1003
Anal. Chem. 1987, 59, 1003-1006
Resonant Two-Photon Ionization of Small Peptides Using Pulsed Laser Desorption in Supersonic Beam Mass Spectrometry Roger Tembreull and David M. Lubman* Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109
Pulsed laser desorption Is used to desorb neutral small peptides Into the gas phase. The resultlng plume Is entralned Into a supersonic jet that carrles the molecules Into a tlmeof-filght mass spectrometer where resonant two-photon ioniratlon (RPPI) is produced by an ultravlolet laser source. RPPI occurs readily In peptides containing tyroslne, tryptophan, and phenylalanine aromatic groups through the X-T' transitlon. Thus, 280 and 266 nm can be used to produce ioniratlon by RPPI. I n the tyrosine and tryptophan peptides molecular Ions are often observed although other fragments such as (M OH)' may also result. I n phenylalanine peptldes, whlch requlre higher energy for lonizatlon, more extensive fragmentation may result In almost all cases, though the fragmentatlon Is rather llmited and is characteristic of the structure of the parent molecule.
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Resonant two-photon ionization (RBPI) has received a great deal of attention recently (1-9) as an ionization source with unique properties in chemical analysis. In particular RBPI has been shown to be a soft ionization method for mass spectrometry (1, 6-8). In this process the sum of the two photons must exceed the ionization potential for ionization to be produced. However, the total energy placed in the molecule is still relatively low (7-13 eV) so that only the parent ion or minimal fragmentation results. Although other soft ionization methods exist, RBPI can produce molecular ions with efficiencies that may range up to several percent or higher within the laser beam during the pulse (1-3). In addition, as the power density is increased, extensive fragmentation can be produced (5). Thus, as the power varies either molecular ions or fragments, some as small as C', are generated and can be detected, making RBPI a versatile ionization source. The truly unique property of RBPI is the ability to obtain wavelength-selective ionization. The ionization cross section is related to the absorption of the first photon by an intermediate state. Thus, although ions are produced as a final product the ionization signal reflects the absorption-excitation spectrum of the So SI transition. The result is that one can now wavelength-selectively produce ions prior to mass analysis. This selectivity is limited due to the broad absorption spectrum characteristic of thermal rotational congestion in most polyatomic systems. In recent work, supersonic jet expansions have been used to increase selectivity by ultracooling of the internal modes of large molecules to produce sharp spectral features (I, 7). The RSPI method has been generally limited to systems that are fairly volatile or can be heated without significant decomposition. A significant class of molecules for which the unique properties of R2PI could have great impact are nonvolatile or thermally labile systems. These include a large spectrum of important biological molecules and pharmaceutical compounds that may be important to detect for clinical
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0003-2700/87/0359-1003$01.50/0
or quality control purposes. However, upon heating, many of these compounds will rapidly decompose. Therefore, in this work we use laser desorption of neutrals as a means of volatilizing these labile species into the gas phase before pyrolysis occurs (8, 10-14). The desorbed neutrals are then entrained in a supersonic jet pulse and swept into a timeof-flight device where RBPI can be studied. In particular, we demonstrate RBPI of small peptides and that ionization can be obtained through the a-A* absorption of these aromaticbased peptides in the near-UV region. Although soft ionization is not always obtained under the conditions of our experiment, generally simple fragmentation, characteristic of the structure of the parent molecule, is observed.
EXPERIMENTAL SECTION The experimental apparatus consists of a supersonic beam time-of-flightmass spectrometer (TOFMS) similar to that used in previous work (8, 10, 11). A pulsed molecular beam crosses the acceleration region of the TOF device where it is ionized by an ultraviolet laser source. The system in this study has been modified with a liquid Nzcold shield around the TOF acceleration region which virtually eliminates background ionization due mainly to pump oil contaminants. In addition, differential pumping is used to maintain the flight tube at a lower pressure than the main chamber and to keep it free of contamination. The main vacuum chamber is pumped by a 6-in. liquid Nzbaffled diffusion pump stack and the TOF drift tube by a 4-in. liquid Nzbaffled diffusion pump station. The background pressure is initially between lo4 and lo-' torr. Laser desorption is performed by using a COz laser at 20-40 mJ/pulse softly focused onto the sample with a 10-cm focal length germanium lens (power density C7 X lo6 W/cm2). The sample is situated on a Macor machinable ceramic rod located about 4-mm downstream of the orifice and about 4 mm from the molecular beam axis. The material of interest is deposited on the face of the rod by dissolving it in benzene or another suitable solvent, coating the surface with a spatula, and then evaporating the solvent, leaving a relatively thick film.The samples studied here ranged generally from 100 pg to 1 mg. Sensitivity is not the aim of this study but rather an examination of R2PI of these peptides so that a sample large enough to observe a sizable signal was used. Upon desorption, the plume of organic neutral molecules is entrained into a supersonic jet pulse of C 0 2 carrier. The supersonic beam source is a magnetic repulsion type pulsed valve (R. M. Jordan Co.) which provides gas pulses of about 55 p s at 10 Hz. The use of this pulsed valve allows a reduction in the duty cycle needed for pumping by a factor of almost 2000X compared to that needed for a continuousjet so that a 0.5-mm orifice can be used. The large orifice provides a high on-axis density for correspondingly increased sensitivity since the on-axis density varies as the orifice diameter squared, i.e. 0'. The other rarely considered advantage of this fast pulsed source is that approximately 20% of the molecules in the jet pulse can be probed on each laser pulse whereas only 2-4% can be interrogated when other longer pulsed sources are used. C 0 2 is used as the carrier gas since it provides an increased collisional rate at longer distances from the orifice than a simple monatomic carrier (15). The desorption occurs 4-mm downstream from the orifice due to the constraints of the present nozzle design. Thus, the use of COz increases the effective collisional cooling in the jet. In previous work, extensive trans0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987
Table I. Laser RZPI of Tryptophan-Based Peptides at 266 and 280 nma 280 nm
266 nm
Trp peptide
mlz
peak
m/z
peak
Trp-Trp
373 130 367 350 130 334 130
(M - 17)
373 130 367 350 130 351 334 130
(M - 17)
Trp-Tyr Trp-Phe Trp-Gly Trp-Gly-Gly Gly-Trp Trp-Ala Ala-Trp Trp-Leu
261 244 244 26 1 244 130 258
Qb
M+ (M - 17)
Q
( M - 17)
Q M+
Q
M+ (M - 17)
(M - 17)
Leu-Trp
258 258 300 130 300 130
Phe peptide
M+ (M - 17)
Phe
NSb
Phe-Ala
NS
Phe-Leu
NS
Phe-Tyr
311 208 120 107
(M - 17) (M - 17)
Phe-Phe
NS
(M - 17)
Pro-Phe
Q
(M - 17)
Q
Leu-Phe
Laser R2PI fragmentation patterns obtained at threshold laser energy needed to observe a signal. These patterns may vary considerably as the laser energy is increased. b Q = quinolinium ion. Corresponds to a loss of glycine.
Gly-Gly-Phe
Table 11. Laser RZPI of Tvrosine-Based Peptides a t 266 and 280 nmo
Leu-Gly-Phe
266 nm
Tyr peptide
m/z
peak
mlz
peak
Tyr-Phe
311 208 107 238 221 179 132 107
( M - 17) ( M - 120) R' M+ ( M - 17)
328 311
M+ (M - 17)
238 221 179 132 107 189 116
M+ ( M - 17) (M - 59) (M - 106)
238 221 278 179 107 277
M+ ( M - 17) M+
Tyr-Glg
(M-59) ( M - 106)
R
Tyr-Gly-Gly Gly-Tyr Pro-Tyr Tyr-Leu-Gly Tyr-Leu Tyr-Ala Tyr-Tyr Tyr-Tyr-Tyr Leu-Tyr
R ( M - 17)
R (M - 106)
207
(M - 71)
277 107
(M - 17) R
NSb 327
(M - 17)
NS 294 86
280 nm peak
(4
a
280 nm
m/z
Q
(M - 17) (M - 74)' M+ (M - 17)
Q
Table 111. Laser R2PI of Phenylalanine-Based Peptides a t 266 and 280 nma
M+ A
Laser RZPI fragmentation patterns obtained at threshold laser energy needed to observe a signal. These patterns may vary considerably as the laser energy is increased. *NS = no detectable signal under the conditions of the experiment. 'R = p-hydroxybenzyl cation produced by simple cleavage p to the phenyl ring in a tvrosine-based DeDtide. d A = aldimine fragment ion. lational cooling was obtained as evidenced by the narrow ion peak widths obtained in the TOFMS, thus indicating a correspondingly narrow energy spread in the beam. However, although rovibronic cooling was shown to be incomplete, it was sufficient for this work since we are interested in the R2PI products without the use of wavelength selectivity. The ionization source is a Quanta-Ray DCR-2A Nd:YAG pumped dye laser which provides a 280-nm wavelength by frequency doubling dye R590. Wavelengths a t 245 nm and 222 nm were generated by mixing the doubled dye output with the 1.06 pm fundamental in KD*P. In addition, the fourth harmonic of
P he-Gly-Gly Phe-Gly-Gly-Phe Phe-Gly-Phe-Gly
( M - 17) ( M - 120) AC
266 nm
peak
m/z 165 120 236 219 145 120 278 261 187 120 311
M+ (M-45)
M+ ( M - 17) (M-91) A M+ (M - 17) (M-91) A ( M - 17)
R 312 295 262 217 192 70 278 129 279 164 149 119 115 335 290 119 279 205 120 206 205 120 206 205 120
M+ ( M - 17) M+ (M-45)
M+ M+ ( M - 115) ( M - 130)
M+ (M - 4 5 ) M+ (M-74)d A
A A
'Laser R2PI fragmentation patterns obtained at threshold laser energy needed to observe a signal. These patterns may vary considerably as the laser energy is increased. b N S = no detectable signal under the conditions of the experiment. C A = aldimine fragment ion. dCorresponds to a loss of glycine. the Nd:YAG laser provided 266-nm radiation. The typical laser energy varied from 3-5 mJ up to 10 mJ used to induce fragmentation. In most experiments the ionizing laser beam was collimated by a positive-negative lens combination to a 2-3mm-diameter beam. The actual sequence of events is controlled by several delay generators where the pulsed COBlaser 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 into the jet expansion of COPand carried into the acceleration region of the TOFMS. The flight time of the jet from the pulsed valve to this region is about 300 ws and the laser is set to pulse as the gas pulse arrives. Laser R2PI is produced and a LeCroy 9400 digital oscilloscopewas used to record the spectrum.
RESULTS AND DISCUSSION In this work we have focused on the study of the R2PI of mainly di- and tripeptides. In particular, these peptides are selected based upon the presence of either phenylalanine, tyrosine, or tryptophan and the results tabulated in Tables 1-111 are arranged according to the aromatic amino acid present. In each case the peptide has been volatilized as neutrals by laser desorption and swept by the supersonic jet into the TOFMS for examination by R2PI. The f i s t key point is that these compounds generally absorb radiation a t either 280 or 266 nm through the T-T* transition of the aromatic
ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987
0
40
50t L-Phenylalanyl-L-Tyrosine
6ot TIME (ps)
Figure 1. Timeof-flight mass spectrum of Phe-Tyr obtained by using a 10.6-pm laser beam as a means of volatilizing a neutral molecular plume into a supersonic jet and a 280-nm laser beam for resonant photoionization. Only a pseudomolecular parent ion is produced under the experimental conditions employed.
moiety and subsequently are ionized by R2PI. Each of the aromatic groups is known to have a strong origin absorption in the region around 280 nm. It must be emphasized that RBPI is a spectroscopic ionization tool since it depends upon the presence of an intermediate absorbing electronic state. Thus, it appears that the presence of small linear amino acids such as glycine, alanine, leucine, etc. serve as substituent
0
5
2
4
6
8
I
I
I
I
IO 12 I
I
16
I
I
18 20
groups on the aromatic center which may shift the absorption center or change the absorption cross section of the aromatic group. However, liquid-phase spectra show that the basic origin absorption transition still occurs between 260 and 290 nm in most of the peptides studied. Thus, in these studies the 280- or 266-nm wavelength is absorbed by most of the small peptides. Although 245- and 222-nm wavelengths were tested as ionization sources, they were generally found to be less effective than the longer wavelengths. We suspect that as the lasing frequency increases, the molecule is excited higher into the SI manifold and the radiationless transition rate increases dramatically thus reducing the RSPI efficiency. The second key result is that in many cases R2PI is able to produce the molecular ion or characteristic fragment ions such as (M - OH)+ or (M - COOH)+. An example of simple fragment ion production is shown in Figure 1 for Phe-Tyr which loses a .OH radical to give a pronounced (M - 17) peak. In general, parent or pseudoparent ion production appears to be more easily achieved in tryptophan- and tyrosine-containing compounds. Phenylalanine ionizes very weakly and only at increased laser energy (- 10 mJ, 266 nm). This could be due either to a small absorption cross section or an ionization potential that is too high for R2PI. However, the addition of an -OH group onto the phenylalanine ring as in the tyrosine molecule strongly releases electrons into the aromatic center, enhancing the transition probability and lowering the I P by strong stabilization of the ionic state. Therefore, tyrosine strongly absorbs and ionizes in R2PI a t 0
2
4
6
8
10
12
14
16
18
20
I
1 120
io
14
1005
(M-120)
mV 15 20
> c -
25
c
0
m z w
OH L - Phenylalanyl- L-Tyroslne
30
262 M t
(M3'
40t
b)
i
1
70
-1
w mV 15 LT
20
io -
25
15-
L- Phenylalanyl-L- Alanine
30 236 M ? 2
4
-
mV 2 0 -
120A t
35
-
I30
6 8 10 12 14 16 FLIGHT TIME ( p s )
18 20
25 -
30 -
H
35 40 0
Glycyl -L-Tryptophan
I
I
I
I
I
I
I
2
4
6
8
10
12
14
(M-17) 24 4 16
18
20
FLIGHT TIME ( p s ) Figure 2. Time-of-flight mass spectra obtained by using laser desorption as a means of volatilizing the neutral peptides (a) Phe-Tyr, (b) Phe-Ala, (c) Pro-We, and (d) Gly-Trp. The ion currents were produced by using a 280-nm (a, d) or a 266-nm (b, c) laser beam for resonant photoionization. These spectra demonstrate the production of diagnostic fragment ions in dipeptides by carefully controlling the input laser energy.
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987
280 nm. Thus, peptides that contain tyrosine and tryptophan have a considerably greater TT* transition moment and ionize at much lower laser energy than those peptides containing phenylalanine. In the former groups of compounds ionization could often be achieved with 2-3 mJ of energy at 280 or 266 nm. In the case of the phenylalanine compounds laser energies in excess of 10 mJ at 266 nm were needed for ionization. Thus, the lower laser energies required for R2PI of tyrosine and tryptophan compounds make parent ion or pseudoparent ion production much easier to achieve in these systems as shown in Tables I and 11. Loss of a hydroxyl radical plays a prominent role in these species and, aside from the (M - 17) peak, the only other prominent fragments are those at 130 u in the case of tryptophan-containing peptides and a peak at 107 u in tyrosine peptides. The latter two fragments are characteristic of these aromatic residues (16);the peak at mass 130 is the quinolinium ion while that at mass 107 is due to simple bond cleavage p to the phenyl ring in a tyrosine moiety. Examples of these characteristic cleavages are illustrated in Figure 2. The R2PI mass spectra of small peptides containing one or more phenylalanine residues are tabulated in Table 111. R2PI in these systems requires the use of relatively high laser power density due to the difficulty of ionizing phenylalanine. Although the liquid-phase absorption spectra of these peptides have broad absorption contours from 285 to 222 nm and frequently exhibit absorption maxima near 222 nm, ionization was generally not observed at either 222 or 280 nm. However, ionization is readily achieved in most of these peptides at 266 nm with the fourth harmonic of the Nd:YAG system since the energy of this beam can be very easily increased compared to the energy of the frequency doubled dye output at 280 or 222 nm. For production of the parent ion of phenylalanine itself, it was found that a beam of at least -8 mJ was required. Furthermore, ionization becomes increasingly difficult when small amino acids such as glycine are added to the phenylalanine residue, i.e. Gly-Gly-Phe and Leu-Gly-Phe ionize weakly. In addition, there appears to be increased fragmentation at the ionization threshold in the phenylalanine-based peptides than for the tyrosine and tryptophan cases. The higher laser power needed to ionize the phenylalanine peptides probably causes absorption of additional photons by the ion initially produced, resulting in subsequent fragmentation. The R2PI method may be useful for detection and identification of small peptides since a limited number of diagnostic fragments are formed. In many cases either the molecular ion or simple fragment ions are obtained, especially at (M - OH) (M - 17) and (M - COOH) (M - 45). Additional peaks observed are also generally characteristic of the parent structure as demonstrated in Figure 2 where the aliphatic secondary amine in the proline residue cleaves from Pro-Phe to give a prominent peak at m/z 70. In addition, as mentioned earlier peptides that contain tyrosine and tryptophan yield prominent ions at 107 and 130 u, respectively. However, the most diagnostic fragments, particularly in larger peptides, are associated with rupture of the peptide backbone. Thus, Phe-Ala, Phe-Leu, and Phe-Tyr cleave adjacent to the carbonyl group to form stable aldimine ions whereas other peptides such as Gly-Gly-Phe preferentially undergo cleavage on the opposite side of the carbonyl function to form acylium or C-terminal ions. In general, the aldimine ion is the favored ion (16). The various cleavages and the corresponding masses of the fragments are shown in Tables 1-111. In conclusion, laser desorption can be used to volatilize neutral small peptides into the gas phase, which are subse-
quently ionized by R2PI in a TOFMS. R2PI can often produce molecular or pseudomolecular ions, and a limited number of fragments characteristic of the parent peptide molecule are also often obtained. In phenylalanine, peptide fragmentation is more likely due to the higher laser power needed for ionization than for the tyrosine or tryptophan peptides. It is not entirely clear whether absolute soft ionization can be obtained in some of these compounds without any accompanying fragment peaks since the molecular ion when formed may rapidly decompose to a thermodynamically more stable species. Facile rupture of labile substituent groups is known to occur in many groups of molecules ( 1 7 ) . In addition, the mechanism for the observed fragmentation depends upon both the desorption and ionization steps, so that at least three plausible explanations may account for the results observed herein: molecular decomposition occurs during the desorption step with subsequent R2P1, photodissociation precedes ionization during RPPI, or efficient parent ion dissociation occurs immediately after R2PI. At this point though it is not possible to distinguish the effects of each of these processes. The ultracooling obtained in jet expansions may still be expected to decrease fragmentation in the R2PI process. Only incomplete cooling has as yet been achieved in our experiments; however, such cooling increases the population of low rovibronic states. The excitation of transitions from these states is therefore frequently much more effective than transitions from higher lying rovibronic states and lower laser power can be used for ionization. In earlier supersonic jet R2PI experiments in aromatic molecules typically 0.2-0.5 mJ was used for ionization (7). In equivalent thermal beam experiments ( 1 ) laser input energies of 1-2 m J were used for R2PI which are more typical of the laser energies needed to ionize tyrosine- and tryptophan-based peptides in this work. Thus, fragmentation may yet be minimized by jet techniques; however, it may never actually be eliminated. Nevertheless, laser R2PI produces reasonably simple spectra by using near-ultraviolet light absorbed through the characteristic aromatic x-x* transition.
LITERATURE CITED Lubman, D. M.;Kronick, M. N. Anal. Chem. 1982, 54,660. Frueholz, R.; Wessel, J.; Wheatley, E. Anal. Chem. 1980, 52, 281. Boesl, U.; Neusser, H. J.; Schlag, E. W. Chem. fhys. 1981, 55, 193. Klimcak, C.;Wessel, J. Anal. Chem. 1980, 52, 1283. Zandee, L.; Bernstein, R. B. J. Chem. fhys. 1979, 71,1359. Sack, T. M.; McCrery, D. A.; Gross, M. L. Anal. Chem. 1985, 57,
1290. Tembreull, R.; Lubman, D.M. Anal. Chem. 1984, 56, 1962. Tembreull, R.; Lubman, D. M. Anal. Chem. 1986, 58, 1299. Dobson, R. L. M.;DSilva. A. P.; Weeks, S. J.; Fassel, V . A. Anal. Chem. 1988, 58, 2129. Lubman, D. M.;Tembreull, R. Anal. Instrum. (Advances in Time-ofFlight Mass Spectrometry; Campana, J.. Ed.), In press. Tembreull, R.; Lubman, D. M. Appl. Spectrosc., in press. Engelke, F.; Hahn, J. H.; Henke, W.; Zare, R. N. Anal. Chem. 1987,
59, 909. Grotemeyer, J.; Boesl, U.; Walter, K.: Schlag, E. W. Org. Mass Spectrom. 1988, 21, 595. Grotemeyer, J.; Boesl, U.; Walter, K.; Schlag, E.W. Org. Mass Spectrom. 1988, 21. 645. Lubman, D. M.; Rettner, C. T.: Zare, R. N. J. fhys. Chem. 1982, 86,
1129. Arpino, P. J.; McLafferty, F. W. Determination of Organic Structures by Physical Methods, 6 ; Nachod, F. C., Zuckerman, J. J., Randall, E. W., Ed.; Academic: New York, 1976;Chapter 1. Tembreull. R.; Lubman, D. M. Anal. Chem., in press.
RECEIVED for review September 17,1986. Accepted December 17,1986. We acknowledge financial support of this work under NSF Grant CHE 8419383 and NSF Grant DMR 8418095 for acquisition of the Chemistry and Materials Science Laser Spectroscopy Laboratory.
