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Anal. Chem. 1986, 58, 1076-1080
RECEIVED for review August 12, 1985. Accepted November 13,1985. The FF portion of the work was one of the activities of the Rockefeller Extended Range Mass Spectrometric Research Resource supported by the Division of Research Re-
sources, NIH. The magnetic sector mass spectrometric determinations were carried out a t the Mid Atlantic Mass Spectrometry Laboratory-NSF shared instrumentation facility (NSF PCM8209954).
Fast Atom Bombardment-Tandem Mass Spectrometry Studies of Organo-Alkali Metal Ions of Small Peptides Larry M. Mallis and David H. Russell*
Department of Chemistry, Texas A&M University, College Station, Texas 77843
Tandem mass spectrometry (TMS) comblned with fast-atom bombardment (FAB) Ionization Is potentlally useful for the structural characterizationof blomolecules. The advantages of the FAB-TMS experiment have been discussed In previous works, with the major advantages usually relatlng to mlxture analysls. I n thls work, FAB-TMS Is used to study the dlssoHI' and [M Ne]' Ions of a clatlon reactlons of the [M small peptide, i.e., hippuryl-L-hlstldyl-L-leucine. Thls work shows that addltlonal lnformatlon can be gained by comparing the dlssoclatlon reactions of these two dlfferent lonlc forms. Owlng to the specific blndlng of Na' In the organo-alkall metal Ion, the collision-Induced dlssoclatlon spectrum of the [M 4- Na]' Ion of hlppuryl-L-hlstldyl-L-leuclne shows characterlstlc dlssoclatlon reactlons not observed for the [M H]' ion.
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+
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Although fast-atom bombardment (FAB) ionization has proven to be a versatile and sensitive ionization method for many biological compounds (1-10), the FAB-MS spectra are frequently complicated by the presence of impurities and/or adduct ions of the sample and liquid matrix (11-13). For these reasons, several workers have proposed the use of tandem mass spectrometry (TMS) in combination with FAB ionization for structural characterization of large molecules. Several reports dealing with the use of FAB-TMS for structural characterization of biomolecules have appeared in the literature (8, 14-1 7). Specific examples of such work are the cyclic peptide studies by Gross et al. (16) and the work of Amster and McLafferty on cobalamines (17). We wish to make a specific point regarding the reported collision-induced dissociation (CID) spectra of large biomolecules, viz., the low relative abundances of many of the structurally significant CID product ions, e.g., fragment ions characteristic of the amino acid sequence of peptides (15). The low abundances of the structurally significant ions in the FAB-TMS spectra of peptides are related to the large number of reaction channels available to the collisionally activated ion. During the development of mass spectrometry, several methods for enhancing specific dissociation reactioiis have been proposed. These methods rely on producing dissociating ions with a narrow range of internal energies, e.g., ion/molecule reactions or chemical ionization (18-22), studies of metastable ions (23, 24), photodissociation methods (25), and angle- and energyresolved CID (26-28). Alternatively, chemical derivatization reactions of specific functional groups can be employed to enhance specific dissociation reaction channels (29). 0003-2700/86/0358-1078$01.50/0
A potentially useful and simple method is described in this paper for enhancing the structural information obtained by FAB-TMS. The method relies upon the comparison of the FAB-TMS spectra of [M + H]+ ions and organo-alkali metal ions of the form [M + A]+ or [M xA - ( x - l)H]+, where A is the alkali metal and x = 1-3. Owing to the fact that molecules such as peptides and/or sugars contain highly polar functional groups and that these functional groups have different H+ and A+ ion affinities, it follows that the binding sites of H+ and A+ may differ. Thus, the site of interaction of the H+ or A+ ion with the molecule as well as the nature of the ionic complex may give rise to significant differences in the FAB-TMS spectrum. Although such changes have been observed in secondary ion mass spectrometry (SIMS) spectra (30),the data reported in this paper for the FAB-TMS of the hippuryl-L-histidyl-L-leucine (HHL) [M + HI+ and [M + A]+ ions is a striking example of this behavior. In this paper, FAB-TMS spectra for the [M + H]+ and [M A]+ ions (A = Li, Na, K, Rb, and Cs) of hippuryl-Lhistidyl-L-leucine (HHL) are reported. First the striking differences in the FAB-TMS spectra for the [M + H]+ and [M Na]+ ions of HHL are interpreted in terms of structural differences of the ionic complex. Second, the FAB-TMS spectrum of [M Na]+ ion is compared with the FAB-TMS spectra for the other [M A]+ ions and these resultskare discussed in terms of the stability of the ionic complex. Last, the FAB-TMS spectrum of the [M 2Cs - H]+ ion is discussed and compared with the spectra of the [M + A]+ ions.
+
+
+
+
+
+
EXPERIMENTAL SECTION The studies reported here were performed with a Kratos MS-50 triple analyzer (31), in the fast atom bombardment (FAB) ionization mode. The FAB ion source used for these studies was the standard Kratos system, equipped with an ION TECH 11-NF saddle field atom gun. Xenon was used for the bombarding fast atom beam; typical operating conditions were beam energies of 6-8 keV and neutral beam currents equivalent to 20-30 PA measured on an ION TECH (Model B 50) current and voltage regulator/meter. Collision-induced-dissociation(CID) studies were performed in the mass-analyzed ion kinetic energy (MIKE) scan mode, with helium target gas and an incident ion potential of 8 kV (31). All CID spectra were recorded with a collision gas pressure corresponding to a 50% attenuation of the molecular ion beam. A signal-to-noise ( S I N )ratio of 5:l for small peaks and greater than 75:l for larger peaks in the CID spectra was obtained by signal averaging 16 scans (at a rate of 20 s/scan), using a Nicolet Instrument Corp. 1170 (Model 172/2) signal averager. Spectra were plotted on a standard X-Y recorder. A solution of HHL was prepared by dissolving ca. 1.5 mg of sample (obtained from Aldrich Chemical Co.; 85,905-2)in 500 p L 0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
1077
Table 11. Mass, Relative Abundance and Proposed Composition of Observed Ions in the [M Na]' CID Spectrum of HHL
+
m/za
338
[M+Na]'
\
284
Na' c
0 OE
0 5E
1.OE
ESAVoltage
Figure 1. Collision-Induced dissociation (CID) spectra of the (a) [M HI+ (mlz 430) and (b) [M Na]' ion ( m / z452) of HHL.
+
+
Table I Mass, Relative Abundance, and Proposed ComDosition of Observed Ions in the [M HI' CID Spectrum of HHL
+
m/za
re1 intens, %
430 413 385 373 341 316 299 269 251 224 209. 193 176 161 133
20 14 8 9 6 40 39 4 4 4 6 3 7 8
7 100 39 8
121
110 105 93 81 18
17
6
structural assignmentb [M + HI' [M + H - OH]+ [M + H - COZH]' [M + H - (CH&H(CHJ,]' [M + H - CH(CHJ2 - COzHz]' [c + HI' [fragment d]+ [b(U + HI+ [b(l) + H - OH]' [b(l) + H - COZH]' [b(l) + H - CH&H(CH,),]' [b(l) + H - CH&H(CH3)2 - OH]' [fragment fl+ [M - W l + [f - NH - CO]+ [a + NH]+ [(b(l) + b(2)) + HI+ [fragment a]+ + b(2)) - "1' [(b(l) + b(2)) - NH - CHI+ [a + H - CO]'
re1 intens, %
structural assignmentb
[M + Na]+ [M + Na - OH]+ 25 [M + Na - COzH]+ 8 [M + Na - CH2CH(CH3),]' 31 [I + Na]' [c + Na]+ 100 50 [d + Na]' [e + Na]+ 64 [b(l) + Na - CHzCH(CH,)2]t 8 10 [f + Na]' 6 [b(l) + Na - 114(c)]+ [M + Na - b(1) - Colt 12 13 [(b(l) + b(2)) + Nal+ 20 Walt a Masses were calculated by using the equation mi = ml(E,/Eo), where Ei is the measured ESA voltage corresponding to mi (the mass of the fragment ion) and E, is the measured ESA voltage corresponding to m, (the mass of the parent ion). The calculated masses are accurate to m / z i l . bLettersb(l), b(2), c, d, e, f, apd I correspond to those CID product ions described in the Results and Discussion section and displayed in Figure 2.
452 435 407 394 363 338 321 294 232 200 177 157 132 23
5
l,.-mes were calculated by using the equation mi = ml(Ei/Eo), where Ei is the measured ESA voltage corresponding to mi (the mass of the fragment ion) and E,,is the measured ESA voltage corresponding to m, (the mass of the parent ion). The calculated masses are accurate to m/z il. bLetters a, b(l), b(2), c, d, and f correspond to those CID product ions described in the Results and Discussion section and displayed in Figure 2. an
of HPLC grade methanol (Fischer Chemical Co.; A-452). Typically, 2 KLof this solution was placed on a gold-plated copper probe tip and air-dried. To this sample, ca. 2 pL of a 1:lmixture of thioglycerol/glycerol (liquid matrix) and ca. 1pg of the alkali metal chloride salt were admixed on the probe tip.
RESULTS AND DISCUSSION The dissociation reactions of several sugar molecules ionized by FAB-TMS, including both the [ M HI+ and [ M + xA - ( x - 1)H]+ (where x = 0-4), have been reported (32). For these sugar molecules the dissociation reactions of the [M H]+ and [M Na]+ ions are quite similar, with the mass of the fragment ions being shifted by the mass of the alkali metal. Conversely, the dissocation reactions of the [M + Na]+ ions of hippuryl-L-histidyl-L-leucine (HHL), are markedly different from the dissociation reactions for the [ M H]+ ions. The FAB-TMS spectra for the [M + H]+ and [M + Na]+ ions are shown in Figure 1,parts A and B, respectively. For convenience we have tabulated the mass, relative abundance, and structural assignment of the major CID product ions observed
+
+
+
+
Proposed origin of fragment ions observed in the CID spectra of (a) [M + HI+ and (b) [M 4- A]+ (where A = Na) of HHL. Flgure 2.
(in the [M + HI+ and [M + Na]+ ion FAB-TMS spectra) in Tables I and 11, respectively. In the FAB-TMS spectrum of the [M H]+ ion (mlz 430), two dominant ions are observed at m / z 105 and m/z 110. The m / z 105 ion is indicative of a benzoyl group (e.g., C,H,O+) and the m / z 110 ion is characteristic of peptides containing histidine residues (e.g., C,H8N3+) (8). These two ions correspond to fragments a and (b(1) + b(2)), respectively (Figure 2A). Two additional structurally significant fragment ions are also observed at m / z 269 and m / z 299. The m / z 269 ion
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ANALYTICAL CHEMISTRY, VOL. 58,
NO. 6, MAY 1986
is assigned to loss of the benzoyl glycine (hippuryl) portion of the molecule, while the mlz 299 ion is formed by loss of the leucine moiety, e.g., cleavage of the Leu-His peptide bond. These two ions correspond to fragments b(1) and d, respectively (Figure 2). The FAB-TMS spectrum of the [M Na]+ ion (mlz 452) of HHL is markedly different from the corresponding spectrum for the [M H]+ ion. In terms of structural significance, the most important ions are observed at mlz 363,338, 321, and 294. These ions account for ca. 70% of the total CID product ion current. Conversely, the two most structurally significant CID product ions (i.e., loss of hippuryl and leucine residues), in the FAB-TMS spectrum of the [M H]+ ion, account for less than 25% of the total product ions. The mlz 363 ion in the FAB-TMS spectrum of the [M + Na]+ ion is assigned to the simultaneous loss of CH(CH& and COzH from the leucine portion of the molecule (fragment I in Figure 2B). The mlz 338 ion (fragment c in Figure 2B; corresponding to loss of 114 amu) is explained as a hydrogen transfer from the leucine residue (R group) followed by cleavage of the NH-CH bond in the leucine moiety. Mechanistic details of this reaction are currently being studied further. The two remaining ions at mlz 321 and mlz 294 correspond to loss of the leucine residue and loss of leucine plus the adjacent (histidyl) carbonyl group (fragments d and e, respectively, Figure 2B). The CID product ions a t mlz 436 and m / z 407 correspond to loss of OH and COOH, common C-terminus dissociation reactions of amino acids and small peptides (6). Attention is also drawn to the mlz 132 ion, which is assigned as the histidyl moiety plus the Na+ ion (i-e.,fragment b, Figure 2A, plus Na+). Owing to the highly basic nature of the imidizole nitrogen, it is not surprising that the Na+ ion is preferentially attached to this position, viz., the Na+ ion is not bound to the carboxy terminus of the HHL molecule. It is also important to note that in all cases the CID product ions retain the Na+ ion. These combined results suggest that formation of an ionic complex, such as that shown in Figure 2B, would direct dissociation reactions involving the leucine residue, e.g., fragment ions I, c, d, and e shown in Figure 2B, to occur. Based on this reasoning, we suggest that specific binding sites in peptides, e.g., highly basic amino acid residues, can be employed to enhance specific and/or characteristic dissociation reactions. Preliminary investigation of the [M Na]+ ion of larger peptides (Le., Angiotensin I and 11) containing both histidine and arginine indicates preferential attachment of Na+ at the arginine residue (33). This attachment is not surprising since the R group of arginine is ca. lo6 times more basic than the R group of histidine. Highly specific attachment of Na+ and K+ ions is also indicated in the [M Na]+ and [M K]+ ions formed by Cs' desorption ionization of gramicidin S (34). In this case, the alkali metal ion is preferentially bound to the ornithine residues; ornithine being the most basic site of the molecule. I t is interesting to note that the relative enhancement of specific CID reactions is dependent upon the alkali metal ion. For example, the FAB-TMS spectra for [M + A]+ ions (A = Li, Na, K, Rb, Cs; see Figure 3) are quite different. In particular, the relative abundance of the A+ ion in the FAB-TMS spectra increases in the order Cs+ > Rb+ > K+ > Na+ > Li+. A graph of relative abundance of A+ vs. ionization energy (Figure 4A) yields a nonlinear decreasing plot on going from Cs to Na. Although the plot is nonlinear, it follows Stevenson's rule, i.e., formation of the lowest energy product ions is favored. Dissociation of the ionic complex to give structural information about the molecule is favored for A+ = Li+ and Na+, whereas, when A+ = K+,Rb+ or Cs', the favored product of dissociation of the ionic complex is A+. These results suggest that the ionic complexes involving Li+ and Na+ ions
CS'
+
+
+
+
+
+
I
I
Na
+
I I
/ 0 DE
0 5E
1 OE
ESA Voltage
+
Figure 3. CID spectra of the [M A]+ ion of HHL where A = Li (mlz 436), Na (m/ z 452), K (m/ z 468), Rb (rn l z 5 14), and Cs (m/ z 562).
Scheme I [M t A(AX),It
- HX
-1M
t A(AX)n-1 t A - H I *
I EM t 2 A - H l t
t (AX),.,
are intrinsically more stable than the ionic complexes involving the K', Rb', and Cs+ ions; Le., the stability of the ionic organo-alkali metal complex follows the order Li+ > Na+ > K+ > Rb+ > Cs+. Since the structure of the HHL molecule (Figure 2B) is so well tailored (in size) to the smaller alkali metals, the observed increase in the relative abundance of A+ in the FAB-TMS spectra may be attributed to steric factors. This is supported by the linear correlation for the Pauling scale of ionic radii for the alkali metal ions vs. the relative abundnace of A+ in the FAB-TMS spectrum (Figure 4B). Therefore, smaller alkali metals (in terms of ionic radius) with higher ionization energies would be more effective for investigating the dissociation reactions of [M + A]+ ions. Owing to the differences observed for the [M + H]+ and [M A]+ ions, it is of interest to examine the FAB-TMS spectra for the [M 2A - H]+ ion of hippuryl-L-histidyl-Lleucine. Several dissociation reactions are enhanced in the FAB-TMS spectrum of this ion. This could be explained by the results from earlier studies performed in our laboratory, which led us to believe that the mechanism for formation of [M 2A - H]+ differs from that of the [M + A]+ ion (35). We have proposed that [M 2A - H]+ ions are formed by dissociation of the [M A(AX),-l + A - HI+ ion (X = halide), which is formed from [M + A(AX),]+ via loss of H X (see Scheme I) (36). It is, therefore, interesting to note that, although the favored product ion in the FAB-TMS spectrum for the [M Cs]+ ion is the Cs+ ion (ca. 85% of the total product ions), several structurally significant ions are found in the FAB-TMS spectrum for the [M + 2Cs - H]+ ion (see
+
+
+
+
+
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
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Table 111. Mass, Relative Abundance, and Proposed Composition of Observed Ions in the [M 2Cs - HI' CID Spectrum of HHL
+
cs
m/za
re1 intens, %
structural assignment*
[M + 2Cs - HI' [M + 2Cs - H - (C02)l' [C + 2Cs - HI' [b(l) + 2Cs - HI' [M + 2Cs - H - CO~CS]' [M + 2Cs - H - CO&S - CH(CH,)2]' [M + 2Cs - H - (a + NH + Cs)]' [e + Cs - C3H3N2It [f + CSI' [M + 2Cs - H - (d + CS)]' 100 [CSI+ aMasses were calculated by using the equation m, = ml(E,/Eo) where E, is the measured ESA voltage corresponding to mi (the mass of the fragment ion) and Eo is the measured ESA voltage corresponding to ml (the mass of the parent ion). The calculated masses are accurate to m/z il. *Letters a, b(l),c, d, e, and f correspond to those CID product ions described in the Results and 694 650 581 533 518 475 443 337 309 264 133
5 3 3 3 4 5 6 19 5
Discussion section and displayed in Figure 2. 133 CS'
694 [M+ZCs-H]+
309
I.E. (eV)
0 OE
1 OE
0 5E
ESA Voltage
Figure 5. CID spectrum of the [M
+ 2Cs - HI+ ion (mlz 694) of HHL.
650 is assigned to the loss of C02. Attention is also drawn to the ions at mlz 475 and m/z 518. Although these ions are of lower relative abundance (as compared to the ions a t m/z 264, 309, 337, and 650), insight toward elucidating the structure of the [M 2A - H]+ ion as well as the [M + A]+ ion of HHL is obtained. The ion a t m / z 475 is assigned to the loss of C02Cs while the ion at m / z 518 corresponds to the simultaneous loss of C02Cs and CH(CH3) from the leucine portion of the HHL molecule. Similar observations for the [M 2Na - H]+ ion provide further evidence for the proposed structural model (Figure 2B) for the [M A]+ ion of HHL; the second alkali metal interacts with the free carboxy terminus of the molecule. One finds, therefore, that in many cases the attachment of an alkali metal can lead to simpler structural interpretations of small peptide molecules. It is possible that this interaction will be limited to the structural identification of small peptides containing basic amino residues such as histidine, arginine, etc. Investigations into the effect of alkali metal attachment on the fragmentation behavior of larger peptides that contain basic amino acid residues (e.g., angiotensin I and angiotensin 11) and peptides that do not contain basic amino acid groups (e.g., leucine-enkephalin) are presently under way. Registry No. HHL, 31373-65-6.
+
+
0.5
0.75
1.o
1.25
1.5
1.75
Ionic Radius (i) Figure 4. (A) Plot of relative intensity of A+ vs. ionization potential. (6)Plot of relative intensity of A+ vs. ionic radius. Figure 5, and Table 111), The most abundant CID product ions in the FAB-TMS spectrum of [M 2Cs - H]+ are mlz 264,309,337, and 650. The ion at mlz 264 is assigned as the leucine moiety plus one Cs+ ion, while the ion a t mlz 309 corresponds to the benzoyl plus glycine plus NH (hippuryl + NH)portion of the molecule (see Figure 2B) plus one Cs+ ion. The m / z 337 ion is formed by the simultaneous loss of the leucine plus CO portion of the molecule with the loss of the histidyl imidizole ring plus one Cs+ ion. The ion a t mlz
+
+
LITERATURE CITED (1) Barber, M.; Bordoli, R. S.; Sedgwlck, R. D.; Tyler, A. N. J. Chem. SOC.,Chem. Commun. 1981, 325. (2) Fenselau, C. "Ion Formation from Organic Solids"; Benninghoven, A,, Ed.; Springer-Verlag: Berlin, Heidelberg, New York, Tokyo, 1983; Springer Series In Chemical Physics, Vol. 25, p 90. (3) Fenwick, R. G.;Eagles, J.; Self, R. Biomed. Mass Spectrom. 1983,
IO, 382.
(4) Gaskeli, S.J.; Brownsey, B. G.; Brooks, P. W.; Green, B. N. Biomed. Mass Spectrom. 1983, 10, 215.
1080
Anal. Chem. 1986, 58, 1080-1083
(5) Williams, D. H.; Bradley, C.; Bojesen, G.; Santikarn, S.; Taylor, L. C. E. J . Am. Chem. SOC. 1981, 103,5700. (6) Williams, D. H.; Bradley, C. V.; Santikarn, S.:Bojeson, G. Biochem. J . 1982, 201, 105. (7) Desiderlo, D. M.; Katakuse, I.Biomed. Mass Spectrom. 1984, 1 1 , 55. (8) Barber. M ; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. Biomed. Mass Spectrom. 1982, 9 , 208. (9) Ulrich, J.; Guy, A.; Molko, D.; Teoule, R. Org. Mass Spectrom. 1984, 19, 585. (10) Cottrell, J. S.; Frank, B. H. Biochem. Biophys. Res. Common. 1985, 127, 1032. (11) Beckner, C. F.; Caprioli, R. M. Anal. Biochem. 1983, 130,328. (12) Puzo, G ; Prom6 J. C. Org. Mass Spectrom. 1985, 20,288. (13) Sweetman, B. J.; Blair, I.A,; Watterson, D. M.; Lukas, T. J. Presented at the 33rd Annual Conference on Mass Spectrometry and Allied Topics, San Diego, CA, May 26-31, 1985. (14) Tondeur, Y. Org. Mass Spectrom. 1985, 20, 157. (15) Heerma. W.; Kamerling, J. P.; Slotboom, A. J.; van Scharrenburg, G. J. M.; Green, B. N.; Lewis, I A. S. Biomed. Mass Spectrom. 1983, IO, 13. (16) Tomer, K. B.; Crow, F. W.; Gross, M. L. Anal. Chem. 1984, 56,880. (17) Amster, J. I.; McLafferty, F. W. A n d . Chem. 1985, 57, 1208. (18) Bombick, D.; Pinkston, J. D.; Allison, J. Anal. Chem. 1984, 56,396. (19) Carroll, D. I.; Nowlin. J. G.; Stillwell, R. N.; Hornlng, E. C. Anal. Chem. 1981, 53,2007. (20) Beuhler, R. J.; Flanigan, E.; Green, L. J.; Friedman, L. J . Am. Chem. SOC. 1974, 96, 3990. (21) Beuhler, R . J.; Flanigan, E.; Green, L. J.; Friedman, L. Blochemistry 1974, 13,5060. (22) Didonato, G. C.; Busch, K. L. Anal. Chim. Acta 1985, 171,233. (23) Katakuse, I.;Nakabushi, H.; Ichihara, T.; Sakurai, T.; Matsuo, T.; Matsuda, H. Int. J . Mass Spectrom. Ion Proc. 1984, 62, 17. (24) Morgan, T. G.; RabrenoviE, M.; Harris, F. M.; Beynon, J. H. Org. Mass Spectrom. 1984, 19, 315.
(25) Krailler, R. E.; Russell, D. H. Anal. Chem. 1985, 57, 1211. (26) McLuckey, S.A.; Cooks, R. G. "Tandem Mass Spectrometry"; McLafferty, F. W., Ed.; Wlley: New York, 1983; p 303. (27) LaPack, M. A.; Pachuta, S. J.; Busch, K. L.; Cooks, R. G. Int. J . Mass Spectrom. Ion Phys 1983, 53,323. (28) Singh, S.;Thacker, M. S.; Harris, F. M.; Beynon, J. H. Org. Mass Spectrom. 1985, 20, 156. (29) Lippstreu-Fischer, D. L.; Gross, M. L. Anal. Chem. 1985, 57, 1174. (30) Kambara, H.; Ogawa, Y.; Sekl, S. "Secondary Ion Mass Spectrometry SIMS IV"; Benninghoven, A., Okano, R., Shimizu, R., Werner, H. W., Eds.; Springer-Verlag: Berlin, Heidelberg, New York, Tokyo, 1984; Springer Series in Chemical Physics, Vol. 36, p 383. (31) Gross, M. L.; Chess, E. K.; Lyon, P. A,; Crow, F W.; Evans, S.;Tudge. H. Int J . Mass Spectrom. Ion Phys. 1982, 42,243. (32) Castro, M. E.; Mallis, L. M.; Russell, D H. J Am. Chem. SOC. 1985, 107,5652. (33) Mallis, L. M.; Russell, D. H., unpublished results. (34) Castro, M. E.; Mallis, L. M.; Russell, D. H., unpublished results. (35) Castro, M. E.; Russell, D. H. Presented at the 33rd Annual Conference on Mass Spectrometry and Allled Topics, San Diego, CA, May 26-31, 1985. (36) Mallis. L. M.; Russell, D. H. Presented at the 33rd Annual Conference on Mass Spectrometry and Allied Topics, San Diego, CA, May 26-31, 1985.
.
RECEIVED for review September 16,1985. Accepted December 2,1985. This work was supported by grants from the National Science Foundation (CHE-8418457) and the National Institute of Health-General Medical Sciences (GM33780-01). Partial support for LMM and funds used for the purchase of equipment for these studies were obtained from the TAMU Center for Energy and Mineral Resources.
Determination of Km and Vm,, for Tryptic Peptide Hydrolysis Using Fast Atom Bombardment Mass Spectrometry Richard M. Caprioli* and Laurence Smith' Department of Biochemistry and Molecular Biology and the Analytical Chemistry Center, The University of Texas Medical School at Houston, Houston, Texas 77030
Fast atom bombardment mass spectrometry was used to provide quantitative analytical data for the calculation of kinetic parameters for the action of trypsin on several small peptides. The method provides a unique capability in that spectfic molecular species may be followed independently of other components'inthe mixture. The analysis takes place within minutes of sampling the reaction and does not require any cleanup or chemical derivatiratlon procedures prior to analysis. The method is illustrated by determining K, and k,, for the hydrolysis of several peptides by trypsin. These results are compared with values for similar peptides that have been reported in the literature.
The determination of kinetic constants of enzymes for specific substrates has involved the use of a variety of analytical methods, which usually include spectroscopic and chromatographic techniques as primary quantitative tools. Much of this information has been obtained through the study of substrate analogues and chemically modified natural substrates in order to provide compounds that can be followed in the enzyme reaction with high sensitivity and specificity. Present address:
Proctor a n d Gamble Corp., Cincinnati, OH.
For example, kinetic constants for trypsin have been determined by using tosylarginine methyl ester and benzoylarginine ethyl ester, as well as various analogues. Although these data serve a valuable role in providing comparative kinetic constants for small substrates and the convenient assay of these enzymes, they do not necessarily represent constants that may be reasonably applied to estimate those for natural substrates. Work done thus far in the determination of K , for trypsin using specific peptide substrates has given values that are much greater than those for many synthetic substrates (1-3). For other enzymes, such data are sparse due to the difficulties encountered with the appropriate analytical methodology. Fast atom bombardment mass spectrometry (FABMS) is emerging as a powerful analytical tool for biomolecular analysis in studies involving the determination of ionic constituents in aqueous solutions (4). For enzyme kinetic studies, FABMS provides several basic capabilities. First, it has a very high degree of molecular specificity in that the mass spectrum can identify individual molecular species in mixtures, including specific charged forms of a given compound. Second, it can analyze most compounds directly in reaction mixtures without chemical derivatization and separation steps. Third, it is a very sensitive technique, which most often requires only a few nanomoles of sample in order to provide an analytical profile of the reaction of interest. Previous work has shown that FABMS can be used to follow enzyme reactions (5) and,
0003-2700/86/0358-1080$01.50/00 1986 American Chemical Society
