptides were desorbed to ascertain the effect of amino acid sequence on the relative sample-specific ion abundances. Then four- and five-component mixtures of tripeptides were analyzed to assess the influence of solubility and surface activity on ion formation. The LD conditions that were utilized In the analysis of the 15-component amino acid mixture were employed throughout these investigations. Most of the sample-specific ions observed are of either the (M - H 2Na)+ or (M + Na)+ type, the former being generally more abundant. Each of the tripeptides Ala-Pro-Gly (APG) and Pro-Gly-Ala (PGA) was mixed with Val-Gly-Gly (VGG) to give two binary mixtures, and the relative sample-specific ion abundances, (VGG)/(APG) and (VGG)/(PGA), were compared for amino acid sequence effects on ion formation. The ion abundance ratios should be equal if no sequence effects exist. These ratios, 0.28 i 0.03 and 0.39 f 0.05 for (VGG)/(APG) and (VGG)/ (PGA), respectively, (12 determinations each) show a small but significant difference. This difference may be

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ANALYTICAL CHEMISTRY, VOL. 61, NO. 17, SEPTEMBER 1. 1989

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attributed to the changes in the conformation of the peptide as a function of the position of the proline residue. Proline is known to cause bends in peptide chains (29). Sublimation enthalpies may also play an important role in determining abundance differences, but these enthalpies are not known for tripeptides. Sensitivity of sublimation enthalpy to temperature is theoretically proportional to the difference in the heat capacities of the material in the gas and solid states: Cp(g)- Cp(s)(30). Because the heat capacity difference should increase with the number of atoms (number of vibrational modes), sublimation enthalpies of peptides should decrease more rapidly as temperature is increased than those of amino acids. This effect forecasts opportunities for large peptide analysis by laser desorption. A four-component mixture of tripeptides was also desorbed to see if differences in solubility and surface activity influence the observed ion abundances, as does occur for FAB (4). Bull-Breese indices (free energies) have been employed to describe relative differences in amino acid and peptide surface activities (4,5,31). The percent total ion abundances of the peptides employed in this study and their Bull-Breese indices are given in Table V. Good precision is seen for these relative ion abundances. The good reproducibility is taken to indicate that the composition of the solid is homogeneous across the probe surface, and a selective crystallization upon evaporation of the solvent is not apparent. A typical spectrum is given in Figure 4A. The larger percent total ion abundances associated with the larger tripeptides are consistent with the sublimation enthalpy theory proposed above. The large relative ion abundance of PGA with respect to GFA, however, suggests other mechanisms of desorption may also be operative.

Table V. Bull-Breese Indices and Relative Percent Ion Abundances for the Four Tripeptides Used in This Investigation tripeptide Gly-Phe-Ala Gly-Pro-Ala Val-Gly-Gly Gly-Gly-Gly

B

+ B indices:

cal

-843 -393 -520 0

a Calculated in the same manner as in ref 4. tions.

% ion abundanceb

0.25 f 0.01 0.47 f 0.04 0.22 f 0.02 0.05 f 0.01

* N = 8 determina-

A decidedly more surface-active peptide (FFF) was added

to the four-component mixture, and the mixture was desorbed to see if the precision of the relative total ion abundances is maintained. Triphenylalanine (FFF) has a Bull-Breese index of -2.33 kcal, which indicates that the solubility properties of FFF are different from those of the four tripeptides in the original mixture. Desorption of the five-component mixture gave relative ion abundances that are much more erratic than those observed for the four-component mixture. Some observations, however, are consistent with those seen in the analysis of the four-component mixture. The largest total ion abundances are due to either PGA or FFF; when the latter is dominant, the other components are of greatly reduced abundance. The trend in relative ion abundance among the other three peptides is the same as for the four-component mixture analysis. All five tripeptides were observed for each laser shot. The erratic relative ion abundances may be due to the selective crystallization and segregation of FFF as the solvent is pumped away. To verify this hypothesis, the five tripeptides

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were mixed in water, rather than in a 60:40 mixture of water and acetonitrile, because FFF is insoluble in water alone when added in the same proportions as the other tripeptides. The mixture to be deposited on the probe was withdrawn while it was stirred to maintain homogeneity. This mixture should simulate that formed as acetonitrile evaporates from the acetonitrile/water solution. The LD results are similarly as erratic as those obtained with the first solvent mixture, thus providing evidence for the selective precipitation of the FFF from the solvent. Because of the greater surface activity of FFF, it may aggregate in islands on top of the solid phase. FAB spectra of this five-component mixture were also acquired, and the best results were obtained when a thioglycerol matrix was used. All five tripeptides gave (M + H)+ ions, and less discrimination was found than for the LD determinations (Figure 4B) when equal amounts of the mixture were analyzed. In glycerol, the discrimination expected from the previously proposed surface activity hypothesis was observed, but thioglycerol appears to be a less discriminating matrix for peptide analysis, at least on the basis of the limited number of measurements made here. There may be at least two ways of improving the utility of LD/FTMS for peptide mixture analysis. Increasing the total laser power and area irradiated should minimize the effects that an inhomogeneous solid phase has on the observed ion abundances. Another approach is to use a nitrocellulose binder as is employed for PDMS (IO). Rinsing this binder with dilute peptide solutions may yield a more representative and uniform distribution of the peptides on the sample surface.

CONCLUSION LD/FTMS can be used for the mixture analysis of amino acids and small peptides at higher sensitivity than can be obtained by standard FAB MS. Variations in sublimation enthalpy and inhomogeneous solid phases formed by selective precipitation of peptide components of widely varying surface activity are problems. When peptides of widely differing solubilities (surface activities) are in the mixture, as in peptide mapping, mass spectra from several laser shots need to be acquired across the substrate surface to ensure that all peptides in the mixture are desorbed. The correlation of relative abundances of desorbed amino acid (M - H + 2Na)+ ions with sublimation enthalpies of the pure amino acids can be rationalized as follows. The amino acids exist in the solid state as zwitterions, +H,NCHRCOO-. In the presence of excess NaC1, the most likely species to desorb is not the zwitterion but neutral H,NCHRCOONa. This neutral salt molecule is cationized with gas-phase Na+ in the selvedge region close to the surface. The desorption tendencies of neutral HzNCHRCOONa species correlate with sublimation enthalpies of the amino acids themselves because the sublimation enthalpy of HzNCHRCOONa can be viewed as a sum of the sublimation enthalpy of the amino acid and that of the reaction to exchange a sodium ion for H+. The latter quantity is approximately a constant for the amino acids.

As a result, the former quantity accounts for the variations. As the laser power is decreased, there is insufficient gas-phase Na+ for the cationization reaction, and other species such as (M + Na)+ and (M + H)+ become important, as was observed.

ACKNOWLEDGMENT We thank Enrico Davoli and Kenneth Caldwell for their assistance in obtaining the FAB spectra, Don Rempel for his design of the vespel guide cone for the laser desorption studies, and Richard Grese for the artwork. LITERATURE CITED Barber, M.: Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. J . Chem. Soc., Chem. Comm. 1981, 325-327. Biemann, K.; Scoble, H. A. Science 1987, 237, 992. Lehmann, W. D.; Kessler, M.; Konig, W. A. Biomed. Mass Spectrom. 1981, 7 1 , 217-222. Clench, M. R.; Garner, G. V.; Gordon, D. B.; Barber, M. Biomed. Mass Spectrom. 1985, 72, 355-357. Naylor, S.; Findeis, A. F.; Gibson, B. W.; Williams, D. H. J . Am. Chem. SOC. 1986, 708, 6359-6363. Pettit, G. R.; Cragg, G. M.; Holzapfel, C. W.; Tuinman, A. A,; Gieschen, D. P. Anal. Biochem. 1987, 762, 236-241. Caprioii, R. M.; Fan, T.; Cottrell, J. S. Anal. Chem. 1886, 5 8 , 2949-2954. Biemann, K.; Martin, S. A. Mass Spectrom. Rev. 1887, 6 , 1-76. Shimonishi, Y.; Hong, Y-M.; Kitagishi, T.; Matsuo, T.; Matsuda, H.; Katakuse, 1. fur. J . Biochem. 1880, 172, 251-264. Jardine, 1.; Scanlan, G. F.; Tsarbopoulous, A,; Liberato, D. J. Anal. Chem. 1988, 60, 1086, 1087. Cotter, R. J. Anal. Chem. 1988, 60. 781A-793A. Steffens, P.; Niehius, E.; Friese, T.; Benninghoven, A. I n Secondary I o n Mass Spectrometry, SIMS I V ; Springer Series in Chemical Physics; Benninghoven, A., Ed.; Springer Verlag: New York, 1983; Vol. 25, pp 111-117. Karas, M.; Bachmann, D.; Hillenkamp, F. Anal. Chem. 1965, 5 7 , 2935-2939. Parker, C. D.; Hercules, D. M. Anal. Chem. 1886, 58, 25-30. Karas, M.; Hillenkamp, F. Anal. Chem. 1888, 6 0 , 2299-2301. Karas, M.; Bachmann, D.; Bahr, U.;Hilienkamp, F. Int. J . Mass Spectrom. Ion Processes 1887, 7 8 , 53-68. Tabet, J.-C.; Cotter, R. J. Anal. Chem. 1984, 5 6 , 1662-1667. Wilkins, C. L.; Wiel, D. A.; Yang, C. L. C.; Ijames, C. F. Anal. Chem. 1885, 57, 520-524. Ledford, E. B., Jr.; White, R. L.; Ghaderi, S.;Wilklns, C. L.; Gross, M. L. Anal. Chem. 1980, 5 2 , 2450, 2451. McCrery, D. A.; Ledford. E. B., Jr.; Gross, M. L. Anal. Chem. 1982, 5 4 , 1435-1437. Gross, M. L.; Chess, E. K.; Lyon, P. A.; Crow, F. W.; Evan, S.; Tudge, H. Int. J . Mass Spectrom. Ion Phys. 1982, 4 2 , 243-254. Cotter, R. J.; Van Breemen, R. 8.; Snow, M. I n t . J . Mass Spectrom. Ion Phys. 1983, 4 9 , 35-50. Sabbah, R.; Minadakis, C. Thermochim. Acta 1881, 43, 269-277. Nguav, S . N.; Sabbah, R.; Laffiie, M. Thermochim. Acta 1977, 2 0 , 371-380. Sabbah. R.; Laffitte. M. Bu//. Chim. SOC. 1978, 7 - 2 , 50-52. Ready, J. F. Effects of High Power Laser Radletlon; Academic Press: New York, 1971; 405 pp. Goldsmith, A.; Waterman, T. E.; Hlrschhorn, H. J. Handbook of Thermophysicalproperfies of SMMaterlels; MacMillan: New York, 1961. Svec, H. J.; Clyde, D. D. J . Chem. fng. Data 1865. 70, 151, 152. Zubay, G. Biochemistry; Addison-Wesley: Reading, MA, 1983; 1268 PP. Berry, R. S.; Rice, S. A.; Ross, J. Matter in Equilbrium: StatisNcal Mechanics; Wiiey and Sons: New York, 1980; p 804ff. Bull, H. B.; Breese, K. Arch. Biochem. Biophys. 1974, 761, 665-670.

RECEIVED for review December 6, 1988. Revised manuscript received May 11, 1989. Accepted June 1, 1989. This work was supported by the Midwest Center for Mass Spectrometry (NSF Grant CHE-8620177).