Anal. Chem. 1988, 6 0 , 1409-1415
1409
Resonant Two-Photon Ionization of Enkephalins and Related Peptides Volatilized by Using Pulsed Laser Desorption in Supersonic Beam Mass Spectrometry Liang Li and David M. Lubman* Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109
Pulsed laser desorption Is used to desorb neutral underbatired peptides including Leu- and MBtsnkephalin into the gas phase. The desorbed plume Is entrained into a supersonic jet that carrks the sample into a timed-fllght mass spectrometer where resonant two-photon ionlratlon (RPPI ) Is produced by an ultraviolet laser source. RPPI occurs readily in peptides containing tyrosine, tryptophan, and phenylalanine aromatlc groups through the T-T* transition at 266 nm. At the laser power used in thls study ( ( 1 4 ) X lo7 W/cm2) subsequent absorption of photons by the molecular ion occurs which resuits in extenslve fragmentation for structural analysis. The fragments produced include characteristic acyiium and aidimine ions as well as @ cleavage at the aromatic moiety. I n addition, a substantial molecular ion, M”, is observed In every case and (M OH)’ or (M COOH)’ ions are often also observed. N
-
-
Resonant two-photon ionization (RPPI) is a technique with several unique properties as an ionization source for mass spectrometry (1-9). In RPPI, ionization occurs following the absorption of two photons by a molecule in the presence of an intense light field. The absorption of the first photon excites the molecule to a real resonant excited electronic state (So S,) and the second photon ionizes the molecule. Thus, the sum of the two photons must be greater than the ionization potential (IP) of the molecule although the two photons may be either of the same or two different frequencies. Since the total amount of energy placed in the molecule is relatively low (7-13 eV), RZPI has generally been shown to be a soft ionization tool where either the parent ion only or minimal fragmentation results. Thus, RZPI can be used to obtain molecular ion information for identification in a mass spectrometer. Although other soft ionization methods exist, R2PI can produce molecular ions with efficiencies that may range up to several percent or higher within the laser beam during the pulse (I). In addition, as the laser beam power density is increased and additional photons are absorbed, extensive fragmentation is produced which can be varied as a function of the laser power. Thus RBPI/MPI (multiphoton ionization) provides greater control over fragmentation as compared to chemical ionization (CI), electron impact ionization (EI), and other classical ionization methods. In fact, as the laser power is varied, ions ranging from the molecular ion to C+ can be observed. A second unique feature of RZPI is the wavelength selectivity. In order to observe efficient resonance-enhanced ionization, the first photon must be in resonance with a real electronic state. Although ions are produced as the detected species, the signal observed reflects the absorption-excitation spectrum of the So SI transition. The result is that molecules can be selectively ionized in a mass spectrometer based upon their broad absorptions in the near-ultraviolet or visible region. More recently, supersonic jet expansions have been
-
-
used to increase selectivity by reducing the population of the internal modes of large molecules resulting in highly resolved features in the wavelength spectrum (1, 9). In this work, however, complete cooling is not obtained and selective absorption is based upon the excitation of molecules with aromatic groups. Previous R2PI studies have generally been limited to reasonably volatile systems and have only recently been applied to nonvolatile and thermally labile molecules using pulsed laser desorption (2-8). The laser desorption method utilizes a high-powered infrared laser pulse to rapidly desorb molecules from a surface before they have time to kinetically decompose. In this process both ions and neutrals are desorbed in a ratio that depends on the transient heating effect induced on the surface (10). Most studies to date have examined the small fraction of ions produced directly by laser desorption (LD) in a mass spectrometer (11-13). However, unless precharged ions exist on the surface, the yield of ions is generally very small compared to the large number of neutrals desorbed from the surface. In this work we focus on the R2PI of the neutral fraction provided by the desorption process. A pulsed COP laser provides desorption of neutral species which are entrained into a supersonic jet pulse and swept into a tirneof-flight mass spectrometer. R2PI is then produced by an ultraviolet laser. Thus,the desorption and ionization processes are now separated in time and space and the energies required for these two processes are independent of one another. In previous work, we have illustrated the use of the pulsed laser desorption method followed by laser multiphoton ionization for study of small peptides and other important biological and pharmaceutical molecules (3,4,14). In other work, Grotemeyer and co-workers (7) have used a similar technique for study of angiotensin I (a decapeptide) using a high-resolution V-shaped reflectron mass spectrometer in which the fragmentation pattern was studied as a function of laser power. In their work, isotopic peaks could be observed even for the molecular ion at m/z 1295. In the present study we use this technique to examine clinically important underivatized pentapeptides such as Leu- and Met-enkephalin and other related smaller peptides which make up the structural units of these species in order to understand the resulting fragmentation patterns for possible use in peptide sequencing. We demonstrate that RPPIIMPI can proceed through the H-H* transition of the aromatic moiety in the near-UV and that molecular ion information as well as fragmentation characteristic of the peptide is obtained. This fragmentation is induced by increasing the laser power density so that further absorption of photons by the molecular ion occurs (to a dissociative ionic state) following the initial RZPI step. The nonaromatic groups absorb further in the UV and ionization through these groups can occur only via nonresonant MPI at 266 nm. The cross section of this process has been shown to be negligibly low ( I ) , and therefore peptides lacking an aromatic amino acid would provide a spectrum of negligibly low intensity at this wavelength. Thus, efficient MPI can only
0003-2700/88/0360-1409$01.50/00 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60,NO. 14, JULY 15, 1988
be induced in these peptides with the use of vacuum UV or far-UV light or through absorption by the aromatic group in the near-UV region. In addition, a comparison with available data on other methods such as fast atom bombardment (FAB), FAB-MS/MS, direct laser desorption, and field desorption is discussed.
EXPERIMENTAL SECTION The experimental apparatus consists of a supersonic beam time-of-flight mass spectrometer (TOF-MS) which has been described in detail in previous work ( 4 ) . Laser desorption is performed with a COzlaser (20-40 mJ/pulse) focused onto the surface on which the sample is coated with a 10 cm focal length germanium lens. In order to desorb the pentapeptides, a slightly tighter focus is needed than for the di- and tripeptides (3). We estimate a power density on the surface of -5 x lo7 W/cm2 from the known temporal pulse profile of the laser (temporal pulse profile is an -80-ns spike followed by a 1-2-ps fall-off as provided by Spectra Physics, Inc., 19851, laser input energy, and spot size (2 mm). The sample is situated on a Macor machinable ceramic rod located about 4 mm downstream of the orifice and 4 mm from the molecular beam axis. The peptide material is deposited on the face of the rod by dissolving 100 Fg in methanol, placing the liquid or paste on the surface of the rod, and evaporating the solvent. These studies have not been optimized for sensitivity or quantitation since the aim of this study is an examination of the R2PI (MPI) mass spectra of these peptides and a sample large enough t o observe a sizable signal was used. Upon desorption, the plume of organic neutral molecules is entrained into a pulsed supersonic jet pulse of C 0 2 carrier gas. The actual sequence of events is controlled by several delay generators where the pulsed COz laser fires first to produce desorption followed by the pulsing of the valve. The two events are synchronized in time so that the desorbed plume is entrapped in the jet and carried into the acceleration region of the TOF-MS. Another delay generator is timed to pulse the laser as the jet reaches the TOF-MS. Laser R2PI is produced and the mass spectrum is recorded by a LeCroy 9400 digital oscilloscope. Carbon dioxide is used as the carrier gas in this experiment rather than argon for several reasons. The ability to use liquid N, to cryopump COz allows for a very low background pressure in the mass spectrometer. At 20 psi reservoir pressure and 10-Hz repetition rate (55 ws pulse) the pressure in the mass spectrometer remains below 5 X lo4 Torr. Also COP provides an increased collisional rate at longer distances from the orifice than a simple monatomic carrier (15). The desorption event occurs 4 mm downstream from the orifice which allows greater penetrability into the jet without producing shock waves as compared to performing desorption directly outside the orifice. The use of COz increases the subsequent effective collisional cooling in the jet. In previous work ( 2 ) , extensive translational cooling was obtained in the jet as demonstrated by the narrow ion peak widths obtained in the TOF-MS which result in a resolution of up to -600 depending upon whether the desorption is performed without inducing shock waves. More recently extensive rovibronic cooling has been demonstrated by using pulsed laser desorption entrainment into supersonic expansions for tyrosine and related metabolites (16). In this work though, we are interested in the products of MPI so that the cooling required for wavelength selectivity is not needed here. The ionization source is a frequency quadrupled Nd:YAG laser which generates 266-nm radiation. The typical laser energy is 10-15 mJ (- (1-5) X lo7 W /cm2) used t o induce fragmentation. In most experiments the ionizing laser beam was collimated by a positive-negative lens combination to a 2-3 mm diameter beam.
-
RESULTS AND DISCUSSION In this work we have studied the laser-induced MPI of Leuand Met-enkephalin which are two important neurotransmitters in the brain (27). Laser ionization was performed a t 266 nm which is absorbed by the aromatic moieties in the peptide chain. High laser power (1.5 X lo7 W/cm2) is used to induce extensive fragmentation for these compounds as demonstrated in parts a and c of Figure 1, which include the corresponding structural assignments for each peak in the
mass spectrum. There are several salient common fragmentation characteristics observed in these compounds and also the smaller di-, tri-, and tetrapeptides examined in the work. In each case a molecular ion M'+ is observed without any apparent cationization in the mass spectrum. In both compounds -COOH elimination occurs and for Met-enkephalin a small (M - OH)+ ion is observed. The (M - OH)+ often is observed as a major peak in many of the smaller peptides and in some cases may even be of greater relative abundance than the molecular ion ( 3 ) . In other cases, such as in the enkephalins, (M - OH)+ may be a very weak ion or may not be observed a t all under the conditions of this experiment. In addition, ions of relatively high abundance due to A, B, C and X, Y, Z peptide cleavages are observed (18). In laser RBPIIMPI though the fragments produced result from fragmentation of a M" ion and not MH'. In the case of Leu-enkephalin cleavage occurs a t the 4 0 - N H peptide bond with a resulting series of acylium ions a t mlz values 278, 221, and 164. In Met-enkephalin such cleavage is observed with acylium ions resulting at m / z values of 425,278, and 221. In addition, a t 28 mass units lower than the acylium ions, the corresponding acy1immoniu:n ions are observed due to loss of CO a t m / z values of 397 and 136 for both Leu- and Met-enkephalin, respectively. A second degradation pathway begins from the N-terminal end and results in formation of aldimine ions a t m / z values of 391, 335, and 277 in the laser-induced fragmentation of Leu-enkephalin. In Met-enkephalin a similar series of aldimine ions are observed a t mlz values of 352, 295, and 148. Such aldimine ions are often difficult to produce with electron impact ionization. In the R2PI/MPI induced mass spectra these ions are formed by bond cleavage of the -CO-NH bond in the molecular cation with a resulting even electron ion. -No H migration is observed as in FAB-MS (19) except for m / z 335 in Leuenkephalin where a H has been added to the Y cleavage product to form an odd electron ion. In Met-enkephalin the analogous fragment is a full-scale peak a t m / z 352 with no hydrogen rearrangement. This difference in hydrogen rearrangements between Met- and Leu-enkephalin is unexpected and a t present we have no explanation to account for this phenomenon. A series of fragments due to cleavage a t every N-C bond is observed for Leu-enkephalin where charge retention can occur on the N or C fragment. Charge retention on the N results in a series of fragments a t m / z values of 179, 236, and 441 whereas charge retention on the C results in ions a t m / z 376,319, and 262. The formation of the fragment at m / z 441 formed by cleavage of the N-C bond with retention of the charge a t the N-containing fragment involves the addition of one H to form an odd electron fragment so that C' is formed. However, the fragments a t mlz 179 and 236 result in even electron fragments with no migration of the H. It should be noted that the positive charge is due t o a sextet of electrons a t N, which is energetically very unfavorable and is not observed to any appreciable extent from M'+ in E1 spectra. However, we presently have no explanation for this unexpected behavior. In the case of Met-enkephalin cleavage of every N-C bond also occurs with charge retention on the N resulting in ions a t m / z values of 179, 236, 294, and 440. As in the case of Leu-enkephalin, there are two possible mechanisms present where H migration may occur as in the ion a t m / z 294 or may not as in the ions formed from the other N-C bond cleavages. However, these hydrogen rearrangements occur for the m / z 441 ion in Leu-enkephalin and not a t the analogous m / z 440 ion in Met-enkephalin and also a t the m /z 294 ion in Met-enkephalin with no analogous fragment detected in Leu-enkephalin under the same conditions. Once again, these differences in hydrogen rearrangements
ANALYTICAL CHEMISTRY, VOL. 60, NO. 14,JULY 15, 1988
1411
VI VI Ln
NH-CH2-CO-NH-C,H-CO-NH-ClH-COOH P 2
OH
Tyr-Gly-Gly-P he-Leu
Tyr-G ly-Gly-P he-Leu
Mf in VI
i z
Ln
I
I
I
1
I
I
I
100
I
I
200
I
1
l l l l l I l l l l l l l l l 1 l L
300
400
500
60(
MIZ
Tyr-Gly-Gly-Phe-Met
-m C
0:
iij C
.-0
C
.-
Q N C
0
i
I I
100
d
Tyr-Gly-Gly-Phe-Met
r
N /
I
I
I
I
I
I I I I I I I I I 1 l l l l l l l l l l l L
200
300
400
500
600
M/Z
Flgure 1. LD-MPI Ionization-fragmentation pattern at A = 266 nm of (a) Leu-enkephalln at high laser ionization energy (P = 1.5 X lo7 W/cm2) and (b) at low ionization energy (P = 1.0 X 10' W/cm2) and (c) Met-enkephalln at high laser lonlzatlon energy ( P = 1.5 X lo7 W/cm2) and (d) at low laser energy (P = 1.0 X 10' W/cm2). The asterisk denotes that a H migration has occurred.
between Met- and Leu-enkephalin are observed b u t remain unexplained. Other fragmentation pathways include simple @-cleavage of the aromatic side chain in tyrosine with charge retention on either fragment resulting in a m / z of 107 if the charge
remains on the hydroxybenzyl ion or in a m / z of 448 for Leu-enkephalin or 466 for Met-enkephalin if the charge remains on the remaining fragment. Also, a strong peak is observed at a mJz of 120 in both compounds due to an internal fragment resulting from the formation of an immonium ion
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 14, JULY 15, 1988
Table I. Comparison of LD-MPI Fragmentation of Leu-Enkephalin to Ionization-Fragmentation Obtained by Other Methods m/z
LD-MPI" ion genesis
mI2
FD-CADb ion genesis
555
Me+
556 538
MH+ MH - H,O
510
M-COOH
512 499
MH MH
465 449
448 441
M - Tyr (sc) Cql
431 425 397 391
A,
376 335 319
Z4
Y, Y,' Z,
397 392
mlz
FAB-CAD' ion genesis
556 540 524
MH+ MH-OH+H
556
MH+
CO, Leu (sc)'
511
MH - COOH
511 499
MH - COOH MH - Leu (sc)
MH - Phe (sc) MH - Tyr (sc)
465 449 442
MH - Phe (sc) MH - Tyr (sc)
465 449 443 436
MH - Phe (sc) MH - Tyr (sc) C/'
B4
425
B4
425
B4
A,
397 393
A4 Y,"
409 397 393
377 336 321
Z,' Y3/'
-
Y,'
336 321
FAB' ion genesis
ml2
Y," 231'
C4"
m/z
LDd ion genesis
556 538
MH' MH-HZO
48 1 465
MH
425 419
B,
397
A,
-
Phe (sc)
380 376 336 320
Zl"
318 307 295 278 277 262
Y, Z,
B3
245 236 221
Cz B,
204 179
C1
279
295 278
290 279 278
B,
z2
262
262
Gly-Gly-Phe
B2
221
221
B,
B,/Y," 262 251 221 215 205
B2
205 578 594 616 362
4This work. *Reference 20.
Reference 21.
M + Na M +K M - H + Na,
Reference 11. e Side chain.
characteristic of the phenylalanine group (19). It should be noted that when the laser energy is lowered to ~ ( 1 - 2 )X lo6 W/cm2, the molecular ion can be obtained with reduced fragmentation as demonstrated in parts of b and d of Figure 1 for Leu-enkephalin and Met-enkephalin, respectively. The ability to reduce fragmentation will be of importance in analysis of complicated mixtures. However, this occurs a t the expense of sensitivity by an order of magnitude or more. The laser-induced mass spectra of the enkephalins can be compared to previous results that use other techniques (see Table I). In the work of Wilkins et al. (11)direct LD-MS was used to study Leu-enkephalin in a Fourier transform mass spectrometer. In this case a small MH+ and large cationized peaks (M + K+, M + Na+) were observed as opposed to our experiment where the ionization by MPI is separated from the desorption step. Although some common ions are observed at m j t 397 and mjz 278, generally the fragmentation patterns obtained are very different. In the work of Katakuse and Desiderio (21)Leu-enkephalin was studied by fast atom bombardment (FAB) and a linked-field (B/E) mass spectrum derived from collision-activated dissociation products from the protonated molecular ion produced by FAB. The spectra obtained by these two methods are similar in some aspects to that obtained by LDMPI in our experiments although there are numerous distinct differences. In the FAB spectra MH+ is observed whereas in our case only M'+ is observed in all the peptides studied. This is true not only both in this and in previous work in our
laboratory but also in using LD-MPI in the work of Schlag (7,8) and Zare ( 5 , 6 )where M + is observed. In addition, where there are common fragments generally addition or subtraction of hydrogen atoms is observed in the FAB spectrum, for example at the carboxy terminus which contains one or two extra hydrogen atoms. In comparison between the FAB spectrum of Leu-enkephalin (21,22)and our LD-MPI spectrum, although there are some common ions or fragments as shown in Table I where the LD-MPI, FAB, FAB-CAD (collision-activated dissociation) and FD-CAD product ions are compared, clearly the majority of ions in the FAB and LDMPI spectra are not the same. Thus, although there are certain characteristic products which are easily formed in both cases, the mechanisms in these two processes are clearly different. This occurs since the FAB-CAD spectrum depends upon fragmentation of MH+ whereas MPI depends upon fragmentation of the molecular radical cation Ma+. The fragmentation obtained may also vary as shown previously depending upon the wavelength and power density used for ionization ( 2 , 4 , 1 4 ) . Nevertheless, the key point here is that the information obtained from the two techniques complement each other for structural analysis. One obvious advantage of LD-MPI versus the FAB technique is that a glycerol matrix is not needed for desorption thus avoiding the problem of background matrix peaks at low mass in the mass spectrum. The LD-MPI method also avoids the sample preparation problems of field desorption (FD) mass spectrometry. One possible disadvantage here may be the ultimate sensitivity of the technique. By use of successive dilutions of
ANALYTICAL CHEMISTRY, VOL. 60, NO. 14, JULY 15, 1988
1413
Glv-Phe
v OH
Gly-Gly-Phe
0 OH
Glv-Glv-Phe-Leu Tyr-Gly-Gly-Phe
v OH
v
Phe-Met
Phe-Leu
GIv-P he- Leu
Flgure 2. LD-MPI fragmentation patterns of tetra-, tri-, and dipeptides related to the enkephaiins at 266 nm at high laser ionization energy used for fragmentation (P = 1.5 X lo7 W/cm2). The asterisk denotes that a H migration has occurred.
Met-enkephalin in methanol/benzene (1:l) and laser desorption followed by laser MPI at 266 nm (P = 1.5 x lo’ W/cm2), a detection limit of -250 ng ( S I N = 2) was determined by monitoring the molecular ion peak. In previous work, Zare and co-workers had determined a detection limit in the picogram range for PTH-derivatized amino acids and subfemtomole detection of a porphyrin (5,6) by using laser desorption followed by R2PI at 266 nm. Recently Li and Lubman have demonstrated detection limits for CBZ (carbobenzoxy)-derivatized amino acids (23)between 50 and 100 pg by using the technique described in the present work. The detection limit for CBZ tripeptides was found to be typically between 500 pg and 1 ng, and as the peptide size increases further the detection limit correspondingly becomes higher. The detection limit determined for Met-enkephalin herein and other peptides studied appear to be affected mainly by the laser-induced ionization efficiency. The latter will be influenced by the absorption coefficient of the molecule and the competing radiationless processes. These will depend on the structure of the particular molecule and thus sensitivity in this technique may be expected to vary significantly with the sample. However, in these nonrigid peptides there are many active modes which can promote internal conversion thus resulting in a decreased ionization efficiency. The entrainment efficiency of the sample into the jet and the re-
sulting jet cooling may also affect the detection limit although the relative contribution of these processes in limiting the sensitivity is still unknown. In addition, we have examined the LD-MPI mass spectra of smaller peptide units found within the pentapeptide enkephalins. The interpreted fragmentation results in terms of the structure of the peptide are listed in Figure 2. In every case a strong M ’ ion is observed whose relative contribution to the spectrum can be varied as a function of laser input power. A peak due to the (M - OH)+ ion is observed in some cases and may sometimes be dominant even over M’+. In addition, a (M - COOH)+ is often but not always observed. In peptides, where there is a NH2-CH2 terminal group with no aromatic ring attached, (M - 30)’ is also sometimes observed. In addition, peptide cleavages similar to Leu- and Met-enkephalin are observed due to R2PI/MPI as shown in Figure 2. In the dipeptides studied CO-NH cleavage generally results in formation of aldimine ions of low abundance with no migration of H atoms as also observed in earlier work ( 3 ) . In the tri- and tetrapeptides studied CO-NH bond cleavage occurs and results in a series of acylium and aldimine ions. Acylium (ac) and aldimine (ald) ions are formed respectively for the following: Try-Gly-Gly a t m / z 221 (ac) and 131 and 74 (ald); Tyr-Gly-Gly-Phe at m / z 164, 221, and 278 (ac) and
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ANALYTICAL CHEMISTRY, VOL. 60,NO. 14, JULY 15, 1988
a
(A). Phe-Gly-Gly-Phe
Gly-Gly-Trp
I
i
b
Gly-Trp-Gly I
-CH ,-COOHI
H
MIZ
MiZ
(E). Phe-Gly-Phe-Gly
Trp-Gly-Gly
C
I
Trp-Gly-Gly
100
200
300
400
500
Mi2
Figure 3. LD-MPI mass spectrum of (a) Gly-Gly-Trp, (b) Gly-Trp-Gly, and (c) Trp-Gly-Gly at 266 nm ( P = 1.5 X lo7 W/cm2). 278 and 221 (ald); Gly-Gly-Phe-Leu a t m / z 58,115, and 262 (ac) and 334,277, and 130 (ald); Gly-Phe-Leu a t m/z 205 (ac) and 277 (ald); and Gly-Gly-Phe-Met at m / z 115 and 262 (ac) and 252,295, and 148 (ald). A series of rather strong peaks due to formation of acylimmonium ions are also observed. In the dipeptides these are observed as intense peaks at the expense of the acylium ion precursor as reported in earlier work (3). Such acylimmonium ions are observed for the following dipeptides: Tyr-Gly a t m / z 136; Phe-Leu a t 120; and Phe-Met at 120. In addition, cleavage of the N-C bond is often observed with charge retention on the nitrogen or carbon. Retention of the charge on the N occurs in Tyr-Gly a t m / z 179, Tyr-Gly-Gly-Phe at 180, Gly-Phe at 73, GlyGly-Phe at 130, Gly-Gly-Phe-Leu a t 73, 130, and 277, GlyPhe-Leu at 220, Phe-Met at 163, and Gly-Gly-Phe-Met at 130. Only in Tyr-Gly-Gly-Phe is H migration observed. In all other cases the charge is retained on the N with formation of even electron fragments. It is not clear at present why H migration is observed in Met- and Leu-enkephalin but not in the analogous fragments from Gly-Gly-Phe-Leu and Gly-GlyPhe-Met. Also simple P-cleavage is observed a t the phenylalanine and tyrosine side chain where fragments a t m / z 107 and 91 are observed from the aromatic portion of tyrosine and phenylalanine, respectively, and (M - 107)' and (M - 91)+ are correspondingly also observed. These smaller structural units of Leu- and Met-enkephalin are often important to identify in themselves as the products of degradative processes in peptide analysis. Although the mass spectra of these smaller peptide units do exhibit certain fragments common to those of pentapeptides, one cannot expect the mass spectra of these compounds to be similar. The
100
200
300
400
500
MIZ Flgure 4. LD-MPI fragmentation patterns of the two isomeric tetra-
peptides at 266 nm (P= 1.5 X io7 W/cm2).
mechanism of MPI of organic molecules (24) is such that the molecular ion is produced by R2PI and then the molecular ion subsequently absorbs additional photons which causes fragmentation and results in formation of fragment ions and neutrals. The fragment ions can absorb additional photons to undergo fragmentation to additional fragments. The fragment ions produced by this process would be different from the pure compounds studied in Figure 2 by a terminus group, and thus the resulting subsequent fragmentation would not be expected to be the same. Nevertheless, these results provide important information on the typical fragmentation units obtained in MPI mass spectrometry of peptides. Since the MPI fragmentation process involves fragmentation of M + , it might be expected that the laser-induced fragmentation patterns would have close similarities to E1 spectra. This has indeed been observed in an earlier work on smaller biomolecules (4);however, in the case of the enkephalins, generally E1 data are available on only derivatized compounds (25). However, one would expect differences in the fragmentation patterns since the selection rules involved in 70-eV E1 are
Anal. Chem. 1900, 60, 1415-1419
different than those of multiple photon absorption in the near-ultraviolet region (1). In this work we have studied several other interesting cases of MPI in aromatic-based peptides. One example is shown in Figure 3 which is a MPI mass spectrum of three isomeric peptides. In each cases a strong M'+ ion is clearly observed as well as an ion a t m / z 130 due to simple @-cleavagea t the tryptophan moiety. Although the preferred cleavage under the conditions of the experiment appears to be @ cleavage with a resulting acylium ion fragment, in each isomer a different fragment is obtained depending on the initial structure of the molecule. For example, the ion a t (M - 74) (244 u) is present in configurations 2 and 3 due to the cleavage as indicated. However, the same cleavage will not provide this ion when as in configuration 1in Figure 3 the tryptophan is on the other side of the bond. In general the fragments obtained from the three species are quite different. The MPI of the isomers of the tetrapeptide Phe-Gly-GlyPhe w Phe-Gly-Phe-Gly also provides different fragmentation patterns as shown in Figure 4. In this case M'+ and (M COOH)+ peaks are observed at 426 and 381 u, respectively. The fragments arising from the Phe-Gly portion of the peptides are similar for both isomers. Ions at m / z 91 and (M 91) (335 u) are observed due to simple @-fragmentationat the phenylalanine side chain. Cleavage a t the CO-NH bonds produces acylium and aldimine ions at m / z values 148 and 205 (ac) and 278 and 221 (ald). Acylimmonium ions are observed at m/z 120 and 177. N-C bond cleavage occurs with charge retention on the C producing a m / z of 263 in both cases but charge retention on the N to produce a m / z of 163 occurs only in Phe-Gly-Phe-Gly. The difference between the two isomers occurs due to the third amino acid depending on whether it is Gly or Phe. The CO-NH cleavage produces an acylium ion of m / z 352 or 262 depending on whether Phe or Gly is present, respectively. The corresponding acylimmonium ions can be found a t 28 mass units lower at 324 and 234 respectively, thus providing distinctly different fragmentation patterns. Registry No. Leu-enkephalin, 58822-25-6;Met-enkephalin, 58569-55-4; Tyr-Gly, 673-08-5; Tyr-Gly-Gly, 21778-69-8; TyrGly-Gly-Phe, 60254-82-2; Phe-Leu, 3303-55-7; Gly-Phe-Leu,
1415
15373-56-5; Gly-Phe, 3321-03-7; Gly-Gly-Phe, 6234-26-0; GlyGly-Phe-Leu, 60254-83-3;Phe-Met, 15080-84-9;Gly-Gly-Phe-Met, 61370-88-5;Gly-Gly-Trp, 20762-32-7; Gly-Trp-Gly, 57850-28-9; Trp-Gly-Gly, 20762-31-6; Phe-Gly-Gly-Phe, 40204-87-3; PheGly-Phe-Gly, 59005-83-3.
LITERATURE CITED Lubman, D. M. Anal. Chem. 1986,59,31A. Tembreull, R.; Lubman, D. M. Anal. Chem. 1986,58, 1299. Tembreull, R.; Lubman, D. M. Anal. Chem. 1987,59, 1003. Tembreull, R.; Lubman, D. M. Anal. Chem. 1987,59, 1082. Engelke, F.; Hahn, J. H.; Henke, W.; Zare. R. N. Anal. Chem. 1987, 59, 909. (6) Hahn, J. H.; Zenoba, R.; Zare, R. N. J. Am. Chem. Soc. 1987, 109, 2842. (7) Grotemeyer, J.; Boesl, U.; Walter, K.; Schlag, E. W. Org. Mass Spectrom. 1986.21.595. (8) Grotemeyer; J.; Boesl, U.;Walter, K.; Schlag, E. W. Org . Mass Spectrom. 1988,21, 645. (9) Rizzo, T. R.; Park, Y. D.; Peteanau, L.; Levy, D. H. J. Chem. Phys. 1985,83, 4819. (10) Posthumus, M. A.; Kistemaker, P. G.; Meuzelaar, H. L. C.; Ten Noever de Brauw, M. C. Anal. Chem. 1978,50, 985. (11) Wilkins, C. L.; Weil, D. A.; Yang, C. L. C.; Ijames, C. F. Anal. Chem. 1985. 57. 520. (12) McCrery, D.A.; Gross, M. L. Anal. Chim. Acta 1985, 778,91. (13) Shomo, R. E.; Marshall, A. G.; Wersenberger, C. R. Anal. Chern. 1985.57. 2940. (14) Tembreuli, R.; Lubman, D. M. Appl. Spectrosc. 1987,47,431. (15) Lubman, D. M.; Rettner, C. T . ; Zare, R. N. J. Phys. Chem. 1982,86, 1129. (16) Li, L.; Lubman, D. M. Appl. Spectrosc. 1988,42,418. (17) Lynch, D. R.; Snyder, S. H. Annu. Rev. 6ioChem. 1986, 55, 773. (18) Roepstorff, P.; Fohlman, J. 6iorned. Mass. Spectrom. 1984, 11, 601. (19) Biemann, K.; Martin, S. A. Mass Spectrom. Rev. 1987, 6, 1. (20) Desiderio, D. M.; Sabbatini, J. 2. Biomed. Mass Spectrom. 1981,8, 565. (21) Katakuse, I.; Desiderio, D. M. I n t . J. Mass Spectrom. I o n Processes 1983,54, 1. (22) Lippstreu-Fisher, D. L.; Gross, M. L. Anal. Chem. 1985, 57, 1174. (23) Li, L.; Lubman, D. M., Appl. Spectrosc. 1988,42,411. (24) Boesl, U.; Neusser, H. J.; Schlag, E. W. J. Chem. Phys. 1980, 72, 4327. (25) Hughes, J.; Smith, 1.W.; Kosterlltz, H. W.; Fothergill. L. A,; Morgan, B. A.; Morris, H. R. Nature (London) 1975,258,577. (1) (2) (3) (4) (5)
RECEIVED for review May 18, 1987. Accepted March 1,1988. We acknowledge financial support of this work under NSF Grant CHE8419383 and NSF Grant DMR8418095 for the acquisition of the Chemistry and Materials Science Laser Spectroscopy Laboratory. David M. Lubman is an Alfred P. Sloan Foundation Research Fellow.
Computer-Based Linear Regression Analysis of Desorption Mass Spectra of Microorganisms J. A. Platt and 0. M. Uy T h e Johns Hopkins University Applied Physics Laboratory, Laurel, Maryland 20707
D. N. Heller,* R. J. Cotter, and Catherine Fenselau' Department of Pharmacology and Molecular Sciences, T h e Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 The deslgn and appllcations of a computer-based system for determlnlng relatlve proportions of library spectra in desorptlon mass spectra are reported. The system can perform correlation and llbrarymatchlngfunctions on slngle unknowns as well as deconvolute mMures of unknowns. The algorithms for thls system were developed for the rapid characterlzation of microorganlsms through desorptlon lonizatlon of their lipid blomarkers.
Current address: Chemistry Department, University of Maryland Baltimore County, Catonsville, MD 21228.
The discovery (1) that intact polar lipids can be desorbed selectively and reproducibly from microorganisms by laser desorption, plasma desorption, and fast atom bombardment provides a rapid and sensitive approach to characterizing these important biomarkers. The presence and relative molarities of the various acylglycerides, phospholipids, glycolipids, and other lipids have been shown by chemotaxonomic studies (2, 3) to be characteristic of microorganisms. Their observation from lysed cells by desorption techniques provides a profile analogous to that obtained by gas chromatography-mass spectrometry analysis of fatty acids ( 4 ) or pyrolysis-mass
0003-2700/88/0360-1415$01.50/00 1988 American Chemical Society
