Anal. Chem. 1900, 60, 2723-2729
2723
Particle Beam Induced Reactions between Peptides and Liquid Matrices Chhabil Dass*J and Dominic M. Desiderio'** Charles B. Stout Neuroscience Mass Spectrometry Laboratory and the Departments of Neurology and Biochemistry, University of Tennessee-Memphis, Memphis, Tennessee 38163
Irradlation of peptides dlssolved In glycerol, ethylene glycol, l,Cbutanedlol, or 1,l-pentanedlol by a high-energy (klloelectronvoits) beam of Xe atoms results In the formation of several adducts havlng masses greater than the MH' Ion, which were characterlzed by mass spectrometry In the posltlve Ion mode. The most abundant of those adducts corresponded to the addltlon of 12 u to the protonated molecular ion (UH') of the peptides. Several other adducts were also visible In the mass spectrum. The correspondlng thlols (athioglycerol and dlthlothreitol/dithloerythrltol) demonstrated poor afflntly for the adduct formatlon with the peptides. The adduct formath was facilitated In dilute and in baslc sdutlons of the peptides and was reduced drastlcally In acldlc solutions. Prolonged Irradlatlon also favored the adduct formation. The mass spectrometry/mass spectrometry spectra obtalned by uslng the B/€ linked-field scan mode revealed that the slte(s) of attachment of glycerol to the peptlde was the most bask site in the mdecule-the free N-termlnus amlno group or the primary amlne group of a basic (Lys, Arg) amho acid resklue. Electrophlllc attack of the Ionized glycerol and Its oilgomers onto the N atom of the highly basic amlno group and subsequent fragmentation of the resutllng adducts appear to be the favored mechanlsm for the formatlon of the adducts. Evldence of the addltlon of up to at least three molecules of glycerol was revealed.
The use of a fast atom beam in obtaining mass spectra of a large number of highly polar, thermally labile, and nonvolatile compounds has been highly successful. In this soft ionization technique, which was introduced by Barber et al. ( I ) , a compound of interest is dissolved or dispersed in a liquid matrix and impacted by a beam of high-energy (several kiloelectronvolts) atoms. Ionization is rationalized to occur by desorption of the preformed ions and/or by gas-phase ionmolecule reactions (2). Although several different types of liquid matrices have been employed (3), glycerol is by far the most commonly used matrix in fast atom bombardment (FAB) mass spectrometry (MS) for a large number of polar and fragile compounds because of its excellent solvent properties, putative chemical inertness, and low volatility. The use of a liquid matrix, however, often complicates a FAB mass spectrum because of the presence of several undesirable matrix ions, which may interfere in the unambiguous characterization of the analyte. Although it was indicated by Barber et al. that the sample lifetimes are longer (4) and the sample is not measurably damaged by the fast atom beam (5), a considerable amount of irradiation damage to glycerol was reported by Field (6) and Keough (7). Prolonged irradiation of glycerol was shown to result in the formation of radicals, which then combined to form several new compounds. Charles B. Stout Neuroscience Mass Spectrometry Laboratory and Department of Neurology. *Department of Biochemistry. 0003-2700/88/0360-2723$0 1.5010
Chemical reactions between solute and FAB solvents were also reported (8-11). For example, (MH + 12)+ and (MH + 14)' ions were observed by Lehman et al. (9) in the positive ion FAB mass spectrum of angiotensin I1 when either glycerol or glycerol-d5,respectively, was employed as the FAB matrix. The reason for the formation of the (MH + 12)+ ion was attributed to a Schiff base reaction between the oligopeptide and the formaldehyde that was produced by FAB-induced fragmentation of the matrix, although no experimental evidence was provided to support that hypothesis. Pang et al. (10) reported the occurrence of (MH + 12)+and other related ions in the FAB mass spectra of some primary and secondary amines that were dissolved in glycerol. They interpreted those adduct ions to be the result of interaction between the solute molecules and glycerol. During the investigation of FAB-MS fragmentation characteristics of several opioid peptides, we also observed a similar phenomenon (11). An interesting feature of the positive ion FAB mass spectra of those peptides was the presence of several abundant ions having masses above the MH+ ions. The particle-beam-induced transmethylation of carnitine was observed by Liguori et al. (12). The interaction of an analyte with the matrix can play a significant role in determining the quality of the FAB mass spectrum. The use of metal ion-solute adducts has been shown to enhance the utility of FAB-MS (13-18). Reduction of a disulfide bond in peptides (19) and of azo dyes (20),and addition of more than one H atom to the molecular ion of the analyte (21-23) are widely observed reactions during FAB ionization. The relative contributions of either the matrix or primary (in the beam) and secondary (in the matrix) electrons in these reactions are not fully understood. Considering the widespread applications of FAB-MS (2) and its rapid growth in the analysis of peptides (24), it is appropriate and necessary to investigate the FAB-induced interaction between peptides and FAB solvents. These interactions may interfere in the quantification of peptides by FAB-MS. More importantly, the products of those interactions in the spectra of unknown compounds must be identified and rationalized so that they can be used analytically. In this paper, we report results of a detailed study on the adduct formation between peptides and FAB matrices. The specific aim of the study was to explore the experimental conditions that facilitate those interactions, to characterize the resulting product ions, and to elucidate the mechanism of their formation. The research was conducted by acquiring positive ion FAB mass spectra of a number of peptides of diverse chemical properties using different solvent matrices such as 1,2-butanediol,1,5-pentanediol,ethylene glycol, glycerol (G), glycerol-d6,a-thioglycerol (TG), and a mixture of dithiothreitol (DTT) and dithioerythritol (DTE) (3:l (w:w)). The mass spectrometryfmass spectrometry (MS/MS) spectra of the peptides and the corresponding adduct ions were compared to establish the site(s) of glycerol attachment to the peptide. The MS/MS spectra were acquired by using a B f E linked-field scan (B = magnetic and E = electric fields), which furnishes the product ion spectrum of the mass-selected parent ion (25). 0 1988 American Chemical Society
2724
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
Table I. Adducts of Proctolin with Glycerol and Glycerol-d5 and Their Probable Compositions m/z
649 661 673 691 705 723 735 753
ion
glycerol composition
MH' (MH + 12)' (MH - 2H + CH2)' (MH + 24)' (MH - 4H + 2CHz)' (MH + 42)' (M + CH2CHO)+ (MH - 2H + CH2 + CH20)' (MH + 56)' (MH - 3H + CH2 + (CH2CH20H))' (MH + 74)' (M + (CH,CHOHC&OH))' (MH - 2H-+ CH2 + CH20HCH20H)' (MH + 86)' (MH - 3H + CH2 + (CH2CHOHCH2OH))' (MH + (MH - 2H + CH2 + (CH20HCHOHCH20H))' 104)'
765 (MH-+ 116)' 783 (MH +
m/z
ion
649 663 677 694
MH' (MH + 14)' (MH + 28)' (MH + 45)"
711 728 742 760
glycerol-d, composition
(MH - 2H + CD2)" (MH - 4H + 2CDz)' (M + CDZCDO)' (MH - 2H + CD2 + CHDO) (MH + 62)' (MH - 3H + CD2 + (CD2CD20H))' (MH + 79)' (M + (CD,CDOHCD,OH))+ (MH - 2H-+ CD2 + CHDOHCD20H)' (MH + 93)+ (MH - 3H + CD2 + (CDZCDOHCD20H))+ (MH + 111)' (MH - 2H + CD2 + (CD20HCDOHCDZOH))+
(MH - 4H + 2CHz + (CHzOHCHOHCH20H))' 774 (MH + 125)' (MH - 4H + 2CD2 + (CD20HCDOHCDZOH))+ (M + CHzCHO + (CH20HCHOHCH~OH))'
134)'
EXPERIMENTAL SECTION The experiments reported here were performed with a VG 7070E-HF (Manchester, UK) double-focusingforward geometry (E$) mass spectrometer outfitted with a Digital PDP 11/24 minicomputer-basedVG 11-250M+ data system. The ion source used was a standard VG FAB system equipped with an ION TECH BllNF saddle-fieldatom gun. Xe atoms of approximately 7 keV impact energy and an emission current of 1mA were used as the ionizing beam. The FAB-desorbed ions were accelerated to a potential of 6 kV. The mass spectra were acquired by use of computer-controlled magnet scans over the appropriate mass range at a resolution of approximately2000 (10% valley definition) and a scan rate of 5 s/decade. The spectra reported in Figure 2 were acquired by using magnet scans over the narrow mass range 640-850 u and averaged (five to seven scans) by using the multichannel analysis (MCA) mode of data acquisition. The B / E linked-field scan technique coupled with the collisionally activated decomposition (CAD)of the mass selected MH+, (MH + 12)+,and other adduct ions (vide infra) was used to obtain the product ion spectra in the appropriate mass range at a scan rate of 5 s/decade. Several (7-10) spectra were averaged by using the MCA. CAD was performed in the first field-free region using He as the collision gas, the pressure of which was adjusted to reduce the precursor ion intensity to two-thirds of its initial value. To ascertain the genesis of the (MH + 12)' ions, the parent ion spectra were acquired by using a B 2 / E linked-field scan (25). The data reported in Table I1 and in Figures 3 and 4 were acquired by scanning the magnet over a narrow mass range (20 u wide) encompassing the MH' and (MH + 12)' ions, and recording the data on UV chart paper using a Thorn EM1 Datatech 6150 MK I1 (Lancashire, UK) UV oscillographic recorder. The data reported are the average of three measurements. The mass spectrometer was calibrated over the appropriate mass range of the analysis with the ions from glycerol, glycerol/CsI, and LiI/NaI reference files. Ethylene glycol, 1,4-butanediol,and 1,5-pentanediol were procured from Aldrich Chemical Co. (Milwaukee, WI). Glycerol-dswas purchased from Merck, Sharpe and Dohme (Rahway,NJ). Glycerol and the peptides used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). Most of the peptides were in the form of their acetate salt, and were used without further purification. The peptides (2-4 pg) were dissolved in the appropriate matrix to obtain the spectra reported in Figures 1, 2, 5, 6, and 7. RESULTS AND DISCUSSION To demonstrate the phenomenon of adduct ions, the positive ion FAB mass spectra of Pro-Phe-Gly-Lys and leucine enkephalinamide (LE-NHa acquired by using glycerol as the matrix are depicted in parts a and b of Figure 1,respectively. Clearly, the FAB irradiation of these two peptides resulted in the formation of a number of abundant ions having masses greater than the MH+ ions. The most dominant adduct ion corresponded to the addition of 12 u to the respective MH' ions of the two peptides. In fact, the (MH + 12)' ion in Figure
791 (MH + 142)' (M + CDzCDO + (CD20HCDOHCD20H))+
Flgure 1. Positive ion FAB mass spectrum of (a) ProRwGIy-Lys and (b) leucine enkephalinamide, both dissolved in glycerol.
l a is more abundant than the MH+ ion. Other adduct ions of measurable intensity corresponding to the addition of 24, 42, 56,74,86, 104, 116, and 134 u to the MH+ ion were also noted. A more expanded set of data is seen in Figure 2a, which collects data acquired in the narrow mass range (640-850 u) for proctolin, a pentapeptide of 648 u molecular weight (M,). A detailed analysis of that data and the data obtained by using glycerol-d, is presented in Table I, which also lists the probable composition of all of the observed adduct ions. The use of glycerol-d5shifted all of the adduct ions to correspondingly higher m / z values (Figure 2b). For example, the (MH + 12)+ [ m / z6611 and (MH + 12 + 92)' [m/z7531 ions in Figure 2a appeared at m/z 663 and 760 in Figure 2b and corresponded to (MH + 14)' and (MH + 14 + 97)+ions, respectively. Those mass shifts suggested that the ions that are observed above the mass of MH+ ion of the peptide are the result of an interaction between the peptide and glycerol, consistent with the conclusions of previous studies (9, 10). A similar observation was made when l,4-butandiol and 1,5-pentanediolwere used as the FAB matrices for proctolin (see parts c and d of Figure 2, respectively). FAB irradiation of proctolin dissolved in these two solvents also produced (MH + 12)+ and [MH + 12 + S(=solvent)]+peaks but with less facility than with the corresponding reaction of the peptide with glycerol. The other prominent ions corresponded to the addition of 70 u ( m l z 719 in Figure 2c) or 84 u ( mf z 733 in Figure 2d) to the MH+ ion of proctolin. The use of ethylene glycol also produced similar data (Figure 2e). In contrast, when sulfur-containing solvents (TG or DTT/DTE) were employed as the FAB matrices, these reactions were practically absent (see parts f and g of Figure 2, respectively). These data indicated that specific peptide-matrix effects were in operation and prompted us to conduct an in-depth investigation of the mechanisms and chemical features of those
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
2725
2 3 4 Time of Irradiation (min) Flgure 3. Effect of particle beam irradiation time on the intensity ratio 0
(MH
Y9
T
+ 12)+/MH+. 1.64
T 1' MI
1
i
0.8
0
2
4
6
8
10
12
Conc. of ME-Lys (nM) Figure 4. Effect of the peptide concentration on the intensity ratio (MH 12)+/MH+. 6Es
681
lo8
721
7k8
16s
7rn
+
8(9
Flgure 2. Positive ion FAB mass spectrum of proctolin dissolved in (a) glycerol, (b) glycerol-d,, (c) 1,4-butanediol,(d) 1,5-pentanedioi, (e) ethylene glycol, (e) a-thioglycerol,and (f) DTTIDTE.
FAB-induced reactions of peptides with different liquid matrices. Considering the potential analytical implications of these adduct reactions on the FAB-MS analysis of peptides, it is important to know the origin of the ions observed at masses grater than the MH+ ion of a peptide. To rationalize the formation of peptidematrix adduct ions, we have chosen a number of peptides (listed in Table 11), which are grouped into three different categories. The Nterminally blocked peptides are assembled in group A. Group B peptides are relatively more hydrophobic in nature compared to the group C peptides, which contain basic amino acid residues. Because of its wide applicability and greater proclivity for interaction with the peptides of interest studied here, glycerol was seleded as the FAB solvent for the following, more detailed studies. Because the (MH + 12)+ ion was always the most abundant adduct ion, it was chosen as the diagnostic probe to gain further insight into the formation of the adducts. The abundance ratios, (MH + 12)+/MH+,for peptides of interest are listed in Table I1 (neat), which also includes the corresponding data acquired in basic and acidic solutions of glycerol (columns 4 and 5, respectively). The data in column 3 demonstrate clearly that the proclivity of the basic amino acid containing peptides for adduct formation is much higher than that of the group A and group B peptides. Whereas no adduct formation was observed for group A peptides (except for the hexapeptide N-formyl-nLeu-Phe-nLeu-Tyr-Lys, which contains Lys, a highly basic amino acid residue), the group B
peptides exhibited a significant abundance of the (MH + 12)+ ion. From these data, it can be concluded that one of the most important factors influencing the relative intensity of the (MH 12)+ion is the presence of a highly basic nitrogen moiety, i.e. the N-terminus amine group, or the primary amine group of the basic amino acid residues (Lys, Arg). The ratio data in group A also suggest that the peptide amide group (-CONH-) is not the primary site of glycerol attachment. Also, the absence of the (MH + 12)+ion in the FAB mass spectrum of pGlu-His-Pro-NH2implies that the C-terminus amide group alone does not participate in the adduct formation, although that group may facilitate the interaction of the peptide with glycerol when it is present together with another basic amino group (compare the data for LE and LE-NHP in Table 11). Effect of FAB Irradiation Time, Peptide Concentration, and pH. In their study on adduct formation with amines and glycerol, Pang et al. (IO) observed that the abundance of the adduct ions increased with the time of exposure to the FAB irradiation. At a given exposure time to the irradiation, the abundance ratio (MH + 12)+/MH+was found to increase with the dilution of the amines in glycerol. The pH of the glycerol solution of those amines was shown to have a very minor effect on that intensity ratio. It was necessary to ascertain whether a similar effect of those experimental variations would be observed in the FAB mass spectra of peptides. The ratio (MH + 12)+/MH+was also found to increase with exposure time to the irradiation of ME-Lys and ME-Arg-Phe (Figure 3) and dilution of the peptide concentration of ME-Lys (Figure 4). The increased ratio (MH + 12)+/MH+at increasing dilution suggests that the rate of formation of the (MH + 12)+ ions was influenced by the number of glycerol molecules available for reaction with
+
2726
0
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
Table 11. Relative Abundance Ratio (MH + 12)+/MH+ in the FAB Mass Spectra of the Peptides Dissolved in Glycerol peptide
M,
neat NHIOH HC1
Group A
pGlu-His-Gly 303.1 a pGlu-Asn-Gly 300.1 a pGlu-His-Pro-NH2 362.1 a formyl-nLeu-Leu-Phe-nLeu-Tyr- 823.5 0.027 LYE
0.045
-
0.085
0.290
0.011
0.081
0.303
0.013
Flgure 5. BIE CAD mass spectrum of (a) the MH+ ion and (b) the (MH 12)' ion of leucine enkephallnamide dissolved in glycerol. The intensities of all of the ions in parts a and b were normalized with
0.115
0.222
0.011
respect to mlr 425 and 437, respectively.
0.568
0.437
0.022
Scheme I. Fragmentation Pattern of LE-NH2
4.00 0.559 0.690 1.60 0.202 0.440 0.917 0.917
1.45 0.375 0.663 0.490 0.640 0.815 0.352 0.510
0.017 0.076 0.015 0.030 0.028 0.025 0.025 0.057
Group B
leucine enkephalin (LE) 555.3 (Tyr-Gly-Gly-Phe-Leu) methionine enkephalin (ME) 573.2 (Tyr-Gly-Gly-Phe-Met) @-casomorphin 789.4 (Tyr-Pro-Phe-Pro-Gly-Pro-Ile) LE-NHS 554.3 Group C Pro-Phe-Gly-Lys 447.2 kentain (Thr-Pro-Arg-Lys) 500.3 proctolin (Arg-Tyr-Leu-Pro-Thr) 648.4 ME-Lys 701.3 ME-Lye-Lys 829.4 ME-Arg-Phe 876.4 ME-Arg-Gly-Leu 899.4 LE-Arg-Lys-Arg 995.6 a
+
A3
(MH + 12)+ ion intensity was < 1%.
each molecule of that peptide. However, a major role of pH of the solution was noted in our study on the formation of adducts (Table 11). The data in Table I1 were obtained by adjusting the solution to either an acidic or basic pH value by the addition of either 200 nmol of HC1 or 9 pmol of NH40H, respectively, to the probe tip that contained a constant amount (2 nmol) of the peptide dissolved in 0.5 pL of glycerol. However, we did not attempt to measure that exact pH of the solution because of the practical difficulties associated with that experiment. The mass spectra were acquired within 20 s of activating the fast atom beam. Addition of NHIOH to group B peptides increased the abundance ratio (MH 12)+/MH+ vs a neat solution, whereas it had less effect on the group C peptides. On the other hand, the abundance ratio (MH + 12)+/MH+ of the acidic solution of the group B and C peptides was diminished drastically. One plausible explanation of this pH effect is that, in an acidic solution, the amine group is fully protonated and is not available for reaction with the matrix ions. In contrast, in basic solutions, the lone pair of electrons on an amino group nitrogen is free to participate in the reaction with the matrix. Site of Glycerol Attachment. The data presented in the earlier section have indicated that the most favorable site for glycerol attachment to the peptide is the free N-terminus amino group or the amine group of Lys and Arg. However, a further piece of corroborative evidence was obtained by comparing the MS/MS spectra of the (MH + 12)+ and the MH+ ions of selected peptides. CAD and a B / E linked-field scan were employed for these experiments. Parts a and b of Figure 5 compare the CAD-B/E spectra of the MH+ and the (MH + 12)' ions, respectively, formed by FAB irradiation of LE-NH2 dissolved in glycerol. The fragmentation pattern of that pentapeptide is depicted in Scheme I. The C-terminus sequence ions are designated as X, Y, and Z and the N-terminus sequence ions as A, B, and C (26). The N-terminus sequence ions ( m / z 136,221,278,397, and 425) dominate the CAD-B/E spectrum of the MH+ ion of LE-NH2 (Figure 5a). A few of the C-terminus sequence ions ( m / z 262,278,319,335, and 376) are also present, although these sequence ions are
+
I 1 . L161
83 C3
554
i4
relatively weak. Comparison of the data in these two figures demonstrates that all of the N-terminus-containing sequence ions in Figure 5a have shifted by 12 u to a higher mass in the spectrum of the (MH + 12)+ ion of LE-NH2 (Figure 5b); those shifts support our suggestion that the reaction of the peptide and glycerol involves preferentially the N-terminal primary amine group. No strong evidence existed for the shift of the C-terminus ions, implying that the C-terminus amide group of LE-NH2has poor affinity for glycerol. The possibility of the adduct formation at the peptide amide bonds is also precluded on the basis of the following reasoning. For example, if the amide group between the Phe and Leu residues participated in the adduct reaction, then we would have expected abundant peaks due to the shift of 12 u in all of the C-terminus sequence ions, except in the Z1ion. Furthermore, all of the N-terminus ions except the C4 ion should not shift to higher masses, which is not the case as shown by the data in Figure 5b. By use of similar arguments, the participation of other amide bonds of the peptide chain is also excluded. Further support of this conclusion is derived from the observation that the immonium ion of m / z 120 formed from the Phe residue (H2NCHCH2C6H5)(22) and the "internal fragments" of mlz 176 and 120 ions present in Figure 5a remain unchanged in Figure 5b. A second example chosen to demonstrate the site of glycerol attachment is the group C tetrapeptide Pro-Phe-Gly-Lys. The (MH + 12)+ ion of that peptide at m / z 460 was the most abundant ion in the FAB mass spectrum with glycerol as the FAB solvent (see Figure la). An ion of m / z 461 due to the protonated glycerol pentamer (GhH') could have contributed to the CAD-B/E spectrum of the (MH + 12)+ ion due to the inherent poor mass resolution of the precursor ion selection in the BIE linked-field technique on a two-sector instrument. To discount the possible interference of that GSH+ ion on the CAD-B/E spectrum of the (MH + 12)+ ion of Pro-Phe-GlyLys, glycerol-d5was employed as the FAB matrix to shift the adduct ion to m l z 462 [(MH + 141'1 and the G5H+-d5to m / z 486. The CAD-BIE spectra of the MH+ and (MH + 14)' ions from Pro-Phe-Gly-Lys are shown in parts a and b of Figure 6, respectively. The fragmentationpattern of Pro-Phe-Gly-Lys is shown in Scheme 11. Because of the presence of a Lys residue in this peptide, the interaction of glycerol at the free N-terminus and at the t-NH2group of the Lys side chain both
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
Scheme 11. Fragmentation Pattern of Pro-Phe-Gly-Lys 317
349
202 1 8 7
2727
Scheme I11
447
1
145
Scheme IV
334
(M+&H)+
3
3(M+CHzCHO+S)+ %o[MH-2H+CH2&)t 1.'H3w
( M H + 1 3 v
(MH+104)
(MH-~H+CH~+CHZ CHCJHCHsCH)' (YH186)
4-
CHzO
(MH-2H+CHz+C~CtCH20H)i (MH-ZH+CHz)* (MHtl2)
1-
(MH+74)
CYOH
(MH-2H+CH2+CH20)+ (MH+42)
Figure 6. B I E C A D mass spectrum of (a) the MH+ lon and (b) the (MH 12)' Ion of Ro-phe-oly-Lysdissolved in glycerol-d,. The intensities of all of the ions in parts a and b were normalized with respect to m / z
+
351 and 365, respectively.
may contribute to the (MH + 14)+ion. The data shown in Figure 6 revealed that the C-terminus sequence ions (mlz 147, 204, and 351) were quite abundant (Figure 6a) and shifted to m / z 161,218, and 365, respectively, in the corresponding mass spectrum of the (MH + 14)+ion (Figure 6b). Those mass shifts indicated that adduct formation occurred a t the e primary amine group of the Lys side chain. It is further apparent that the A2, B2, B,, and Cfl N-terminus sequence ions, which appeared at m / z 217, 245, 302, and 319, respectively, in the CAD spectra of the FAB-desorbed MH+ ion of Pro-Phe-Gly-Lys (Figure 6a), have shifted 14 u to m / z 231, 259,316, and 333, respectively, in the correspondingspectrum of the (MH 14)' ion (Figure 6b). Those data suggest that the secondary amine group of the N-terminus Pro residue was also a site of the adduct formation. By use of the arguments presented above, the participation of the less basic peptide amide bonds was ruled out. The preponderance of the (Y" 14)' ions (mlz 218 and 365) and the near-absence of the Y"-type ions in Figure 6b implied that a larger population of the (MH + 14)+ions was derived from the reaction involving the e primary amine group of the Lys side chain, an observation consistent with the strong basic character of that amine group compared to the secondary amine group of Pro residue. A significant abundance of the A2,B2,B,, and Cg" N-terminus sequence ions in Figure 6b supports further that statement. Mechanism of the Formation of the (MH + 12)+ and the Other Related Adduct Ions. Because it was concluded earlier that the ions observed at masses greater than the MH+ ion in the FAB mass spectra of the peptides are the product of the FAB-induced interaction of peptides with glycerol, it was expected that those adduct ions may be formed by one or more of the following plausible mechanisms: (i) the formation of the peptide-G,H+ adducts and their subsequent dissociation to yield the intermediate fragment ions and ultimately the (MH 12)+ ion; (ii) ion-molecule reactions between the peptide and different fragment ions of glycerol that were formed initially by FAB irradiation; and (iii) the initial formation of the (MH + 12)' ion by the Schiff base reaaction of the peptide with formaldehyde that was generated by FAB irradiation of glycerol, and subsequent addition of glycerol, its oligomers, and neutral fragments to the (MH 12)+ion to account for the remaining adduct ions. We cannot presently rule out any of these possible mechanisms because all of them would yield products of identical masses, and it is likely that
+
+
+
+
/MH-~H+CHZ+CHZCHZOH)' (MH+55)
4.
(MH-4H+ZCH2)* (MH124)
the adducts observed in this study may be formed by multiple mechanisms. However, an examination of the data (Table I) suggests a preference for mechanism i. Because the addition of HC1 suppressed considerably the (MH + 12)+ ion intensity (Table II), it is argued that the neutral peptide was the reactive species. This conclusion is supported further by the fact that the (MH 12)+ion formation was favorable in dilute solutions of a peptide. Under those conditions, more ionized glycerol species are formed because of less mutual suppression. The formation of the (MH 12)+ion according to the fist mechanism can be explained as follows: As a first step, the interaction of a glycerol ion or its oligomers with the peptide forms an adduct. Ion-molecule adducts are generally unstable because of the exothermicity of those reactions. Here also, those initially formed adducts dissociate within the mass spectrometric time frame (1-10 ps) and are not observed. Scheme I11 rationalizes the formation of the adduct ions a t masses (MH + 74)+, (MH + 42)+, and (MH + 12)+from the transitory [ (MH + CHI+] adduct (all the short-lived ions are placed in brackets). The loss of a molecule of water from that short-lived adduct yields the (MH 74)+ ion. The sequential loss of a molecule each of methanol and formaldehyde from the (MH + 74)' ion forms ultimately the (MH + 12)+ion. In this overall scheme of the decomposition of the [(M GH)+] adduct, two hydrogen atoms, which did not belong to the glycerol molecule, are lost, and resulted in the net addition of 12 u from glycerol. Support for this hypothesis and of the other fragmentation reactions shown in Scheme I11 was derived from the FAB mass spectrum of proctolin by using glycerol-ds as the FAB solvent (Figure 2b). The (MH + 12)+ ion was shifted to (MH + 14)+,and the (MH + 42)+ and (MH + 74)' ions appeared at (MH + 45)' and (MH + 79)+, respectively (see also Table I). The losses of neutral 31 and 34 u are rationalized to involve the DCOH and HD2COH moieties, respectively. Because two H atoms of the glycerol skeleton are involved in the formation of the (MH + 12)+ion, its structure is rationalized to be that shown in Scheme 111, and is consistent with the structure reported by Pang et al. (IO) for the corresponding adduct formed between cyclic amines and glycerol. Likewise, the oligomers of ionized glycerol may also add to a peptide. The fragmentation of those transitory adducts would explain the formation of several intermediate adducts that occur in the FAB mass spectra of that peptide. For example, Scheme IV rationalizes a potential route to the formation of (MH + 24)+, (MH + 42)+, (MH + 56)+, (MH + 74)+,(MH + 86)+, (MH + 104)+,and (MH + 134)' ions from the [(M + G2H)+]adduct. This tentative proposal was corroborated by the product ions formed when glycerol-ds was substituted for glycerol. The structure of the (MH + 42)' and (MH + 74)+ ions formed in this reaction scheme may differ from the corresponding ions shown in Scheme I11 (see Table I). The weak signals at masses higher than the (MH + 104)'
+
+
+
+
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
+ a)+, (MH + 104)+,and other high mass adduct ions cannot be explained by the reaction of a peptide and the glycerol fragment ions (mechanism ii) , because the corresponding fragment ions either are absent or are of relatively very low abundance (
