Anal. Chem. l988, 60, 1791-1799 (25) Magnante, P. C.; Stroke, H. H. J . Opt. SOC. Am. 1969, 5 9 , 836. (26) Chuckrow, R.; Stroke, H. H. J . Opt. SOC.Am. 1971, 67, 218. (27) Marino , C. A.; Fuiop, G. A.; Groner, W.; Moskowitz, P. A,; Redi, 0.; Stroke, H. H. Phys. Rev. Left. 1975, 34, 625. (28) Poulsen, 0.; Hall, J. L. Phys. Rev. A 1978, 78, 1089. (29) Barboza-Flores, M.; Redi, 0.;Stroke, H. H. 2.Phys. A 1085, 321, 85. (30) George, S.; Munsee, J. H.; Verges, J. J . Opt. SOC. Am. 8 : Opt. Phys. 1985, 2 , 1258. (31) Fearey, B. L.; Parent, D. C.; Keller, R. A.; Miller. C. M., LOS Alamos National Laboratory, unpublished resuks, 1987. (32) Parent, D. C.; Fearey, 6. L.; Miller, C. M.; Keller, R. A. Proceedingsof the 35th Conference of the American Society for Mass Spectrometry,
1791
May 1987; p 1006.
RECEIVED for review October 8, 1987. Accepted March 27, 1988. B.L.F. thanks Los Alamos National Laboratory for postdoctoral fellowship supportduring the performance of this work. Also, M.W.R. is grateful for support from the Robert A. Welch Foundation and the Associated Western Universities. This research was supported by the U S . Department of Energy under Contract W-7405-ENG-,36.
Daughter Ion Mass Spectra from Cationized Molecules of Small Oligopeptides in a Reflecting Time-of-Flight Mass Spectrometer Xuejun Tang,’ Werner Ens,’ Kenneth G . Standing,’ and John B. Westmore*B2
University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
The use of a reflecting time-of-flight mass spectrometer to analyze daughter Ion mass spectra of metastable Ions Is brlefly descrlbed. Representatlvespectra are presented for decomposltlon of cationlzed molecules of glycylglycylphenylalanlne, leuclne-enkephalin, and methionlne-enkephalln; these were produced by cesium ion bombardment of thin solid sample deposits. The spectra Illustrate the current performance and capabilities of the Instrument. The daughter ion spectra, which can be analyzed wlth greater simplicity and less ambiguity than “dlrect” spectra, are strongly Influenced by the identlty of the bound catlon (H’, Na’, K’, or Ag’). Many of the daughter Ions are formed by known reactions, yielding structural and sequence lnformatlon. I n addition, the [M Na]’ and [M Ag]’ Ions decompose by a previously unreported pathway, namely, rearrangement of a C-terminal carboxyl oxygen (as OH) onto the daughter ion contalning the N termlnal. Thls Is, In fact, the main channel for [M Na]’ Ag]’ dedecay and accounts for a large fraction of [M compositions. Thls assignment Is supported by ‘*O-labelIng studles of the C termlnal of glycylglycylphenylalanine.
+
+
+
+
In recent years, several methods of analyzing daughter ion mass spectra have been developed in order to determine fragmentations of their parent ions. These methods have commonly used multiple sector (various E and B sequences and scanning combinations), triple quadrupole, Fourier transform ion cyclotron resonance (FT-ICR), or hybrid mass spectrometers. The advantages of daughter ion mass spectra for structural analysis of ions have been well documented. Of particular relevance to this paper are a number of studies of small oligopeptides (1-16). I t is also possible to study the metastable decomposition of a parent ion in a reflecting time-of-flight (TOF) mass spectrometer ( I 7-21). A new reflecting TOF instrument has recently been built at the University of Manitoba (21,22). It incorporates an ion mirror that can reflect a charged particle at 177’ back along the flight tube. This instrument (which has a vertical flight tube) is shown schematically in Figure ‘Department of Physics. *Department of Chemistry.
1. An individual ion, desorbed from a sample by a primary ion pulse, is accelerated by a potential difference between the target and a grid; it then travels toward a detector (detector 1)located at the end of the flight tube. With zero electric field in the mirror it passes through the mirror to this detector. Alternatively, when a sufficiently high retarding field (i.e. a potential on the rear grid greater than the ion acceleration potential) is applied within the mirror, the ion is reflected back out of the mirror toward another detector (detector 2) a t the other end of the flight tube. Some ions may decompose after passing through the acceleration grid. With zero potential on the mirror both charged and neutral daughters pass through the mirror to detector 1. A “normal” or “direct” mass spectrum results from the integration of all particles recorded by this detector. Since momentum is conserved in the decay, both daughters arrive there at a time close to the flight time characterizing their parent. They thus appear in the spectrum as a broadened peak at the same mass number as the parent ion. Ions that enter and leave the mirror without decomposing will travel toward detector 2 and will be focused in time on this detector. In this way a “reflected ion” spectrum is obtained, while detector 1 can be used to record a “neutral species” spectrum generated by parent ion decompositions during the first leg of field-free flight. As pointed out above, this spectrum exhibits broadened peaks at positions characteristic of the parent ions. For ions that decompose in this stage of field-free flight, it is possible to record a correlated spectrum, i.e. those reflected daughter ions that belong to the same event as the neutral fragment. At the end of the observation period (ca. 100 p s ) corresponding to a given primary pulse, a computer examines the spectrum of neutral fragments observed in detector 1 during that period (23). If a neutral fragment has arrived during a preselected time (mass) window, the spectrum of charged daughters detected in that observation period is recorded in a dedicated section of computer memory (23). Such spectra are “daughter ion” spectra and correspond, for example, to those obtained by an E / B linked scan in a sector-field mass spectrometer of EB geometry. An advantage of using a TOF mass spectrometer is its high efficiency; no scanning is performed, ion transmission is much higher than for sector instruments, and decay of a number of parent ions can be examined at the same time. Therefore, sensitivity is high. As we shall see, the resolution and sig-
0003-2700/88/0360-1791$01.50/00 1988 American Chemical Society
1792
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
' C
'$7
,
1
Flgure 1. Schematic diagram of the reflecting time-of-flight mass spectrometer, showing typical paths of parent ions (0)and daughter The angle between the secondary ions and the spectrometer ions (0). axis has been exaggerated for the sake of clarity (actual angle = 1.4"). The front grid of the mirror is 56 and 74 cm from the acceleration grid and detector 2, respectively. Detector 1 is 100.4 cm from the acceleration grid.
nal/noise ratio for daughter ion spectra are both good. Although the option was not utilized in this study, it is also possible to generate "parent ion" mass spectra of selected daughter ions. In this case, a time window is selected for detection of a given daughter ion in detector 2 and the correlated neutral fragments detected in detector 1 are recorded. The recorded neutrals, which have approximately the same flight times as their parent ions, thus correspond to a parent ion spectrum. The recorded peaks are broadened by kinetic energy released upon fragmentation. In general this will not be a serious problem, because such spectra normally contain very few peaks, and high resolution is not necessary to resolve them or to measure their time centroids. An ion may also decompose during acceleration, within the mirror, or on the return leg of field-free flight. Ion decomposition during acceleration can cause peak broadening and tailing; it also contributes to background counts (24,25). The probability of ion decomposition within the mirror can be quite high because an ion spends a substantial time there (in the present mirror configuration, a parent ion spends up to 50% of its total flight time in the mirror). The products of such decompositions (charged and neutral) will be formed at different points within the mirror and will therefore have an extensive distribution of velocities. Should they reach a detector, they will not be focused in time so they will contribute to the general background. Ions that decompose outside the mirror during the return leg of field-free flight can cause peak broadening in the reflected ion spectrum. However, the effect on peak shape is usually small because the relative number of ionic decompositions is much smaller than in the first leg (assuming a first-order rate law for unimolecular decompositions). These studies with the reflecting time-of-flight mass spectrometer have shown that the decompositions of metastable [M + HI+, [M + Na]+, [M + K]+, [M + Ag]+, and [M + 2Na + 1]+ions of small oligopeptides exhibit considerable differences. While many of the fragmentations correspond to known processes, we have observed specific fragmentations that appear to be triggered by the bound cation. These studies also illustrate the performance of the instrument.
EXPERIMENTAL SECTION Samples. The peptide samples (Sigma Chemical Co.) were used as supplied. Labeling of the C terminal of glycylglycylphenylalanine with l80was based on a method described in the literature (26). To about 0.1 mg of peptide were added ca. 20 pL of HZ'*O (97 atom % "0; Merck, Sharp and Dohme (Canada) Ltd.) and ca. 1pL of 3 M HC1 in a sealed vial. After 16 h at 42.5 "C volatiles were removed by evaporation and the residue was
analyzed in the TOF mass spectrometer. This analysis indicated ca. 50% incorporation of l80into the peptide. (Note: Success or failure of label incorporation is critically dependent upon experimental conditions. Thus, after 2 days at 37 "C, incorporation of "0 could ilot be detected, while for a similar time at 47 "C, complete hydrolysis of the peptide occurred.) For mass spectrometric study, a few micrograms of each sample, at a solute concentration of ca. 1 mg/mL in methanol, was either electrosprayed onto the metal surface of aluminized Mylar film or deposited from solution to give a thin solid deposit on 12-50 mm2 of a silver planchet that had been etched by dilute nitric acid. (The etching procedure with 1-270 nitric acid, which can be monitored visually, requires 2-5 min and can be accelerated by warming the solution). Mass Spectrometry. Spectra of samples were obtained in the Manitoba reflecting time-of-flight mass spectrometer (21,22). This instrument uses a thermionic cesium primary ion source (27) Torr. and is maintained at an operating pressure of (0.5-5) X Samples are bombarded by a pulsed beam of primary ions at ca. 20" to the target normal (2-ns pulses full width at half-maximum, repeated at a frequency of 2 kHz). The intensity of the primary ion beam is adjusted to give ca. 1secondary ion per primary ion packet. The Cs' ions (generated by an 18-keV ion gun) pass through a grounded grid and then strike the sample 4 mm behind it. Since the target is at +5 kV for the positive ion studies described here, the Cs+ ions strike the sample with an energy of 13 keV. Secondary ions and their decomposition products are detected by microchannel plate electron multipliers located at the ends of the flight tube (see Figure 1). Both detector outputs are processed simultaneously by the data acquisition system (23). Daughter ion mass calibration of the instrument is achieved by measuring flight times for parent and daughter ions from the metastable decompositions of cesium iodide cluster ions under the same accelerating and mirror potentials that are used for the peptide studies [cs(csI),+,l+
-
[CS(CSI),]+ + (CSI), (n 1 2; p 2 1)
When the electric field in the mirror is set for optimum resolution of parent ions, the mass m'and flight time t* of a daughter ion from a parent ion of mass m that has decomposed in the first leg of field-free flight are related by m' = m[2(t*/t) - 11 (la) or
t* = t[(m'+ m)/2m]
(lb)
where t is the flight time of the parent ion (22). Flight times are determined from the time centroids of the peaks corresponding to the ions of interest, and the daughter ion masses are then calculated from eq la. From eq l b it is clear that the flight times of the daughter ions are always less then those of their parents. However, the flight times of the daughter ions are somewhat greater than the flight times t'of ions of the same mass produced directly at the target, e.g. for m'/m = 0.7, t * / t ' = 1.016 (since t 0: m1I2for ions produced at the target).
RESULTS AND DISCUSSION Glycylglycylphenylalanine (GGF). Mass spectra generated by bombardment of thin deposits of solid peptide samples were recorded by using techniques and instrumentation that have been described previously (20-23). Ordinary direct spectra could be observed in detector 1 with zero field in the mirror and compared with reflected spectra observed in detector 2 with the mirror on. For example, Figures 2 and 3 show a comparison between direct and reflected ion spectra of positive secondary ions generated from GGF (1) deposited upon etched silver. Although the resolution in the reflected ion spectrum is better and the background signals are smaller than for a direct spectrum, the reflected ion spectrum is more complicated. The time scale calibrations for daughter ions of metastable decompositions differ from the parent ion time calibration (see eq 11, producing, in effect, several superimposed mass spectra. Reflected daughter ions have flight times
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
1793
5060u/
1
L
FLiGHT TIME (us)
Direct spectrum (positive ion mode) of GGF, deposited on etched silver, accumulated in 8-11stime bins. Nominal masses of ions are shown. Flgure 2.
z
I
I
between those of their reflected parent ions and those of reflected ions, isobaric with the daughter ions, that have been formed promptly (i.e. prior to acceleration). In the high-mass part of the reflected ion mass spectrum the cationized molecules [M + HI+, [M + Nal+, and [M + Agl' are prominent. These are, of course, parent ions. Although daughter ions are also present in this spectrum, they are not usually readily distinguished from other fragments because the strengths of their signals are quite low. However, they are readily observed in the correlated (i.e. daughter ion) spectra. This is illustrated in Figure 4a, which shows, on the same time scale, the daughter ion spectrum for decomposition of [M Ag]+. The daughter ion peaks are well resolved, and although the intensity of the spectrum is -10% of the reflected ion spectrum, the daughter ions are no longer obscured. (Also observed in the correlated spectrum are parent [M + Ag]+ ions. These arise from the occasional desorption of two or more parent ions (i.e. random coincidences) by the same primary ion packet.) A comparison between the daughter ion spectra for decomposition of [M H]+ and [M + Na]+, presented in parts b and c of Figure 4, respectively, shows that [M + Na]+ ions undergo significantly less extensive decomposition and only one daughter ion species from [M + Na]+ is detected; the daughter is not even visible in the overall reflected spectrum (Figure 3). This indicates that [M Na]+ ions surviving the acceleration period (ca. s) are significantly more stable than corresponding surviving [M + H]+ ions. (Note that the observed relative stabilities need not reflect the energetics of ion formation. Thus, even if [M + Na]+ and [M + H]+ are not preformed ions and have differing amounts of excitation energy due to differing exothemicities of cation-transfer reactions leading to their formation, the cationized molecules could be collisionally deenergized in the condensed phase before desorption. In addition, highly excited ions may not survive the acceleration period.) The results are summarized in Table I. Two points can immediately be made. First, considerable variation in the stabilities and decomposition pathways of the different cationized molecules is apparent. Second, information about ion decompositions can be extracted from reflected ion and daughter ion mass spectra with greater simplicity and less ambiguity than from a direct mass spectrum. The second point is particularly important because matrix effects and the presence of impurities in the sample are minimized. In this respect the advantages of the present technique are similar to those of daughter ion studies made by other methods. As we shall see later, structural informtion for other small peptides can also be readily obtained. Before discussing the structural detail that can be derived from the daughter ion spectra, we find it appropriate to assess the confidence that can be placed on the measured masses by comparing experimental and calculated masses (most abundant isotopes) based on the assignments given. (To identify ionic fragments of peptides, we have adopted a symbolism based on that suggested by Roepstorff and Fohlman (28) and subsequently endorsed in slightly modified form by Biemann and Martin (29). A brief explanation is given in the
--1
m
& +
o 26
28
30
32
34
+
38
36
'00 2600 280
I: I
0
+
FLIGHT TIME ius1
Reflected positive ion mass spectrum of GGF, deposited on etched silver, accumulated in 8-ns time bins. Nominal masses of parent ions are shown. Peaks denoted by an asterisk correspond to daughter ions. For example, the peak at t = 34.5 ps corresponds to a daughter ion of m l r 166 formed from a parent [M HI+ ion (see Figure 4b). A s noted in the text, such ions have flight times shorter than their parent ions but longer than ions of the same mass formed at the target, so the peak is displaced by about 1 ps from the normal m l r 166 peak. Similarly, the peaks at ~ 4 3 . 2ps correspond to a daughter ion of m l r 272l274 from [M Ag]+ (see Figure 4a) and appear at a longer flight time than the normal peak for m l r 280. Figure 3.
+
+
0
FLIGHT TIME (us)
Daughter ion mass spectra of parent ions from GGF,accumulated in 32-ns time bins: (a) [M Ag]', (b) [M HI+, (c) [M Na]'. Nominal masses of daughter ions are shown.
Flgure 4.
+
+
+
+
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
Table I. Daughter Ion Spectra of Cationized Molecules of Glycylglycylphenylalanine mass parent ion
[M + HIt
exptl
(calcd)
280.16
(280.13)
[M + Na]+
302.16
(302.12)
[M + lo7Ag]+
386.09
(386.03)
daughter ion mass exptl (calcd)
assignt" (positive ions)
223.31 166.08 120.31 115.07
(223.11) (166.09) (120.08) (115.05)
Y2 + 2 Y1 + 2 I, + 1 B2
155.33
(155.05)
B2 + 17 + Na
272.16 238.94 225.94
(271.99) (238.96) (225.98)
Y1+ 1 + Ag B2 + 17 + Ag I3 + Ag
mass diffb (exptl - calcd) 0.03 0.20 -0.01 0.23 0.02 0.04 0.28 0.06 0.17 -0.02 -0.04
'I, = C,H,N; Bz = C4H,Nz02; Y1 = C9HloNO2;Yz = CllH13N203. *Assumes 1 = H; 2 = 2H.
Appendix.) As shown in Table I, the calculated masses of parent and daughter ions are consistently within 0.3 u of the experimental masses. The largest differences are associated with small peaks. For such peaks, poor counting statistics mean that centroid determination is imprecise. The smallness of the error for known ions means that, for all ions, the nominal masses are not in doubt. (For the relatively low mass ions studied here the concept of nominal mass is still meaningful.) The formation of some daughter ions can be explained by bond fissions accompanied by incorporation of up to two extra hydrogen atoms or a metal cation to give ion structures expected to be stable. Specifically, these are B2,Y, + 2, Y2 + 2, and I3 1 (which are known fragmentations (29) together with the silver analogues Y, 1 Ag and I3 + Ag (see Table I and individual ion structures). Sequence information can be obtained directly from some of these.
+
Scheme I Na+
Na]'
Nat
+ +
0
0
BZ+l7+Na
Y,+2
S'H2 H3kH2CONHCHCOOH Y2+2
?HZ dg+ NHZ-CH- COOH Y,+ltAg
13+1
CH2
I
&+NH=CH h+Ag
A significant new finding is the appearance in these spectra of abundant B2 + 17 + Na and B2 + 17 + Ag daughter ions. These represent a major channel of decay from [M + Na]+ and [M + Ag]+,respectively, and have no analogue among the daughter ions from [M + HI+.These ions are not analogues of C, + 2, i.e. C, + 1 + Na/Ag, that have been reported in peptide mass spectra (29). The latter ions would correspond to B2 + 16 Na/Ag. At the same time, when Na+ or Ag+ is bound to the peptide molecule, the fragmentations leading to B2and Y2 2 (or their Na or Ag analogues) =e suppressed. This implies that the metal ions are bound to the peptide in such a way that peptide bond fission is prevented. Chelation of the peptide to the metal ions offers an attractive mechanism for reducing the number of decomposition channels and diverting the fragmentation along alternative pathways. It is known that, in solution, Naf binds preferentially to peptide carbonyls (30). If this is also true in the gas phase, then the decomposing form of [M Na]+ may be as depicted in Scheme I. Although Na+ attachment to the carbonyl oxygen increases the double bond character of the amide CN bond,
+ +
+
it also makes the carbon atom more susceptible to nucleophilic attack. (The net result of these competing effects may be quite sensitive to the actual cation that is bound.) The hydroxyl of the terminal carboxyl group is suitably placed for just such attack and thereby to trigger the rearrangement shown, which leads also to stable neutral species. Thus, B + 17 + Na/Ag is assigned as B OH Na/Ag. This evenelectron species is to be preferred over an alternative assignment such as the odd-electron species B + NH3 + Na/Ag. The alternative assignment has several problems: it is not apparent where to place the two rearranged hydrogens, even-electron parent ions tend to decompose to even-electron rather than odd-electron daughters, and it is difficult to conceive of stable neutral species that could be eliminated. Support for the mechanism proposed in Scheme I is provided by I80-labeling studies to be described later. The nature of the bonding of Ag+ to the peptide is not well documented. If we assume, from the well-known complexing of Ag+ by amines, that Ag+ binds preferentially to the primary amino group and note that Ag+ has a strong tendency to form collinear bonds, then it is geometrically possible for Ag+ to also bind to the second peptide carbonyl removed from the peptide N terminus, as shown in the structure. (Note that
+
H2N
/ H2C
A :
+
0
Nc/
NH\CHCH2CsH5
/1
'
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988 26000
4132
136
I*
2200
Z
I
1
J
\
1795
'
397
4300
0
FLIGHT TIME (us)
FLiGHT TIME lusi
Flgure 5. Partial reflected positive ion mass spectrum of Leu-enkephalin deposited on etched silver, accumulated in 20-ns time bins. Nominal masses of stable ions are shown. Peaks denoted by an asterisk correspond to daughter ions.
this is not possible for the peptide carbonyl first removed from the N terminus.) Such a structure could undergo a decomposition similar to that depicted in Scheme I for [M + Na]'.
Leucine-Enkephalin and Methionine-Enkephalin. Figure 5 shows a partial reflected ion spectrum for Leuenkephalin (2) deposited on etched silver, and Figure 6 shows the daughter ion spectra for the various cationized molecules. Tables I1 and I11 summarize the daughter ions obtained from cationized molecules of Leu-enkephalin and Met-enkephalin (3), respectively, which differ only in the C-terminal amino
IAI ILL 24 23 x, 22 xi Y1 2 , rrrrrrrrrrrr
I
x4
Y4
x3
Y2
Y3
2 and 3: R I = C H ~ C ~ H ~ O H R,=R3=H; ; Rq=CH2C6H5 2: R5=CH2CH(CH3), (Leu) ', 3: t%5=CH2CH2SCH3 (Met)
acid. Since methionine has a molecular mass 18 u greater than that of leucine, it is easily possible to identify those ions containing the C-terminal amino acid residue. In addition to proton and sodium ion attachment, potassium ion attachment is observed for these molecules. Although the abundance of [M + K]+ in the spectra is never large, this species is less stable than [M + Na]+ and yields a daughter ion spectrum of reasonable quality for both molecules. An attempt to generate [M Cs]+ ions of Leu-enkephalin by spiking the sample with cesium iodide resulted instead in the detection of Cs+, without any evidence in the spectrum for [M + Cs]+ (or any Cs-containing fragment ions). Attachment of Na+ ions was greatly enhanced, producing a significant yield of [M 2Na - 1]+ ions. (Since water was not rigorously excluded during sample preparation, it may compete with the peptide for binding of alkali-metal ions. It has been pointed out (31) that, when water is excluded, Cs+ bonds very strongly to oxygen in ether linkages to form stable [M Cs]+ ions.) The number and yield of daughter ions produced by decomposition of [M + 2Na - 1]+,listed in Table
+
+
+
Daughter ion mass spectra of parent ions from Leu-enkephalin: (a) [M + Ag]' (32-ns bins), (b) [M + H]+ (48-11sbins), (c) [M Na]+ (32-ns bins). Nominal masses of daughter ions are shown.
Flgure 6.
+
+
I1 indicate it to be less stable than [M Na]+. As before, several of the ions in Tables I1 and I11 can be directly explained by a simple bond fission, together with incorporation of hydrogen or metal atoms, if necessary. From these, sequence information can readily be deduced but will not be elaborated upon here, since this is self-evident from the tables. Furthermore, the daughter ions from unimolecular decomposition of [M H]+ ions are similar to those reported by others (2-16) for collisionally induced decompositions. Instead, some other noteworthy features of the daughter ion spectra are now discussed. The hydroxyl transfer mechanism proposed in Scheme I for decomposition of [M + Na]+ and [M Ag]+ ions of GGF can be invoked to explain the formation of B4 17 + (Na/Ag) and B3 17 + (Na/Ag) daughter ions from the enkephalins. For B4 17 + (Na/Ag) this would be analogous to that proposed for compound 1 in Scheme I. The B4 17 + (Na/Ag) ion can be regarded as the [M + (Na/Ag)]+ ion of a tetrapeptide and after reorganization of the metal-peptide bonds could decompose to B3 + 17 + (Na/Ag), again by the mechanism of Scheme I. Scheme I1 depicts an alternative mechanism for the formation of B3 + 17 (Na/Ag) from [M + Ag]+. The presence of B, + 17 + H as a daughter ion of [M + H]+ of Leu-enkephalin (but not of Met-enkephalin) is an indication that an analogous decomposition reaction can occur for protonated molecules, though less readily. Furthermore, the decomposition reaction has not been observed for [M + K]+ of either molecule, suggesting, perhaps, that the carbonyl carbon is less susceptible to nucleophilic attack when K+ rather than Na+ or Ag+ is bound to carbonyl oxygen. In the case of the Ag-containing ions, further ions are observed 18 and 46 u lower than B, 17 Ag. These have been assigned as B, -1 Ag and A, -1 + Ag, respectively. They could be analogues of known B, and A, species that are formed directly from [M + Ag]+ but might be formed from B, + 17 + Ag, by simple losses of H 2 0 or HCOOH, respectively. The mj z 262,205, and 177 ions are interior fragments. The first two have previously been assigned an acylium ion structure (29),consistent with B-type ions as their immediate precursors. They can be represented by a two-letter code, based on that originally suggested (28),as (B4Y4)3 + 1 and (B4Y& + 1, respectively. In these codes the first letter indicates the first bond cleaved (perhaps not implied in the
+
+
+ +
+
+
+
+
+ +
1796
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
Table 11. Daughter Ion Spectra of Cationized Molecules of Leucine-Enkephalin mass
parent ion [M +
(CZ8H38NS07)
(calcd)
556.29
(656.28)
578.20
daughter inn mass
assignment"
mass diffb
(positive ions)
(exptl - calcd)
exptl
(calcd)
443.05 425.08 397.05 336.14 279.4'2 278.20 262.13 220.73 20F, 18 176.43 135.77 120.51
(443.19) (425.18) (397.19) (336.19) (279.17) (278.11) (262.12) (221.09) (205.10) (177.10) (136.07) (1 20.08)
46.5.08 406.01 344..55 31: 97
(465.17) (318.1I )
R,
435.03 332.95 316.43 276.21
(435.14) (333.10) (316.07) (276.07)
Aq-l+K C,+I+K Cq + 1 + K - NH,? C,+l+K
t544.13 531.10 503.02 401.99 384.13 3R5.97 326.81
(549.09) R, + 17 + Ag R, - 1 + Ag (531.08) A4 - 1 + Ag (503.08) R, + 17 + A a (402.02) ~ + . o i ) R? - 1 + Ag (356.02) A ? - 1 + Ag (326.99) R, - 1 + Ag
493.09 436.93 418.90
(493.19) (437.18) (419.17) (380.16) (339.10) (323.13) 1282.08) (225.06) (1 76.07)
0.01 -0.14 -0.10 -0.14 -0.05 0.25 0.09 0.01 -0.38 0.08 -0.67 -0.30 0.43 -0.06 -0.09
(578.26)
+ 17 + Na
(594.23)
594.20
(662.17)
662.17
[M + 2Na - 11' (C28H86Ns07Na= Q)
--
exptl
(600.24)
600.22
379.99
338.86 322.99 281.92 2?5.?9 175 44
original proposal), and the last subscript denotes the number of amino acid residues contained in the ion fragment. (Note that this value, when subtracted from the sum of the subscripts within the parentheses, gives the number of amino acid residues in the intact peptide.) However, if Y3 2 and Y, + 2 are their precursors, then an ammonium ion structure seems more probable. The isomeric ion structures for m / z 205 are as shown. Experimental confirmation of the actual precursors has not been achieved because the correlated spectra for decomposition of these putative precursor ions are too weak.
Q - CH2CeH4OH Y, + 2Na A, - 1 + Na Y, + 2Na C, + 2Na Y, + 2Na Cz + 2Na C, + 2Na Y, + 2Na
-0.14 -0.03 -0.11 -0.15 0.36 0.14 0.00 0.04 0.02 -0.06 -0.03 0.12 -0.05 -0.1 8 -0.02 -0.10 -0.25 -0.27 0.17 - 0.24 -0.14 -0.16
n "? 0.1 3
Most of the daughter ions from [M + 2Na - 1]+retain two sodium atoms. Those in the Y, + 2Na series likely h a w structiires based on that shown. The prominence of C, + 2Nfi ONa
+
A
ONa Y.-2Na CHz I
H2NCH2CONHCH- C-0'
YHZ H,&CH~CONHC=C=O (Y&)z+1
(B1Y3)2+1
m z 205
[M + K]+ ions, upon fragmentation, yield a number of sequence-related daughter ions. Interestingly, these differ from the sequences of daughter ions from [M + HI+.
(m-0
3)
C.+2Na
in the C, + 2Na sequence suggests the involvement of the phenolic group of tyrosine, as shown. However, the phenolic group is not essential because even phenylalanine can form an [M + 2Na - 11' ion (32). '*O-LabelingStudies. Labeling studies with lSOfulfilled two purposes: (a) to provide support for the mechanism proposed in Scheme I and (b) to evaluate the current capahility of the instrument in selecting specifically labeled parent ions for daughter ion analysis. This technique, of course, is
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
1797
1 *:
FLIGHT TIME (p)
Reflected positive ion mass spectrum of '80-labeled GGF, with conditions and notation as for Figure 3. The unassigned peak at m l z 270 may arise from an unidentified compound formed during the Flgure 7.
preparation of the labeled peptide. Scheme I1 CHPCBH5
I
A!3+
FLIGHT TIME (ps) Figure 8. Daughter ion mass spectra of parent ions from '80-labeled GGF, with conditions as for Figure 4: (a) [M 4- Ag]', (b) [M 4- H I + , (c and d) [M Na]'. For (a) and (b) wide time windows were used to include all isotopic members of the parent ion groups. For (c)and (d) narrow time windows were used to select the 180-labeledand ' ' 0
+
parent ions, respectively.
HOCsHdCH2CH
I I
1 HN
CH2
'
CH2-
B3+17+Ag
C
+
{
NH=CHCH2CBHS
5R H: : !
/NH
b
well-known when double (or higher) sector, triple quadrupole, or FT mass spectrometers are used, but to our knowledge, it has not been previously reported for TOF instruments. Labeling of the C-terminal COOH group of peptides with is achieved under acidic conditions that may also promote hydrolysis of the peptide. We did succeed, however, in achieving sufficient incorporation into GGF for these studies. The extent of incorporation was determined from the areas of the peaks in the [M + HI+, [M + Na]+, and [M + Ag]+ regions of the reflected spectrum (Figure 7). The P/(P 2)/(P 4) ratios were 33.9:43.7:22.4 and 44.5:40.3:15.2 for [M H]+ and [M + Na]+, respectively, and 23.446.3:25.3:4.9 for P / ( P + 2 ) / ( P + 4)/(P + 6) of [M + Ag]+. (P represents the isotopic ion of lowest m / z value in the natural abundance ion group corresponding to the indicated ion type.) These ratios lead to calculated incorporations of 43.7,46.3, and 48.2% for a single atoms, and 22.4, 15.2, and 5.0% for two respectively. The [M Na]+ ratios are probably the most reliable owing to the greater signal/background ratio. To study decompositions of isotopically labeled parent ions it is necessary to record daughter ions in detector 2 that correspond to arrival of the associated neutral species in de-
+
+
+
+
tector 1. In principle, a time window should be selected such that only events corresponding to a specific isotopically labeled species are used. In practice, the success of this strategy depends upon the width of the peak corresponding to the arrival of the neutral species. The results described here illustrate three different situations. For decomposition of [M + HI+ ions the neutral species peaks observed in detector 1 with the mirror on are relatively broad. Consequently, a relatively wide time window was selected to include all isotopically labeled components of [M + HI+.The daughter ion spectrum (Figure 8b) confirmed, as expected, that the l8O label is located on the C terminal. The measured P/(P 2)/(P + 4) ratios for the C-terminal Y1 + 2 ( m / z 166/168/170) and Y2 + 2 (223/225/227) fragments were 47.6:44.8:7.7and 51.6:48.4:0,respectively, i.e. close to the values above for l80incorporation into the intact molecule. Incorporation of into the B2 ion ( m / z 115) was not detected, but the signal involved was, admittedly, very is located small. Thus, within experimental error, all of the a t the C terminal. To study decomposition of [M + Na]+, it is possible to open a narrow time window that selects individual isotopically labeled parent ions. The neutral species peak profile is shown in Figure 9a. When a narrow time window centered on 18.56 fis is used to select the m / z 302 parent (unlabeled), the B2+ 17 + Na daughter is observed unshifted a t m / z 155, as expected (Figure 8d). (Actually, ca. 5% of the 180-labeled daughter is recorded at m / z 157 because some neutrals from lSOparents fall within the time window for neutrals from l60 parents.) When a narrow time window centered on 18.64 fis is used to select the neutral from m / z 304 labeled parent (Figure 9a), the daughter ions are recorded a t m / z 155 and 157 in the ratio 55:45 (Figure 8c). Ideally, this ratio should be 50:50 for incorporation of one of the two COOH oxygens
+
1798
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
Table 111. Daughter Ion Spectra of Cationized Molecules of Methionine-Enkephalin mass exptl
(calcd)
574.19
(574.23)
596.19
612.20
680.06
daughter ion mass exptl (calcd)
assignment" (positive ions)
425.17 410.94 397.02 354.32 297.08 278.21 262.43 221.28 205.19 177.03 150.12 136.19 120.10
(425.18) (411.17) (397.19) (354.15) (297.13) (278.11) (262.12) (221.09) (205.10) (177.10) (150.06) (136.08) (120.08)
B, Y,
465.15 419.28 395.95 318.08
(465.17) (419.17)
B, 17 + Na A, - 1 + Na ? B3 + 17 + Na
434.98 348.09 333.08 317.11 314.55 290.28 276.22
(435.14)
A,-l+K ?
(333.10)
C3+1+K
(276.07)
? ? ? C,+l+K
(549.09) (531.08) (517.07) (503.08) (460.05) (402.02) (384.01) (356.02) (326.99)
? B, + 17 + Ag B4 - 1 Ag Y, + 1 + Ag A4 - 1 + Ag Y3 + 1 + Ag B3 + 17 + Ag B3 - 1 + Ag A3 - 1 + Ag B, - 1 Ag ?
(596.23)
(318.11)
+2
A4
Y3 + 2 Y, + 2 B3 (B4Y413 + 1 B, (B4Y3)2 + 1 (A4Y3)Z + 1 Y, + 2 I, + 1 I, + 1
+
(612.19)
(680.13) 610.46 548.92 531.08 516.93 502.79 459.69 401.77 383.92 356.31 326.68
+
+
mass diffb (exptl - calcd) -0.04 -0.01 -0.23 -0.17 0.17 0.05 0.10 0.31 0.19 0.09 -0.07 0.06 0.11 0.02 -0.04 -0.02 0.11 -0.03 0.01 -0.16 -0.02
0.15 -0.07 -0.16 0.00 -0.14 -0.29 -0.36 -0.25 -0.09 0.29 -0.31
A3,(, B2-4, c1-3,(A4Y3)2, (B,Y3)2? and (B4Y4)3compositions as in Table 11. Y, = C5HloN0,S; Y, = Cl,HISN,O3S; Y, = Cl6Hz2N30,S;Y, = ClaH,sNd05S. *Assumes 1 = H; 2 = 2H; 17 = OH.
into the N-terminal daughter. The deviation is ascribed to imperfect selection of the neutral from mlz 304 parent by the time window; i.e. some neutrals from mlz 302 parents are included within it. Only an oxygen transfer to the N-terminal ion fragment, such as that proposed in Scheme I, can account for this result. To study decomposition of [M + Ag]+ ions, it was not possible to adjust the time window to select a neutral from specifically labeled parent ion. The neutral species peak profile from [M + Ag]+ decompositions, shown in Figure 9b, is virtually structureless. Adjusting the time window served only to change the isotopic percentages in the daughter ions (Figure 8a). Nevertheless, the extent of ls0incorporation into
B, + 17 + Ag can be estimated. The mlz 2261228 ratio of 51.2:48.8 gives the ratio of Ag isotopes in the I, + Ag ion. Assuming that this ratio is maintained in other daughter ions, the mlz 27212741276 ratio of 22.0:51.4:26.6 for Y, + 1 + Ag Similarly, then yields a ratio of 42.058.00 for 1602:160'80:1802. the mlz 23912411243 ratio of 34.0:48.9:17.0 yields a ratio of in B, + 17 + Ag. Thus, it 65.7:31.7:2.6 for 1602:1601sO:1s02 incorporates ca. 55% of the l80available in the parent [M + Ag]+ ion. This is very close to the value of 50% required t o support the mechanism of Scheme I. These results can be compared to two recent studies of decomposition of cationized peptide molecules. In the first study, in which a reflecting TOF mass spectrometer was used, [M + Na]+ ions of Leu-enkephalin and Met-enkephalin were reported to give daughter ions of m/z 317 and 464 (19). These were assigned as C,-type fragments (with Na+ addition) but probably correspond to the ions we observe a t m / z 318 and 465 (Tables I1 and 111) formed by oxygen transfer from the C terminal. The second study recorded the decompositions of cationized molecules of hippurylhistidylleucine in a triple sector (EBE) mass spectrometer (16). The daughter ion spectra of collisionally activated [M + HI+and [M + Na]+ ions, produced by xenon atom bombardment of the sample in a thioglycerol/glycerol matrix spiked with the alkali-metal chloride salt, were quite different from each other, in agreement with the present observations. Of particular interest to our study is a daughter ion of [M + Na]+ a t mlz 338 f l that could correspond to B3 + 17 + Na (in the current sym-
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
bolism), for which m/z 339 is required. We may also compare a secondary ion mass spectrum of 2-Ala-5-Leu-enkephalin on etched silver, recently measured in an ion cyclotron mass spectrometer (33). Ions were observed at m/z 332 and 479 and assigned as inner fragments; however, these are, in fact, the values predicted for B3 + 17 + Na and B, 17 Na, respectively. The corresponding doublets for Ag addition also appear in the spectra, although they are not listed in the tables. It therefore appears likely that the type of rearrangement discussed above has been observed previously but was unrecognized because of instrumental limitations. The present technique has provided identification of the parent ion decaying into a given daughter, as well as accurate measurement of the daughter ion mass, both of which are necessary for determination of the decay pathways. Qualitatively, the daughter ion spectra we observe in the reflecting TOF mass spectrometer are similar to those generated by collisional activation in other types of instruments. Our ability to observe daughter ion spectra v.+ithout resorting to collisional activation is a consequence of the earlier observational time frame for decompositions in a TOF instrument (10-'-104 s) compared to that of sector-field (10-5-104 s), triple quadrupole (104-10-3 s), or FT-ICR (10-*-1Oo s) instruments. However, the intensity is rather low for ions with relatively long lifetimes such as the [M + Na]+ ions observed here. In such cases collisionally induced dissociation or photodissociation might give significant improvements in intensity. Experiments using the latter effect are planned.
+ +
APPENDIX Identification of ionic fragments of petides can be illustrated by reference to the pentapeptides 2 and 3. Ions that contain the N terminus result from cleavages denoted by A, B, and C; those that contain the C terminus from cleavages denoted by X, Y, and Z. In both cases, subscripts denote which bond is cleaved (i.e. the number of amino acid residues contained in the ion fragment), counting from the N terminus and C terminus, respectively. By extension, we have used the letter I to denote immonium ions, with a subscript indicating the amino acid residue (numbered from the N terminus) from which it is derived. Because several of the daughter ions that we observe contain sodium or silver, we have added chemical symbols and integral mass values to the letter symbols to indicate modified fragments rather than the primes and double primes of the original proposal (28). For simplicity, the positive charge on the ionic fragment is implied rather than explicit. Note Added in Proof: Professor Michael Gross has kindly sent us a preprint of a communication recently submitted to J. Am. Chem. SOC.It draws similar conclusions about the oxygen migration from the C terminus, although some details of the proposed mechanism differ.
1799
Registry No. GGF, 6234-26-0;GGF (%-labeled), 115289-09-3; leucine-enkephalin, 58822-25-6; methionine-enkephalin,5856955-4.
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RECEIVED for review September 29, 1987. Accepted April 9, 1988. This work was supported by grants from the US. National Institutes of Health (Institute of General Medical Sciences) and from the Natural Sciences and Engineering Research Council of Canada.
