J . Phys. Chem. 1992, 96, 3157-3162 seems an intuitively reasonable result unless the solvent dynamics is massively affected by the presence of the polymer segments in a manner that is not merely an obstruction. The data shown in Figure 6 are that of toluene in 270 000 molecular weight polystyrene at temperatures in the range from 15 to 115 O C 4 0 a system in which one might expect the solvent to have no unusual specific interaction with the polymer. The fact that the simulation can be extended to polymer concentrations exceeding the domain overlap, Figure 6b, and still match the experimental data with only a modest reduction in solvent motion presents a view of solvent diffusion in random coil polymers a t low to moderate concentrations different than that frequently given.
3157
Acknowledgment. R.O. acknowledges support from CONACYT (Mexico). Support from the Midwest Technology Development Institute, the NSF-Minnesota Center for Interfacial Engineering, and the Graduate School, University of Minnesota, is also acknowledged. The hospitality of Prof. Paul Callaghan and the Department of Physics and Biophysics, Massey University, New Zealand during a sabbatical leave (W.G.M.) and the use of the JEOL PGSE N M R spectrometer are gratefully acknowledged. Registry No. PBLG (homopolymer), 25014-27-1; PBLG (SRU), 25038-53-3; PBIC (homopolymer),25067-04-3;DMF,68-12-2; benzene, 71-43-2.
Peptide Sequence Ions Produced by Postionizatlon of Neutral Molecules Formed during Resonant 266-nm Laser Desorption Gary R. Kinsel, Josef Lindner, and Jiirgen Grotemeyer* Institut fur Physikalische und Theoretische Chemie der Technischen Universitat Munchen, Lichtenbergstrasse 4, 0-8046 Garching, Germany (Received: August 29, 1991; In Final Form: December 3, 1991)
A number of examples of peptide mass spectra obtained using 266-nm laser desorption (LD) followed by 255-nm multiphoton ionization (MUPI) of the laser desorbed neutral molecules are presented which show a variety of structurally significant sequence fragment ions. It is believed that most of these sequence fragment ions result from postionization of neutral fragments produced during 266-nm LD. MUPI postionization of the fragment neutrals produced during LD can provide sequence information which is complementary to the sequence information derived from direct ion desorption experiments. In addition, a variety of immonium fragment ions may be produced at higher ionizing laser power densities which give an overview of the amino acid residues contained in the peptide structure. These fragment ions are formed during MUPI positionization step by multiphoton fragmentation of larger intact ions. Finally, a comparision of the sequence fragment ions produced in these experiments with the sequence ions produced using other direct ion desorption techniques illustrates the analytical usefulness of the sequence ions produced in these experiments.
Introduction One of the goals of analytical mass spectroscopists is the development of routine mass analysis techniques for the structural elucidation of small and medium sized peptides. Ideally, a technique should provide both the molecular weight of the intact peptide and sufficient structural information to allow the determination of the amino acid sequence in the peptide molecule. A variety of approaches for generating this information have been reported in the literature and are summarized in a recent review.' The approach taken in this laboratory involves the combination of laser desorption (LD) followed by multiphoton ionization (MUPI) pitionization of the laser desorbed neutral molecules.* This combined technique has been applied with success to a wide variety of b i m o l e ~ u l e s . ~ ~ ~ In the original experiments performed in this laboratory, a COz laser (10.6 pm) was used to perform the LD step. It was believed that the low energy of the C 0 2 laser photons would help in maximizing the number of intact neutral molecules desorbed. In a recent paper we have shown that much higher energy 266-nm photons can also be used to desorb abundant numbers of intact neutral molecules from many of the samples investigated in the earlier experiment^.^ It was determined in this study that the primary requirement for efficient LD of intact neutral molecules was that the sample under investigation absorb strongly at the desorbing laser wavelength. In these experiments an intense signal from the MUPI postionized peptide molecular ion was always observed, even up to the mass gramicidin D (1881 amu). However, it was also noted in these experiments that the resulting peptide mass spectra always contained abundant signals from a wide variety of sequence type fragment ions. It is believed that the To whom all correspondence should be addressed.
0022-365419212096-3157$03.00/0
majority of the sequence ions observed are produced as fragment neutrals during 266-nm LD which are then transported into the RETOF-MS source region for MUPI postionization.6 In all cases, high quality spectra were obtained and in some cases complete sequence information could be determined from the fragment ion signals in the peptide mass spectra. Resonant 266-nm LD followed by MUPI postionization of the desorbed neutral species, therefore, is an attractive method for investigating small and medium-sized peptides since an abundance of structurally significant fragment ions as well as a strong signal from the molecular ion are all observed in the peptide mass spectrum simultaneously. In addition, the peptide structural information obtained in these experiments is often complementary to the information produced using direct ion desorption techniques such as fast atom bombardment (FAB) or PDMS. In this paper we provide detailed information regarding the types and relative abundances of the sequence fragment ions observed in the 266nm-LD/255-nm-MUPI peptide mass spectra.
Experimental Section The instrument used in performing these experiments is the Bruker TOF-1 reflectron time-of-flight mass spectrometer (RE(1) Biemann, K.; Martin, S.A. Mass Spectrom. Rev. 1987, 6, 1.
(2) Grotemeyer, J.; Lindner, J.; Kbter, C.; Schlag, E. W.J . Mol. Struct. 1990, 21 7, 5 I . (3) Grotemeyer, J.; Schlag, E. W.Biomed. Emiron. Muss Spectrom. 1988, 16, 143. (4) Grotemeyer, J.; Schlag, E. W.Org. Mass Spectrom. 1988, 23, 388. ( 5 ) Kinsel, G. R.; Lindner, J.; Grotemeyer, J.; Schlag, E. W.J. Phys. Chem., 1991, 95, 7824. ( 6 ) Kinsel, G. R.; Lindner, J.; Grotemeyer, J.; Schlag, E. W.J . Phys.
Chem., following paper in this issue.
0 1992 American Chemical Society
3158 The Journal of Physical Chemistry, Vol. 96, No. 7, 1992 TABLE I: Sequence Ions" Observed* in the 266-nm-LD/ZSS-nm-MUPI Mass Spectra of Methionine-Enkephalin N terminus C terminus
Kinsel et al. TABLE III: Sequence Ions" Observedbin the 266-nm-LD/ZSS-nm-MUPI Mass Spectra of Leu-Trrt-Met-ArePhe
N terminus A, ... C ,
C terminus
zs' ......
.. (BZ') Ci
A,' ......
Met
.........
I
A,' ......
Phe
.........
"Nomenclature according to ref 8. *Ions listed within parentheses had signal intensities -3% of the molecular ion but > 3 times the signal to noise ratio.
"Nomenclature according to ref 8. *Ions listed within parentheses had signal intensities 3 times the signal to noise ratio.
TABLE II: Sequence Ions" Observed* in the 266-nm-LD/255-nm-MUPIMass Spectra of Leucine-Enkephalin
TABLE I V Sequence Ions' Observed* in the 266nm-LD/ZSS-nm-MUPI Mass Spectra of Substance P
AI
N terminus ('Bl) C I ' )
TYr
I
... 'B2 (C;)
GIY
C terminus Z,' ......
N terminus
.........
Arg
... (Y4))...
.........
Pro
I (A37 (B3') (C10 ('449 (B49
A(
......
c4)
GlY
V3') Y3' ('X3)
.........
Phe
.........
.........
I I
Met
.........
"Nomenclature according to ref 8. *Ions listed within parentheses had signal intensities 3 times the signal to noise ratio.
TOF-MS) which has been modified to allow resonant 266-nm LD using the frequency quadrupled output of a Quanta-Ray DCR-11 Nd:YAG laser. This instrument has been described in detail previously and only a brief description is presented here.' The 266-nm desorbing radiation entered the desorption chamber through a UV grade window opposite the sample introduction port. The sample probe was inserted into the desorption chamber and positioned ca. 1 mm away from the opening of a pulsed jet valve. Typically, the desorbing laser was fired and the jet valve opened to emit a pulse of Ar atoms. The desorbed neutral molecules were entrained in the Ar and transported into the RETOF-MS source region where postionization was performed by 255-nm MUPI. Ionic material produced during 266-nm LD was prevented from entering the RETOF-MS source region by the positive bias on the source region repeller plate. The ionizing radiation at 255 nm was provided by the frequency doubled output of a Nd:YAG (Lumonics H Y 1200) pumped dye laser (Lambda Physik FL 3001). The ionizing wavelength of 255 nm was chosen because all sample peptides absorbed at this wavelength and the photon energy should be sufficient to perform two-photon ionization. Ionizing power densities could be adjusted to a maximum of ca. 50 MW/cm2 by adjusting the focus of the ionizing beam using a 20-cm spherical lens. The positive ions produced by MUPI postionization were separated and detected in the RETOF-MS with a typical resolution of ca. 3500 at mass 300 (fwhm). The peptide spectra displayed in this paper represent the sum of 20 spectra recorded using the dedicated 200-MHz LeCroy transient digitizer. Sample preparation was maintained as simple as possible to improve reproducibility. Typically, ca. 1 mg of dry peptide sample, as provided by the manufacturer (Serva Chemicals, Heidelberg), was compressed into a slight depression machined into the steel probe tip. This method produced a thin layer ca. 4 mm2 in area with varying thickness, Le. thicker in the middle and thinner at the edges. The pure peptide samples were introduced to the LD chamber and analyzed in the manner described. Using this simple preparation method, it was possible to obtain reproducible mass
......... ......... A{
... Cl'
C terminus (Zl1') ......
I
I
LYS
I
Pro
I
Gln
I
Gln I Phe
I
"'
y,; ('XIrJ)
zg' yg' (X9)
... Ys'...
z7/ YII 'X1 z; Y;
'X,
zs' Y,!
'X,
z:
... C{ Ag' ... Cg'
GIY
.........
A1d ... Cld
Leu
.........
Ag'
A,," ......
Phe
I I I
Met
Y( IX,
.........
'Nomenclature according to ref 8. *Ions listed within parentheses of the molecular ion but >3 times the signal had signal intensities 6% to noise ratio.
spectra from all peptide samples investigated.
Results Tables I-V list the peptides that were investigated in this work and the sequence fragment ions typically observed in the 266nm-LD/255-nm-MUPI postionization mass spectra. The nomenclature used to refer to the sequence fragmentation ions is in accordance with that originally proposed by Roerpstorff et ala8 In general the sequence fragment ions listed in the tables without parentheses are those whose intensities exceeded 5% of the base peak intensity and are labeled in the mass spectra shown. The sequence ions given in the tables within parentheses were observed in the mass spectra but at intensities less than 5% of the base peak and are not labeled in the mass spectra shown. All the peptides studied for this paper gave abundant signals from the intact molecular ion, M'+. Under soft MUPI conditions the molecular ion formed the base peak of the mass spectrum in all cases. This result was obtained in spite of the high desorbing photon energy at 266 nm which might be expected to cause extensive ionization and/or fragmentation of the peptide sample molecules during LD. Clearly, using high energy UV photons is an acceptable alternative to using lower energy IR photons produced by a C 0 2 laser for LD of intact neutral molecules. Two other attractive features of the spectra presented here should be noted which are a result of the MUPI postionization of neutral molecules approach. First, no cationized molecular or fragment ions are observed in the mass spectra since all ions
(7) Boesl, U.; Grotemeyer, J.; Walter, K.; Schlag, E. W. Anal. Insfrum. 1987, 16, 151.
(8) Roepstorff, P.; Fohlman, J. Biomed. Mass Specfrom. 1984, 11, 601.
Peptide Sequence Ions from Neutral Molecules
The Journal of Physical Chemistry, Vol. 96, No. 7, 1992 3159
TABLE V Sequence Ions" Observedb in the 266-nm-LD/255-nm-MUPI Mass Spectra of Gramicidin D N terminus C terminus
.........
Val
I
......... .........
GIY
.........
Leu
I
Ala
A'S
-1
M '+
.........
... Y 1 l ... z,; Y,,' ...
I
el;)
Y,l
Phe 4098-1
(Y12/) ..'
..
"Nomenclature according to ref 8. bIons listed within parentheses had signal intensities 3 times the signal to noise ratio.
formed directly during the 266-nm-LD step are prevented from entering the RETOF-MS source region. Although assignment of cationized species usually presents little difficulty when the structure of the sample peptide is known, structural assignment of fragment ions would become more difficult if a complex mixture of cationized and protonated fragment ions made up the observed mass spectrum for a peptide of unknown structure. Therefore, from the standpoint of simplified spectrum interpretation, the elimination of these species from the peptide mass spectrum may be considered an advantage of the approach used in these experiments. The second feature of note is the low background of nonspecific fragment ions. A large nonspecific background is often observed for many direct ion desorption methods which may obscure weak peptide specific fragment ions, particularly in the low mass region. There are primarily two reasons for the low background signals observed in these experiments. First, 266-nm LD of pure sample peptides is performed, thereby eliminating ion signals arising from matrix material. Second, only species containing an absorbing chromophore at the 255-nm-MUPI ionizing wavelength will be ionized and detected in the mass spectrum. Therefore, mass spectrometer contaminants or species resulting from LD of the sample or substrate which do not contain a chromophore are generally not observed as background in the peptide mass spectrum. This second point is important to bear in mind when interpreting the peptide mass spectra, and it will be seen that, in general, only thase peptide sequence fragment ions which contain a chromophore are observed with strong intensities in the mass spectra. 1. Pentapeptides. Tables 1-111 list the sequence type fragment ions observed in the peptide mass spectra for Met-enkephalin (MW = 573 amu), Leu-enkephalin (MW = 555 amu), and Leu-TrpMet-Arg-Phe (MW = 751 amu). Figure 1A shows a typical mass spectrum for Met-enkephalin taken a t a desorbing laser power of 90 MW/cm2 and an ionizing laser power of 20 MW/cm2. It must be emphasized that the abundances of the sequence fragment ions relative to the parent ion are strongly dependent on the experimental conditions used for spectrum acquisition. In par-
Figure 1. Resonant 266-nm-LD/255-nm-MUPI mass spectrum of Met-enkephalin taken at a desorbing laser power of 90 MW/cm* and an ionizing laser power of 20 MW/cmZ. (A) Full scale spectrum with major sequence, side chain, and immonium fragment ions labeled. Sequence ions with intensities greater than 5% of the base peak are labeled. (B) Spectrum e,xpanded by a factor of 4 to show weaker intensity fragment ions listed in Table I. Peaks labeled with an asterisk have been identified as background signals.
ticular, the desorbing laser power and the delay between the desorbing and ionizing laser pulses play an important role. However, the specific types of sequence fragment ions observed and their abundances relative to each other are, in general, independent of these parameters. For the two enkephalins, the abundant sequence fragment ions observed in the mass spectra were very similar and incorporated both the N and C terminus of the peptide. In addition, two relatively intense fragment ion signals were observed resulting from side chain reactions. For Met-enkephalin the fragment ions at m / z = 466 and 467 have been attributed to the loss of the tyrosine side chain with a mass of 106 amu, both alone and with the transfer of an H atom? An additional loss of 16 or 17 amu, most likely due to the loss of N H 2 and NH3,respectively, then yields the fragment ion at m / z = 450. This interpretation is supported by the observation of an analogous pair of intense fragment ion signals in the spectrum of Leu-enkephalin at mlz = 448,449 and at m / z = 431. For the case of Leu-TrpMet-Arg-Phe, somewhat different behavior was observed in the production of an intense set of N terminal C,' (n = 2-4) fragment ions. It is apparent from the sequence fragments listed in Tables 1-111 that a wide variety of weaker intensity fragment ions are produced from the pentapeptides in these experiments in addition to the stronger intensity fragment ion signals discussed above. An inspection of the expansion of the Met-enkephalin spectrum shown in figure 1B indicates that the weaker intensity sequence fragment ion signals are still well above the background noise level. Similar weak intensity signal continuum were observed in the mass spectra of the larger peptide molecules as well. Closer inspection of the spectra showed that the majority of these ion signals were not metastably broadened and were clearly resolved individual masses. In addition, the intensity of the majority of these signals relative (9) Li, L.; Lubman, D. M. And. Chem. 1988, 60, 1409.
3160 The Journal of Physical Chemistry, Vol. 96, No. 7, 1992 to the parent ion signal did not show any dependence on the ionizing laser power density, and this observation suggested that these fragment ions were formed as neutral species during 266-nm LD. It appears that the peptide can fragment to produce a variety of low abundance small neutral fragments during the 266-nm-LD step. For the case of Met-enkephalin show in Figure 1, the number of fragment ion signals between m/z = 150 and the parent ion signal may be reduced to approximately 40 by setting a statistical limit for signal significance of 3 times the average noise level and eliminating the known background signals. Note that uncorrected spectra are displayed in Figure 1. An inspection of the remaining ion signals indicated that ca. 55% could be assigned to sequence type fragment ions resulting from cleavages of the peptide backbone with the addition or substraction of one hydrogen atom. These sequence fragment ions are listed in Table I. An additional 30% of the fragment ion signals could be assigned to side chain fragmentations, for which the loss of one or the other of the chromophore side chains played a dominant role. The remaining 15% of the fragment ion signals most likely resulted from combined peptide backbone and side chain cleavages but were not assigned. Analyses of the low intensity fragment ion signals observed for the other peptides investigated yielded similar results and formed the basis for inclusion of the sequence ion signal in the peptide tables given. For all three pentapeptides, clearly detectable signals from a variety of immonium ions could be observed in the mass spectra at higher ionizing laser powers. These immonium ions are characterized by fragment ion signals 45 amu lower in mass than the mass of the individual amino acids and are useful for obtaining an overview of the types of amino acid residues contained in the peptide. These signals can be used to provide secondary confirmation for a proposed amino acid sequence derived from higher mass sequence fragment ion signals.'Osll The ionizing laser power dependence of these signals indicated that a significant fraction of these ions did not enter the RETOF-MS source region as separate neutral species, but instead arose from MUPI postionization and fragmentation of larger neutral molecules. An example of these ions can be seen in Figure I A , where the low mass fragment ions at m/z = 120 and 136, labeled Phe and Tyr, respectively, are observed in the Met-enkephalin mass spectrum. The chromophore containing immonium ions were generally more abundant in the MUPI mass spectra, but most other immonium ions could be observed in the peptide mass spectra at somewhat higher ionizing laser powers. 2. Substance P. The 266-nm-LD/255-nm-MUPI mass spectrum of substance P, shown in Figure 2, taken at a desorbing power of 200 MW/cm2 and an ionizing power of 5 MW/cm2 is remarkable in the high quality of the spectrum and the strong relative intensity of the sequence specific fragment ion signals (see also Table IV). A clear pattern of fragment ion triplets separated by 15 and 26 amu, respectively, is observed in the peptide mass spectrum which may be assigned to the C terminal sequence ions of type Z,,', Y,,', and X , respectively. This pattern continues from the n = 4 position to the n = 7 position. The breaks at the n = 8 and n = 10 positions with the dominant formation of the Y,' appear to be associated with the proline residue found at these positions. The cyclic structure of the proline residue requires that two bonds be broken for the formation of the Z type sequence fragment ion and would explain a preference for the formation of the Y and/or X type sequence ions. The N terminus fragment ions are observed to begin at the n = 7 position and show a series of A,' and C,' sequence ions separated by 43 amu. As mentioned earlier, it is noteworthy for the cases of both the C and N terminal sequence fragment ions that the intense series of sequence ion signals begin at the position of the first aromatic phenylalanine amino acid residue in the substance P chain, i.e. (IO) Kausler, W.; Schnieder, K.; RBhr, U.; Reiner, J.; Spiteller, G. In Ion Formation from Organic Solids: IFOS 1V; Benninghoven, A,, Ed.; Wiley: Chichester, U.K., 1989; p 7 . ( 1 1 ) Biemann, K. Biomed. Enuiron. Mass Spectrom. 1988, 16, 99.
Kinsel et al.
Figure 2. Resonant 266-nm-LD/255-nm-MUPI mass spectrum of substance P taken at a desorbing laser power of 200 MW/cm2 and an ionizing laser power of 5 MW/cm2. Sequence ions with intensities greater than 5% of the base peak are labeled. Peaks labeled with an asterisk have been identified as background signals.
n = 4 for the C terminus sequence ions and n = 7 for the N terminus sequence ions. Recognizing this behavior was fundamental in leading us to the conclusion that many of these sequence fragment ions are formed as neutral fragments during the 266nm-LD step which are then transported into the RETOF-MS source region for MUPI postionization. Only those neutral fragments containing a chromophore would be expected to be efficiently postionized a t the 255-nm ionizing wavelength, and this would account for the cutoff of the sequence ion series at the position of the last absorbing chromophore. On the basis of this observation, the chromophore can provide an anchor from which to begin the determination of the amino acid sequence by recognizing that the first low mass sequence fragment ion signal from either the N or C terminus is most likely due to an aromatic amino acid residue within the amino acid sequence. This behavior was observed for the series of sequence fragment ions produced in the spectra of other peptides investigated using 266-nm LD/255-nm MUPI as well. As with the pentapeptide mass spectra, it was again observed that a variety of immonium ion signals may be produced in the substance P mass spectrum a t sufficiently high ionizing laser powers. Examples of these low mass ions and the amino acid residues for which they are indicative are labeled in Figure 2. From these fragment ion signals it is possible to confirm the phenylanlanine aromatic amino acid contained in the peptide as well as several of the other amino acid residues in the peptide sequence. 3. Cramicidine D. Figure 3 shows a typical 266-nm-LD/ 255-nm-MUPI mass spectrum of gramicidin D taken at a desorbing laser intensity of 300 MW/cm2 and an ionizing laser intensity of 20 MW/cm2. A rich variety of sequence type fragment ions is again observed in the mass spectrum containing both the N and C terminus of the peptide (see also Table V). Starting from the C terminus, a relatively intense series of Y,,' sequence fragment ions and a partial series of relatively intense 'X, sequence fragment ions is formed beginning a t the n = 1 and 2 positions, respectively. A series of relatively intense C,' sequence ions containing the peptide N terminus is observed to begin at the n = 9 position. These sequence starting positions are in accordance
The Journal of Physical Chemistry, Vol. 96, No. 7, 1992 3161
Peptide Sequence Ions from Neutral Molecules 130 4ow
2008
0
0
"3
CJ
cJac;J
J
la0
aD00 UnU
c;0
Figure 3. Resonant 266-nm-Ld/255-nm-MUPI mass spectrum of gramicidin D taken at a desorbing laser power of 300 MW/cm2 and an ionizing laser power of 20 MW/cm2. Sequence ions with intensities greater than 5% of the base peak are labeled. Peaks labeled with an asterisk have been identified as background signals.
with the earlier observations made for substance P, where the sequence series began at the position of the first aromatic amino acid residue. The position of the first aromatic tryptophan amino acid residue for gramicidin D corresponds to the n = 1 position when starting from the C terminus and to the n = 9 position when starting from the N terminus. Characteristic immonium ions were again observed in the low mass region of the gramicidin D mass spectrum at higher ionizing laser powers, and these are labeled in Figure 3. The immonium ion signals for gramicidin D clearly show that the tyrptophan aromatic amino acid is contained in the peptide structure and that the other two aromatic amino acids, tyrosine and phenylalanine, are not part of the peptide sequence. In addition, immonium ion signals are observed with particularly strong signal intensities for the valine and leucine residues. These signals account for 3 of the 5 amino acid residues contained in gramicidin D. Intense immonium ion signals for the other two amino acid residues, glycine and alanine, may also be observed at somewhat higher ionizing laser intensities.
Discussion The analytical quality of the peptide spectra presented here may be illustrated through comparisons with analogous peptide spectra produced using other common mass spectrometric techniques such as FAB, PDMS, and collision-induced dissociation (CID). It is important to bear in mind that these other mass spectrometric techniques directly analyze the ionic material produced during the desorption event where our experiments analyze the neutral material desorbed from the surface. Comparison of the two enkephalin spectra with the published FAB results show spectra of comparable quality, particularly when the weak intensity sequence fragment ions are expanded for both approaches.I2J3 (12) Barber, M.; Bordoli, R. S.;Garner, G. V.; Gordon, D. B.; Sedgwick, R. D.; Tetler, L. W.; Tyler, A. N. Biochem. J. 1981, 197, 401. (13) Dass, C.; Desiderio, D. M. Anal, Biochem. 1987, 163, 52.
However, it is clear from the reported results that the relative abundances of the various types of sequence fragment ions observed using the two techniques are not the same. Where stronger signals from A and B type sequence ions appear in the FAB spectra, stronger signals from the C and Y sequence ions are observed under 266-nm-LD/255-nm-MUPI conditions. Both approaches produce abundant signals from the immonium ions of the two aromatic amino acids. Spectra containing complementary sequence fragment ion information are obtained in the 266-nm-LD/255-MUPI and FAB mass spectra. For the case of substance P, the reported FAB spectra begin to suffer from the weak sequence ion signal intensity relative to the background noise in the mass spectrum.I4 However, a partial series of C and Y type sequence ions have been identified in the spectrum. A second reported FAB spectrum of substance P shows a partial series of A type sequence ions.I5 The PDMS spectrum of substance P has been shown to contain almost complete series of A and C type sequence ions.I5 The substance P spectrum obtained in the 266-nm-MUPI experiments gives substantially more structurally significant fragment ion signals over the entire peptide sequence than either the FAB or PDMS mass spectra. In addition, a rather dramatic difference is observed in the types of sequence fragment ions observed. Where the substance P spectrum obtained in our MUPI experiments shows a dominant series of C terminus X, Y, and Z type sequence ions, the FAB and PDMS mass spectra show primarily dominant series of N terminus A, B, and C type sequence ions. It is also of interest to compare the substance P spectrum obtained in these experiments with the reported tandem FAB-CID ~pectra.'~J'In the spectrum reported by Scoble et al. where He was used as the collision gas, a dominant series of N terminus A sequence ions is again observed. In the spectrum reported by Desiderio and Katakuse where Xe was used as the collision gas, a variety of sequence ions containing both the N and C termini of the peptide was observed. For the case of gramicidin D, no direct ion desorption mass spectra (FAB, PDMS, or secondary ion mass spectrometry (SIMS)) could be located in the literature. However, the tandem FAB-CID spectrum shows a dominant series of B and Y type sequence fragment ions.I8 The gramicidin D spectrum obtained in the 266-nm-LD/255-nm-MUPI experiments also shows a strong series of Y type sequence ions, and, in addition, a variety of other sequence fragment ions were observed, again with an emphasis on the C terminus containing fragments. However, it is noteworthy that no B type sequence ions are detected. For both substance P and gramicidin D, the structural information obtained using the 266-nm-LD/255-nm-MUPI approach is once again of equal or better analytical quality and generally complementary in nature to the information obtained using other direct ion desorption or CID techniques. As detailed in the previous discussion, a general observation for the 266-nm-LD/255-nm-MUPI postionization mass spectra is that the sequence fragment ions observed in the mass spectra have structures complementary to the sequence ions observed when the directly desorbed ionic material produced in FAB and PDMS experiments is analyzed. It has been proposed that similar desorption mechanisms are in operation for both LD and particle bombardment techniques such as FAB and PDMS,19*20 and this view allows the complementary nature of the observed sequence fragment ion series to be easily rationalized. The particle desorption techniques analyze directly desorbed fragment ions. For (14) Williams, D. H.; Bradley, C. V.; Santikarn, S.;Bojesen, G. Biochem.
J . 1982, 201, 105. (1 5) Vorst, H. J.; van Tilborg, M. W. E. M.; van Veelen, P. A.; Tjaden, U. R.; van der Greef, J. Rapid Commun. Mass Spectrom. 1990, 4, 202. (16) Scoble, H. A.; Martin, S.A.: Biemann, K. Biochem. J . 1987, 245,
621. (17) Desiderio, D. M.; Katakuse, I. Anal. Biochem. 1983, 129, 425. (18) Johnson, R. S.;Martin, S.A.; Biemann, K. Int. J . Mass Specrrom. Ion Processes 1988, 86, 137. (19) Zakett, D.; Schoen, A. E.; Cooks,R. G. J. Am. Chem. Soc. 1981,103, 1295. (20) Hillenkamp, F. In Microbeam Analysis-1989; Russell, P. E., Ed.;San Francisco Press: San Francisco, CA, 1989; p 277.
3162
J . Phys. Chem. 1992, 96, 3162-3166
each ionic sequence fragment produced, it is reasonable to suggest that the complementary peptide fragment will be produced as a neutral species. The postionization approach used in these experiments would be expected to analyze these neutral fragments. Thus, for substance P, where a strong series of N terminus sequence fragment ions are observed under the PDMS direct ion desorption conditions, the neutral products of these fragmentations, the C terminus sequence fragments, form the dominant series under MUPI postionization conditions. This general observation may be considered supportive of the suggestion that similar processes lead to desorption in both LD and particle bombardment desorption techniques. This interpretation is certainly preliminary in nature and further experiments with, if possible, direct comparisons are necessary to confm or deny the apparent mechanistic similarities of the 266-nm-LD and particle desorption techniques.
These neutral molecules, upon MUPI postionization, produced high quality peptide mass spectra containing both strong signals from the peptide molecular ion and numerous sequence specific fragment ions. In m a t cases, sufficient sequence specific fragment ions are observed for deduction of the sequence of amino acid residues contained in the peptide. In addition, low mass immonium ions produced during the 255-nm-MUPI step at higher ionizing laser intensities may be used to provide secondary confirmation of the existence of a particular amino acid residue contained in a proposed peptide sequence. Finally, the complementary nature of the sequence fragment ion series observed in these experiments, when compared to those produced by direct ion desorption techniques such as FAB and PDMS, may be interpreted in support of the suggestion that similar desorption/fragmentation mechanisms are in operation for the different desorption methods.
Conclusion The examples reported here show that 266-nm LD of pure peptide samples followed by 255-nm MUPI postionization of the desorbed neutral molecules provides an effective means for characterizing small and medium sized peptides. The high energy of the 266-nm photons used for LD of the peptides produce both abundant numbers of intact neutral peptide molecules and a wide variety of structurally significant neutral fragment molecules.
Acknowledgment. This work was supported by grants from Deutsche Forschungsgemeinschaft (GR9 17/ 1-2) and the Bundesministerium fur Forschung und Technologie (1 3N5307-2). G.R.K. gratefully acknowledges the fellowship support of the Alexander von Humboldt Stiftung-Bonn, Germany. Registry No. Leu-Trp-Met-Arg-Phe,67201-39-2;Met-enkephalin, 58569-55-4;Leu-enkephalin, 58822-25-6;substance P, 33507-63-0; gramicidin D, 1393-88-0.
Investigations of Neutral Fragment Formation during Resonant 266-nm Laser Desorption Gary R. Kinsel, Josef Lindner, and Jurgen Grotemeyer* Institut fur Physikalische und Theoretische Chemie der Technischen Universitat Munchen, Lichtenbergstrasse 4, 0-8046 Garching, Germany (Received: August 29, 1991; In Final Form: December 3, 1991)
Fragment ions observed in mass spectra produced using 266-nm laser desorption followed by multiphoton ionization of the desorbed neutral molecules may originate as neutrsl fragments formed during LD or be produced from the intact molecule (L-W-M-R-F) was investigated during postionization. The pentapeptide leucyl-trvptophyl-methionyl-arginyl-phenylalanine to determine the origins of the C,' sequence fragment ions observed in the mass spectrum. It was found that the relative abundances of the fragment ion signals depended on both the desorbing laser power and on the ionization probe time in the jet entrained neutral molecule profile but not on the ionizing laser intensity. Neutral fragment formation during 266-nm laser desorption followed by postionization is suggested, but not confirmed, by these observations. Further investigations of the ionizing power and wavelength dependence of the relative fragment ion abundances also supported the neutral fragment formation interpretation. The results of the studies performed for L-W-M-R-F are presented, and the difficulties encountered in interpretation of these results are discussed. In addition, observations made on a larger sample of peptides are reported which suggest that a wide variety of structurally significant neutral fragments may be formed during 266-nm laser desorption.
Introduction In a recent paper we have shown that the 266-nm radiation of a frequency quadrupled Nd:YAG laser can be used to efficiently, resonantly desorb intact neutral molecules from a number of peptides up to the mass of gramicidin D (1881 amu).] The purpose of the previous study was to investigate the influence of resonant absorption by the sample at the desorbing laser wavelength, and the results showed that efficient desorption of large numbers of intact neutral molecules from thermally labile compounds occurred only under resonant laser desorption (LD) conditions. In earlier investigations a C 0 2 laser operating at 10.6 pm was used to perform LD because it was believed that the low energy of the IR photons would minimize both ionization and fragmentation of the sample molecules during d e s o r p t i ~ n . ~Indeed, ,~ under 266-nm-LD conditions, it was noted that a variety of fragment ions were always present in the peptide mass spectra with a broad range of relative abundances. A number of additional observations, which will be discussed, also suggested that many 'To whom all correspondence should be addressed.
0022-3654/92/2096-3 162$03.00/0
of these sequence fragment ions are produced as neutral fragments during the 266-nm-LD step which are then postionized by laser multiphoton ionization (MUPI). There is, at least in principle, no reason not to believe that large neutral fragments may be formed during resonant 266-nm LD. Certainly, direct 266-nm LD of ions from many smaller peptides produce spectra containing a variety of fragment ion^.^,^ In addition, many direct ion desorption techniques, such as fast atom bombardment (FAB) and PDMS, have also been shown to produce a variety of fragment ions.@ If ionic fragments are formed ( 1 ) Kinsel, G. R.; Lindner, J.; Grotemeyer, J.; Schlag, E. W. J . Phys. Chem. 1991, 95, 7824. (2) Grotemeyer, J.; Schlag, E. W. Angew. Chem., Int. Ed. Engl. 1988, 27, 447. . .
(3) Grotemeyer, J.; Lindner, J.; Koster, C.; Schlag, E. W. J . Mol. Strucr. 1990, 217, 51.
(4) Karas, M.; Bachmann, D.; Hillenkamp, F. Anal. Chem. 1985,57,2935. ( 5 ) Van Vaeck, L.; Van Espen, F.; Adams, F.;Gijbels, R.; Lauwers, W.; Esmans, E. Biomed. Environ. Mass Spectrom. 1989, 18, 581. (6) Dass, C.; Desiderio, D. M. Anal. Biochem. 1987, 163, 52. (7) Barber, M.; Bordoli, R.S.; Garner, G . V.; Gordon, D. B.; Sedgwick, R. D.; Tetler, L. W.; Tyler, A. N. Biochem. J . 1981, 197, 401. (8) Williams, D. H.; Bradley, C. V.; Santikarn, S.; Bojesen, G. Biochem. J . 1982, 201, 105.
0 1992 American Chemical Society
