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Fast atom bombardment mass spectrometry analysis of opioid peptides
ANALYTICAL
BIOCHEMISTRY
163,52-66
Fast Atom Bombardment CHHABIL
(1987)
Mass Spectrometry
Analysis of Opioid Peptides
DASS* AND DOMINIC M. DESIDERIO*,?
Charles B. Stout Neuroscience Mass Spectrometry Laboratory, and Departments of *Neurology and tBiochemistry, University of Tennessee, 800 Madison Avenue, Memphis, Tennessee 38163 Received October 13, 1986 Positive and negative ion fast atom bombardment mass spectrometries have been used to determine the amino acid sequence-determining fragment ion information of opioid peptides containing from 5 to 10 amino acid residues. The opioids investigated include several enkephalins, dynorphin A fragments l-7 through 1- 10, and cc-and &neoendorphins. Data obtained in the two ionization polarities provide complementary information and exhibit the C-terminaland the N-terminal-containing amino acid sequence-determining fragment ions that are formed by cleavage of (a) the bond between the carbonyl group and the a-carbon (-CHR-CO-), (b) the peptide amide bond (-CO-NH-), and (c) the amino-alkyl (-NH-CHR-) bond. The C-terminal sequence ions are dominant in the positive ion mode, whereas the C-terminal and N-terminal ions are equally important in the negative ion mode. Detection limits for full mass scans extend down to the picomole range. The apparent role of hydrophobicity of the amino acid residues on the fragmentation characteristics of the peptide is discussed. o 1987 Academic Ress, Inc. KEY WORDS: opioid peptides; mass spectroscopy; fast atom bombardment; enkephalins; dynorphins.
Since the discovery in 1975 of endorphins as endogenous opiates (l), an increasing number of polypeptides with opioid activity has been identified in the central nervous system and the periphery. These endogenous substances bind to opiate receptors and mimic the biological effects of morphine; hence they were termed endorphins (endogenous morphinelike factors). Three classes of endogenous opioid peptides-enkephalins, dynorphins, and endorphins-are known, along with their corresponding precursors, preproenkephalins A and B and proopiomelanocortin (POMC),’ respectively. Furthermore, several types of opioid receptors (mu, sigma, delta, epsilon, kappa) in brain and gut tissue have been pharmacologically defined
(2). However, our understanding of the molecular events that occur in the endogenous peptidergic pathways is still not complete, and toward that end, quantitative measurements must be made in biologic extracts of selected opioid peptides. Before those analytical measurements are made, however, a firm understanding of the detailed mass spectrometric behavior of those peptides must be provided from model studies. Mass spectrometry (MS) has been used for nearly three decades to determine the amino acid sequence of peptides. Chemical derivatization is necessary whenever electron ionization (EI) and chemical ionization (CI) are used for analyzing this very polar class of compounds (3-5, but see 5a). However, underivatized peptides are now analyzed with ,newer desorption methods such as fast atom bombardment (FAB; 6-12), secondary ion mass spectrometry (SIMS; 13), field desorption (FD; 14), Califomum-252 plasma desorption (15), and laser desorption (16). Of
’ Abbreviations used: POMC, proopiomelanocortin; EI, electron ionization; CI, chemical ionization; FAB, fast atom bombardment; SIMS, secondary ion mass spectrometry; FD, field desorption; RIA, radioimmunoassay; RRA, radioreceptorassay; SRM, selected reaction monitoring. 0003-2697187 $3.00 Copyright 0 1987 by Academic Press, Inc. AII rights of reproduction in any form reserved.
sequencing;
52
FAST ATOM
BOMBARDMENT
these desorption techniques, FAB is an ap propriate technique for the analysis of peptides due to its simplicity, reproducibility, and extended mass range. Recently, another soft ionization technique, thermospray liquid chromatography/MS, has been employed for sequencing peptides ( 17). MS sequencing methodology is useful, and in certain cases necessary, as an adjunct to the more common wet chemical (Edman) and gas phase methods. In certain cases for sequence determination of biological peptides, MS offers the only alternative method; for example, when the target peptide has blocked termini, especially the N-terminus, and/or when very little biologic material is available (5b). In this paper, we present the application of FAB MS for the characterization of several selected opioid peptides (listed in Table 1) by acquiring positive and negative ion FAB mass spectra. The peptides investigated are important in our continuing studies on the basic and clinical neurochemistry of neuropeptides, and the data in this paper are important first to identify a particular peptide in a biologic extract and second to quantify that peptide. TABLE THEOPIOID FWTIDESSTUDIEDBY Peptide Group A Leucine enkephalin (LE) Dynorphin A Fragment l-7 Fragment l-8 Fragment l-9 Fragment l-10 8-Neoendorphin a-Neoendorphin Group B Methionine enkephalin (ME) ME-enkephalin-Lys ME-enkephalin-Lys-Lys ME-enkephalin-Lys-Arg ME-enkephalin-Arg-Phe ME-enkephatin-Arg-Gly-Lcu
M, 555 867 980 1136 1233 1099 1227 573 701 829 857 876 899
MASS SPECTROMETRY
53
A few scattered reports of MS studies have appeared in the literature for some of these opioid peptides, such as for leutine enkephalin (LE=YGGFL) (1,9,18-20), methionine enkephalin (ME=YGGFM) ( 1,9,10,19,2 1,22), and cy- and P-neoendorphins (13b). However, to our knowledge, no systematic investigation has yet been carried out to rationalize the fragmentation mechanisms of all of the peptides contained in this present set of biologically important peptides. Moreover, the commonly used methods for quantification of the neuropeptides rely on radioreceptorassay (RRA) and radioimmunoassay (RIA) measurements. These techniques lack the maximum molecular specificity that is offered by MS methods. Because of the continuing interest in neuropeptides in general and the rapid expansion of neurosciences in particular, the need to continue the development of measuring other peptides that derive from the four gene product precursors protachykinin, preproenkephalins A and B, and proopiomelanocortin exists. Our need for measuring these peptides derives directly from our HPLC-RRA and HPLC-RIA methodologies, where those data consistently indicate 1 FAB MASS~PECTROMETRY Amino acid sequence Tyr-Gly-Gly-Phe-Leu-OH TyrGly-Gly-Phe-Leu-Arg-Arg-OH Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Be-OH Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Be-Arg-OH Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Be-Am-Pro-OH Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-OH Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys-OH Tyr-Gly-Gly-Phe-Met-OH Tyr-Gly-Gly-Phe-Met-Lys-OH Tyr-Gly-Gly-Phe-Met-Lys-Lys-OH Tyr-Gly-Gly-Phe-Met-Lys-Arg-OH Tyr-Gly-Gly-Phe-Met-Arg-Phe-OH Tyr-Gly-Gly-Phe-Met-Arg-Gly-Lcu-OH
54
DASS AND DESIDERIO
the presence of other C-terminally extended ME and LE peptides, which must be quantified ( 19).
(St. Louis, MO) and were used without any further purification. RESULTS AND DISCUSSION
EXPERIMENTAL
SECTION
Fragmentation Mechanisms
The positive and negative ion FAB mass For ease in data tabulation and rationalspectra were obtained with a VG 7070E-HF ization of their positive and negative ion fragmentation mechanisms, the peptides double-focusing forward geometry (E,B, are divided into two where E = electric and B = magnetic sector) under investigation mass spectrometer equipped with a standard groups (Table 1) based upon the amino acid VG FAB ion source and an Ion Tech B 11NF sequence of the N-terminus of each peptide. saddle-field primary atom gun. Xe atoms of The peptides containing the amino acid seapproximately 7 keV impact energy and a quence of LE at the N-terminus are classified beam flux of 1 mA were used as the ionizing as Group A, and those with ME at the N-terbeam. The gas flow of Xe was regulated to ,minus are collected into Group B. produce a source pressure of 1 X 1Oe6 Torr as Positive ion FAB mass spectra. The posiindicated by an ion gauge connected to the tive ion FAB mass spectra of Group A and ion source housing. The FAB target con- Group B peptides are listed in Tables 2 and sisted of a stainless-steel probe tip of approxi3, respectively, which include only the amino mately 8 mm* surface area beveled at an ap- acid sequence-determining fragment ions propriate angle of incidence and fitted onto (hereafter called sequence ions). With the the tip of the direct insertion probe. The ions exception of a few low-mass (<200) fragment were accelerated to 6 keV from the source ions, the (M + H)+ ions are the most intense region. The mass spectra were acquired ions in the spectra. Their preponderance faunder computer (Digital PDP 1 l/24) con- cilitates the determination of the molecular trolled magnet scans at a resolution of ap- weight, one of the most important experiproximately 2000 (10% valley definition) mental parameters used in characterizing an and scan rates of 10 s per decade by using VG unknown peptide or corroborating the pres1 l-250 M+ software. CsI was used as the ence of a known peptide in a biologic extract. Similar to the earlier studies (8a,13b,23) on mass calibrant in the positive and negative other oligopeptides, the genesis of the imporionization modes. Glycerol served as the liquid matrix. tant sequence ions from the neuropeptides The test samples were dissolved in under investigation is rationalized in terms H20:CH30H (1: 1) at a concentration of 0.5 of cleavage of (a) the bond between the carpg $‘. An amount of l-2 pg, corresponding bony1 group and a-carbon (-CHR-CO-), (b) and (c) to 2-4 nmol of the peptide, was applied to the peptide amide bond (-CO-NH-), bond (-NH-CHR-). The the FAB probe tip that already contained a the amino-alkyl layer of 0.2 ~1 of glycerol. The methanol was cleavage of these bonds, in many instances, is evaporated by heating under an infrared associated with a transfer of hydrogen(s) lamp, and the probe was introduced into the from the neutral fragment to the charged ion source via a vacuum lock. For the data in species. In designating the sequence ions, the Fig. 3, a stock solution of dynorphin A 1-7 of nomenclature proposed recently by Roepstoti and Fohlman is utilized (24). For ex0.5 nmol ~1~’ concentration was prepared, ample, the N-terminus-containing ions and an appropriate amount (0.25 to 1 nmol) was mixed with 0.2 ~1 of glycerol on the formed according to the aforementioned probe tip. All the peptides listed in Table 1 mechanism are designated A, B, and C and were purchased from Sigma Chemical Co. the corresponding C-terminal ions are de-
FAST
ATOM
BOMBARDMENT
MASS
TABLE AMINO
r
ACID SEQUENCE-DETERMINING
:,
136 A la0 c 117 Z" 132 y" 158 x 136 159 175 201 136
c
at,
02
193 221 236c 264 279 307
7 16 42 5
A 166 z' loo y" 137 x 53 A 215 -
115 y' 141 'X
d-NO0 endorphin
A a Z" y" X"
193 A 221 B 315 2 331 y" 357 x 221 B
a
i
c
a
:
9 11 3 8 25 3
251 279 295 321 336 361,x
A 8' c" Z" y"
5397A 25 425 5 442 4 378 IO 393 2419x
B C" z" y"
26 23
278 B 429 z" 444 y" 470 x 278 B
24 16 IO 33
23 72 14 6 37
397 A 427 8" 576-Y 591 y" 617X 397 A -
c
a 5 3 2 3 5 1
MASS SPECTRA OF GROUP
221 B
480 146
136 A 222 115 Y' 153 141 'x 58
136 A -
-
196
(47 y" 107 174 x' 25
501 35 278 1 B
34
y-j 427
B
:
c
a
5 76 19 7
666A 696 711 690 705 733
:
5-14 555 c" lo6 633 T 21 648 y" IO 674 X IO -
“y;;,539
12
8" c" z" y" x" -
4 4 12 50 11 4
/
B
‘;I-;: 14 696
z' Y
14 II
B
795 F al0 Y"
ig 14 a
193 223
z y" X'
II 7 7
519 z 533Y 561 X
7 11 7
noted as X, Y, and 2 ions, respectively. The number of hydrogens transferred to or lost from the charged moiety is indicated by the corresponding number of apostrophes to the right or left of the letter designation, respectively. Peptides are very complex molecules consisting of the 20 naturally occurring amino acid residues. For n known amino acids, n! sequences are possible; for an unknown with m amino acids, 20” possible peptides exist. Due to the presence of several noncovalent molecular forces, the enkephalins in solution are known to fold to a state of lowest free energy and to exist in several conformations, the more common one being the P-sheet or
c
a
8 b
cla
I
9
b
cl
II0
675 691 719
Z y" x"
7 789 z" 5ao4v 3830x
i
a68 (M+H)
-
263 278 307
391 407 434
b
867 c" 803z" 818~" 644x
193 221
33 46 24
a
556
255 272 298
228 z" 244 y" 272 X-
7
c
A PEPTIDES
(M+H)+
II_‘yy/i
@-NW endorphin
ION FAB
Amino Acid Reaidua a
LE
2
IONS IN THE POSITIVE
T
55
SPECTROMETRY
a 4 3
5 30 a 2
867 C902z" 917 y" 943x 622 A a67 c" 942 Z" 957 y" 985 X"
8 63 15 10
794A a24 8" 839 cm a65z" aao Y" 908x" 794 A a24 B a39 c" 936 z" 951 y" 978 x'
3 5 5 7 4 2 5 13 19 29 11 8
6 33 IO 5 5
extended structure involving intermolecular H bonds and several types of &turns-a lomember intramolecular H-bonded ring involving the ith and (i + 3)th residues (25,26). A few conformations may pedominate, depending upon the particular chemical microenvironment (solvent, ionic strength) in which that peptide is found and the nature (hydrophobicity, charge, size) of the unique amino acid residues in the peptide chain. For example, it was shown recently that the pturn structure is less prevalent in an aqueous solution of ME than in that of LE (26). A specific conformation of a peptide dissolved in the FAB matrix will affect the overall energy content of the molecule, which in turn
56
DASS AND DESIDERIO TABLE 3 AMINO
ACID
SEQUENCE-DETERMINING IONS IN THE POSITIVE OF GROUP B PE~TIDES
/ 1
ME
ME-Lys
2
ME-Lys-Arg
c IME-Arg-Phe
ME-Arg Gly-Lsu
3
I
AminoAcidResidues 4 I
MASS SPECTRA
5
a b c 136 A 173 18Oc' 30 133 2 61 15oy" 92 178 x" 31 136A 201 131 z' 284 146 y' 312 136A 165B'
ME-Lys-Lys
I
ION FAB
64 91
193 A 223 B"
97 89
131 z' 147 y" -
66 46
259 F
73
136 A 165B'
114 54
301 x 193 A 223 B" 287 Z' 303 y" 329 x 193 A 221 B 305 z 322 y" 348 X 193 A
20 23 80
159 175 203 136 165 180 151 166 192 136 18oc' 117 132 158
r 81 y" 77 X" 29 A 281 B' 102 c' 114 z" 90 y" 128 x 75 A 205 66 z" 245 y" 163 x 70
29 22 14 66 54 79 52 46 40
391 406
279 419 434 460x 251 276 293c 438 453 479 251
Z" y" 8' Z" y" A B Z" y" X A'
399A"
24 529A' 442 c" 15 573 39 538 Z" 40 595 13 553 Y" 20 610 581 X" 9638X" 399A"8 20 556 28 566 z" 55 623 12 581 y" 12 636 5609x" 76&4x 53 399 A" 74 52 29 571 80 585 z" 112 642 28 600 y" 23 657 17 685 19 628 x" 31 399 A" 83
C" z" y" S z" y" c z" y" x"
6 9 24 16 6 5 33 16 5
12 52 27 15
721X 684A 712 6 729c" 699z" 714 y"
4 14 13 12 50 27
-
a, b, c. - See Talbls? II
may influence the ease of formation of the (M + H)+ ion and its subsequent fmgmentation. The energy content of an ion is also controlled by the lattice forces associated with solute-solvent interaction. Furthermore, the charge distribution in a peptide may also influence the fragmentation behavior. In view of these complexities and the diverse number and nature of the amino acid residues present in peptides, it is a nearly impossible task to predict a priori the fragmentation behavior of a peptide and the occurrence of all specific sequence ions in their spectra. Nevertheless, from the data in Tables 2 and 3, the following salient features can be summarized for the particular peptides studied here. In general, the C-terminal-containing sequence ions are more abundant than the N-
terminal sequence ions. A likely explanation for this observation is that the charge site in the molecular ion generally influences the fragmentation reactions. Because peptides exist as zwitterions, the initial site of protonation is assumed to be the negatively charged terminal carboxyl group, leaving the amino group at the N-terminal as positively charged during the generation of (M + H)+ ions of the peptides by FAB ionization. The formation of the positively charged sequence ions occurs by sequential migration of the positive charge in the peptide chain from the terminal HJN+ group as depicted in Scheme 1, which rationalizes the production only of the series of C-terminus-containing ions. Alternately, the fragment ions may be formed by transfer of a proton from the terminal HJN+ group to a specific basic site within the pep-
FAST
+,PQ
ATOM
BQ
H,N-CH-C-NH-CH-C-OH
BOMBARDMENT
BQ
MASS
90
---* +C$C-NH-CH-C-OH SCHEME
tide chain, resulting in cleavage of the bond adjacent to the newly created charge site. This mechanism will favor both the N-terminus and C-terminus ions. The charge site within a peptide is affected by the nature of the amino acid side chains constituting that peptide. The basic amino acid residues Arg and Lys play a role in controlling the charge site prior to and/or after dissociation of the positively charged protonated molecular ion. The majority of the opioid peptides studied here contains Arg and Lys at or near the carboxylic group of the peptide, suggesting that formation of the positively charged C-tenninus ions via Scheme 1 is aided by the presence of Arg and Lys at the C-terminus of these peptides. This rationale is further supported by the fact that in the positive ion FAB MS of the opioid peptides ME and LE, in which Arg and Lys are absent, the N-terminus ions are equally important, consistent with the observation of Roepstorff et al. (27) in their study of other peptides of a more hydrophobic nature. The role of Arg and Lys in controlling the type of fragmentation has also been discussed by Schafer (23). The sequence ions containing only the single N- or C-terminus amino acid residue are the most abundant ions in the spectra and even more so when the amino acid residue is hydrophobic in nature (vide infra); compare the Z, , Y, , and X1 ions with the remaining C-terminus ions of a particular peptide in Tables 2 and 3. Cleavage of the -CHR-CObond, with few exceptions (e.g., Ai ions), yields A-type N-terminal sequence ions, whereas the corresponding C-terminus sequence ions are formed with or without H transfer (Tables 2 and 3). In other words, both X and X” ions are observed. However, the ions formed by cleavage of the -CHR-CObond are relatively less important in the spectra cornoared
57
SPECTROMETRY
Q
7 0
--* +C,-NH-CH-C-OH
-+ -+
1
to other sequence ions, except for some Atype ions, which are discussed below. The cleavage of the peptide bond is almost always followed by a transfer of a hydrogen atom from the neutral fragment to form a Y”-type C-terminal ion (quatemary ammonium ion). The N-terminal sequence ions, on the other hand, are formed with or without H transfer following the peptide bond cleavage to yield B” and B ions, respectively. These N-terminal sequence ions are normally weak and in some instances are not observed in the spectrum (see Tables 2 and 3). The data of Tables 2 and 3 reveal that the principal process of ion formation in the positive ion FAB MS of the peptides under study is cleavage of the amino-alkyl bonds of the (M + H)+ ion of the peptides accompanied by H transfer from the neutral moieties to the charged species (the N-terminal or the C-terminal) leading to the formation of amino-amide (C”) and amino-carboxyl (Z”) ions, respectively. This observation is consistent with the other FAB MS studies @a), but is in contrast to the results of EI and CI studies of chemically derivatized peptides (22a). In the latter studies, it was shown that the prominent fragmentation process of the peptide molecular ion is the cleavage of the peptide bond. The discussion so far is consistent with the formation of the six known types of amino acid sequence-determining fragment ions when these opioid peptides are subjected to positive ion FAB. However, not all of the ions are formed with equal facility, and in some instances a few of the fragments are even absent (Tables 2 and 3). On the other hand, some fragments are unusually dominant in the mass spectra. For example, the C-terminal ions that contain three or more amino acid residues are intense in the spec-
58
DASS
AND
n-urn of dynorphin A 1-7 (Fig. 1). Likewise, the fragments that terminate with the hydrophobic amino acids Leu, Pro, and Phe residues are of significant abundance in the spectra of the other neuropeptides being considered here (Tables 2 and 3). Although it is not easy to assign a single factor, from these results it appears that, at least to a certain extent, the hydrophobicity of the amino acid side chain influences the fragmentation pattern of the peptides. Recently, Pelzer and DePauw also attributed the increase in the ion fragment intensity to the hydrophobicity of the solute (28). Because of the complex structural nature of the peptide molecules, it is not easy to rationalize the manner in which the hydrophobic residues act in controlling fragmentation of the peptides. However, the following explanation may account, at least partly, for the role of hydrophobic residues. It is well known that charged side chains such as Lys, Arg, Gly, and Asp are usually located on the outer surface of the three-dimensional conformation of a peptide molecule dissolved in a polar solvent, whereas the nonpolar residues are found within the hydrophobic volume. Therefore, the polar side chains are predominantly involved via H bonds in interaction with the surrounding matrix molecules and also participate in the molecular folding of the peptide itself. Consequently, it is logical to rationalize that the
se
--Arg
-
----
e
FIG.
l-7.
1. Positive
ion FAB
mass spectra
of dynorphin
A
DESIDERIO
energy requirements for cleavage of the bonds connected to the polar versus nonpolar residues are different. We believe that this phenomenon could affect the intensity of the various peptide fragments found in the mass spectra. This hypothesis, indeed, is corroborated by the present results (Tables 2 and 3). For instance, the C-terminus ions having more than two amino acid residues found at the site of Lys or Arg are commensurately weaker than the ions that are formed at the sites of more hydrophobic residues such as Leu, Be, Met, Phe, and Tyr in the positive ion FAB mass spectra of the opioids under study (for example, compare 25 ion intensity with those of Z$ and Z5 and Zg ions in the spectra of Dynorphin 1-8; Table 2). The decreasing order of hydrophobicity based on the Bull and Breese index (29) for the amino acid residues present in these peptides is Leu > Phe > Ile > Tyr % Met % Lys > Arg. Other factors such as charge delocalization of the carbonium ion and the size of the fragment ion also influence the fmgmentation yield. For example, cleavage of the bond between Tyr and Gly residues (m/z 136) and between Phe and either a Leu or a Met residue (m/z 397 or 399) yields intense A-type N-terminus ions. Similarly, the Z-type ions (see Table 2 for Zi, Z’; , ZI; , and Z’; ions from dynorphin A fractions 1-7, l-8, 1-9, and 1- 10, respectively, and Zl; and Z$ from /3- and cr-neoendorphins, respectively; see Table 3 for Z; from ME-Lys, Z$ from ME-Lys-Lys, ME-Lys-Arg, and ME-Arg-Phe, and Z? from ME-Arg-Gly-Leu) generated at the site of cleavage between Phe and Gly are always the dominant C-terminus ions. The dominance of these ions in these spectra may result from charge delocalization by the phenyl ring by H-atom migration as depicted in Scheme 2. The effect of fragmentation size, i.e., the number of amino acid residues present in that fragment, is clearly evident when we compare the relative abundance of Z; ions with the Z’; and Zg ions in the spectra of ME-Lys-Lys, ME-Lys-Arg, and ME-ArgPhe (see Table 3).
FAST ATOM
BOMBARDMENT
Considering all these aspects, we suggest that it is not prudent to assign a single specific factor responsible for the formation of a fragment. We feel that the relative abundance of a fragment ion will depend upon a combination of several factors such as hydrophobicity, charge delocalization, and size. In addition to the sequence ions discussed so far, the presence of other ions characteristic of particular amino acid residues are noted. Most important are the immonium ions, which are produced only from the hydrophobic amino acids. For example, ions of significant abundance occur at nominal masses 104 (H*N=CH-CH2--CHz-SCH3) and 120 (H2N=CH-CH2-Ph). In fact, m/z 120 is the most abundant ion found in the spectra of several of these peptides. The corresponding ions characteristic of the basic amino acid residues (Lys and Arg) were either absent or present in very low abundance. The ions at m/z 107, 116, 131, and 147, presumably having structures a, b, c, and d, respectively (Scheme 3), also occurred in significant abundance. The c’ and d’ ions are also likely candidates for the structures of
r!o
I
P
II
270 (-HC-C-NH-&H,
![(A,Z&],
0 R 0 II II HZN-CH-C-NH-CH-C-[(B6Y$],or
Ill
0
298 (H,C-C-NH-CH-C-
the ions found at m/z 131 and 147, respectively. However, the exact nature of the distonic ions b-d is not known and is the subject of further investigation. The intense signals due to the aforementioned ions are further evidence of the influence of hydrophobicity of the amino acid side chain. The occurrence of these ions in the spectra aid in confirming the presence of the suspected, indicated amino acid residues in the peptides and will probably provide clues to their presence in the spectra of unknown peptides. The ions arising from the loss of the side chain of specific amino acids are also seen at the high mass end of the spectrum and can be helpful in identifying the presence of an amino acid residue. The losses of 15 (CH,), 44 (CO&, 45 (HCOOH), 61 (CH2-S-CH3), and 107 (CH,-Ph-OH) mass units are often noticed (see Fig. 1). However, some ions of undefined origin are also present in the positive ion FAB spectra of the opioid peptides studied here. For example, the ions of ambiguous character (with the probable structure shown in parentheses) seen in the spectra of dynorphin A 1-7 are m/z 229 (A, + G + H, where G = glycerol), 0
R
Q
-H&NH-&H-&-NH,
L
R
59
MASS SPECTROMETRY
[(cl,Z,),],
0 L 0 II II -C-NH-CH-C-NH-CH2
P
[(&X&l),
0
P
II
[(B,Z$] 0 or
0
II
F
L
II
0
R
II
0
II
-C-NH-CH-C-NH-AH-C-H 0
II
L
385 (C, + G), 459 (-C-NH-CH-C-NH-CH-C-NH-CH-C-NH-
0
II
R
[(BbX,),]),
0
II
[(C,X,),]),
5 17 (B4 + G), and 533 (C, + G). However, further work is needed to unambiguously assign structures to these ions. Finally, it was also observed that fragment
ion intensities increase with the time of FAB irradiation, as shown in Fig. 2 for dynorphin
60
DASS AND DESIDERIO 140. 120lOO-
SCHEME
2
A 1-7. The increase in fragmentation is attributed to an increase in the internal energy of the molecular ion caused by changes in the relative\ concentration of the peptides, because with time the solvent is depleted from the probe tip. As a result, fewer solvent molecules are associated with the desorbed clusters, a phenomenon that causes the molecular ions to be preferentially energy enriched because now less energy is dissipated in desolvating the remaining decreased number of solvent molecules. This rationale was confirmed by loading different concentrations of dynorphin A l-7 on the probe tip (see Fig. 3); increased fragmentation was observed with
2%
0I I ;jH +
+Ii)+-CHJ 20-
I , 6
1
10
15
FIG. 2. Increase in the fragmentation of (M + H)+ of dynorphin A l-7 with respect to the time of irradiation.
the more concentrated solution because clusters with fewer solvent molecules were formed. The increase in the fragment ion intensity with concentration was also observed by other workers (30). Negative ion FAB mass spectra. To gain additional structural information and possibly to increase the molecular specificity of quantitative MS measurements, negative ion FAB mass spectra of these opioid peptides were acquired. Because the structural features that stabilize a negative charge are not usually the same as those that stabilize a positive charge (see below), the negative ion
a
180
0 Hi-CH-Cd
&-t-tiH
160 3
LH I *
{H2 140
-
F+ $ a3
c-+ 0 H-&JH Y
b
0.2
:I
0.4
0.6
0.8
1 .o
1.2
Cont. (nM) SCHEME
3
FIG. 3. Increase in the fragment ions intensities with concentration of dynorphin A 1-7.
FAST
ATOM
BOMBARDMENT
MASS
TABLE AMINO
ACID SEQUENCE-DETERMINING
61
SPECTROMETRY
4
IONS IN THE NEGATIVE
ION FAB
SPECTRA OF GROUP
A WITIDES
LE
364 “X Dynorphin A l-7
135 163 179
'A 'B c
70 10 7
191 "A 219 "6 236 C
11 4 8
249 277 293 427 442 470
'A 'B c Z Y x
277 'B 293 c 426 'Z 441 'Y 468 "X 249 A 293c4644oc 426 'Z 441 'Y 468 "X
Dynorphin A 1-a
Dynorphin A l-10
417 "X
4 I
1 3 12 2 3 1
395 "A 425 B 441 c 575 z 590 Y' 617 x
1 1 8 4 3 1
6 14 4 4 2 18
395 "A 425 E 440 c 539 'Z 554 'Y 581 "X 395 -A 425 B
14 11 9
582 598 626
c - Relative abundanceofthe
'A B c Z Y 'X -
2 2538B a 553 c 5 687 z 12 702 Y 2 730 x 9 508 “A IO 538 B 20553c 11 695 Z 4 710 'Y 4 -
2 665'A 1 694 B 8709C 368EZ 3704y' 1 732 x' 3 7 709 c 5 744 z 3 759 Y 1 787 x 8 665 'A 7 694 B 28709C 7 a43 z 7 858 Y -
'B c Z Y x
9 33 IO 8 7
395 "A 425 B 440 c 523 'Z 538 'Y 565 "Z
2 2 IO 6 5 2
509 'A 538 B 553 c 679 'Z 694 'Y 721 "X
3 665'A 5694B 15 709 6 792 2 3
277 293 389 405 433
'B C 'Z Y x
38 38 9 5 5
397 425 440 545 5EQ 587
A B C 'Z 'Y x
12 4 17 8 4 2
395 -A 425 B 440 c 532 'Y 559 "X
30 2 7
508 "A 538 E 554 c' 658 'Z 674 Y 701 'X 508 "A 553 c 673 'Z 715 "X
6 5 26 6 4 3 2
'A 240 'B 12 389 'Z IO 431 "X 2
d-Neo endorphin
'Z Y X
509 538 554 632 647 673
277 293 368 383 411
249 277
a. b - see Tablell,
3
sequence
ions considemg
2 1
3 1 3 4 4 1
866 (M-H)-
4a65c 6 801 Z 4816Y la44X 4 5 13a65c 10 !3N z 4915Y -
c 'Z -
3 3 6 a65 C 39402 955 Y 983 x
B C 'Z Y 'x 'A 7 709 c 2 786 'Z 802 Y 2 -
2 792 "A 3 a22 B 12 a37 C 5 862 ‘Z 2 078 Y 2 5 822 B 8 a37 c 4 934 z 2 950 y' 977 x
665'A 694 709 805 821 848 665
2 4 4 2
4 12 6
4 6 4 1 3 4 7 6 4 3 9 4 6 2
(M-H)- asthe base peak
spectra may provide information complementary to that obtained from the positive ion mode. Tables 4 and 5 list the sequence ions from the negative ion FAB mass spectra of Groups A and B peptides, respectively. The negative ion mass spectra of dynorphin A 1-7 is shown in Fig. 4 and may be compared to the corresponding positive ion spectra in Fig. 1. By comparing the negative ion data in Tables 4 and 5 with the positive ion data listed in Tables 2 and 3, we find that the mechanism of fragmentation of the quasimolecular (M - H)- anions of the peptides generated by negative FAB ionization is comparable to the pattern of the (M + H)+
ions of these peptides. However, by comparing the two ionization modes, some striking differences are found in the data. The most significant difference is seen in the relative fragment ion yields. In the negative ion spectra, the bulk of the total ion current consists of the quasimolecular (M - H)- anion. This difference suggests that (M - H)- anions formed by the FAB desorption process are energetically more stable than the corresponding (M + H)+ cations, consistent with the general observation that the negative ions formed by electron capture are of low internal energy (3 1). Another important difference is that the N-terminal ions, which were
62
DASS
AND
DESIDERIO
TABLE AMINO
5
ACID SEQUENCE-DETERMINING IONS IN THE NEGATIVE OF GROUP B PEPTIDES
ION FAB M.us
SPECTRA
1
Amino AC
1 a
ME
ME-Lys
ME-Lys-Lys
ME-Lyo-Arg
ME-Arg-Phe
b
163 ‘6 7 179c 5 146 Y 12 175 ‘X 5 __135 ‘A 15 163 ‘6 7 130 z 15 145 Y 15 173x 7 135 ‘A 501 163 ‘B 113 13Oz 38 145Y 46 173 X 24 135 ‘A 163 ‘B 173 Y -
2 a
b “A B c z Y X “A
E 4 4 4 7 7 3 7
23s; 260 ‘Z 276 Y 303 ‘X 221 6 236 C 273 Y 301 x
a 6 8 3
T 191
221 236 280 295 323 191
13 19
191 “A 221 B 236 c
20
301 327 191 221 236 303 319 34.8 191
135 ‘A 29 163 ‘B 73 151 z” 130 164 Y 108 191 ‘X 17
135 ‘A 163 ‘6 179C 115 z 13OY 158x a. b. c. - See Talblc? IV
ME-ArgGly-LSIJ
c
16 9 6 18 60 4
236 172 187 213
572 (M-H)277 ‘B 293c 4082 424 Y 451 x
21 26 75 8
293 389 405
C z Y
395 “A 5 1344Oc 4465z 446OY 2 1 506 “X 397 A 425 B 27 440 C 6536Z 4 551 Y
13 14 11
Y 4 “X 3 “A 17 6 190 C 19 “Z 12 ‘Y 9 “X 13 “A 27 C 6 2 21 Y 35 “X 18
ion FAB mass spectra
5 4 4 1 9 5 20 7 16
554 “B 572 c’ 522 z 537 Y 563 “X 554 “El 571 c 593 z @Y3Y 63’X 554 “6 572 c’ 620 ‘Z 636Y 663’X 554 “6 571 c 6402 655Y 661 “X 530
A”
572 ;’ 607z 622 y’ 648,x
rare in the positive ion spectra, are now quite conspicuous in the negative ion spectra (compare C-type ions in Tables 4 and 5).
FIG. 4. Negative l-7.
2
of dynorphin
A
8 12 2 4 2
700 (M-H)-
11 12 7 6 3 13 6 5 5 2
677 ‘Z 693Y 720 ‘X
7 5 2
(M-H)-
26 9 7 9 3 2 4 5 7 2
898 (M-H)-
This observation is explained by the fact that one of the major processes of generation of negatively charged sequence ions of the opioid peptides of Table 1 is the initial charge localization at the terminal carboxy group yielding an (M - H)- anion by donation of the proton from the H3N+ terminal group and subsequent sequential migration of the negative charge in the peptide chain from the carboxyl group in a process that is the reverse of the formation of the positive ions discussed earlier in Scheme 1. Furthermore, contrary to the positive ion data, the Z-type negative ions are not always the dominant ions, and in some cases, cleavage of the peptide bond also gains importance in forming the Y-type C-terminal sequence ions (Tables 4 and 5). The cleavage of
FAST
ATOM
BOMBARDMENT
the -CHR-CObond produces two types of carbonyl-containing fragment ions (X and “X).
Formation of Adduct Ions Previous studies have reported the occurrence of ionic adducts between glycerol and solute molecules containing primary and secondary amines (32). A similar phenomenon was observed in the current study of these opioid peptides. The positive ion spectra include several ions having mass numbers greater than the protonated molecular ion of the peptides. Most prominent is the addition of I2 mass units. Other adduct ions corresponded to the addition to the (M + H)+ ion of 24,29,44, 54,66, 74, and 104 mass units. On the other hand, the formation of adduct ions in the negative ionization mode is not as facile. However, an addition of 98 mass units to the (M - H)- anion was frequently observed. A detailed mechanistic study concerning the adduct formation, presumably between the glycerol and these opioid peptides, is underway and will be reported elsewhere.
SequenceDetermination In the course of describing peptide fragmentation processes, with our specific goal of quantification, some general observations on features relating to amino acid sequence determination were made. For sequence determination of a peptide, identification of the terminal amino acid residues is quite important. In the case of the peptides studied here, the N-terminal amino acid Tyr is readily recognized by considering the intense C-terminal sequence ions of the (n - 1)th amino acid residue, which is formed by the loss of the Tyr residue from the peptide. Another piece of evidence for the presence of Tyr at the N-terminus is the presence of an ion of m/z 136 (A,-type ion). On the other hand, recognition of the Cterminal amino acid is somewhat ambiguous
MASS
SPECTROMETRY
63
when the C-terminal sequence ions such as X1, Y, , and Z1 are considered. This limitation is due to the excessive chemical noise at the lower end of the FAB spectra that arises due to the matrix ions and to the fragment ions from the peptide itself. Furthermore, it is sometimes not possible to unambiguously assign a signal to the specific amino acid residue (33). For example, the ion at m/z 13 1 in the spectra of ME-Lys and ME-Arg-GlyLeu can be due to either Lys (Z’) and Leu (Y’) residues, respectively, or Met (structure b). However, these limitations can be overcome effectively by the careful choice of the solvent matrix and by searching for N-terminal sequence ions formed due to the (n - 1)th amino acid residue. These ions are generally of significant abundance in the FAB MS of the opioid peptides studied here. The remaining amino acid residues can be recognized by considering the most intense C-terminal ion series. Figure 1 demonstrates the efficacy of FAB positive ion mass spectrometry in sequencing these known opioid peptides. The data reported in this study were obtained using l-2 nmol of the peptides. However, a better approach for sequencing the peptides reported here is to use negative ion FAB MS. This polarity advantage arises because (a) the ions belonging to the C-type N-terminal sequence ion series are of significant abundance in the negative ion spectra; (b) compared to the Z-type C-terminal ion series, the Y-type series is equally dominant in the negative ion spectra and, therefore, may be used for sequencing the peptides; and (c) in comparison to positive ion spectra, the negative ion spectra are relatively clean at the upper mass end. Therefore, in the negative ion mode, it is easier to recognize a specific sequence ion and it is possible to use the N-terminus ion series to substantiate the conclusion drawn based on the C-terminus ion series, thereby lending considerable confidence to the structure elucidation of an unknown peptide. The example of the mass spectrum of dynorphin A l-7
64
DASS AND DESIDERIO
(Fig. 4) demonstrates the usefulness of negative ion FAB MS for sequencing peptides.
Detection Limits Although we normally quantify a peptide in a biological extract by MS/MS selected reaction monitoring SRM (19,34), on occasion we obtain a full mass spectrum of an HPLC fraction to survey for the possible presence of a peptide. Toward that latter end, we also investigated the minimum amount of a peptide needed to obtain the meaningful sequence data from a full mass spectrum. This experiment was accomplished by acquiring the spectra of the pentapeptides LE and ME in the positive and negative ionization modes. It was noticed that, in the negative ion mode, the (M - H)- anion of LE could be seen in a single scan with as little as 10 ng (18 pmol) of that peptide. However, the interpretable amino acid sequence-determining data could only be obtained with amounts greater than 25 ng (ca. 50 pmol). The corresponding detection limits in the positive ionization mode were 20 and 40 ng, respectively. These detection limits may be considerably improved with signal averaging techniques. ME, on the other hand, cannot be detected with the same ease. The lowest amounts necessary to obtain useful amino acid sequence data of ME in the negative and positive ion modes were 100 and 200 ng, respectively.
Mixture Analysis In order to test the utility of FAB MS as a tool for the characterization of a specific peptide in the presence of other peptides, which is a situation that could be experienced with biologic extracts even, in our experience, after HPLC separation, a synthetic mixture of four peptides consisting of 120, 350, 150, and 150 pmol of LE, ME, MEArg-Phe, and ME-Arg-Gly-Leu, respectively, was prepared. The choice of this mixture is important from a practical point of
view because these opioids have a common precursor, the preproenkephalin A (35) and have been found together in body tissues (36). It was observed that the (M + H)+ and sequence ion signals due to ME, ME-ArgPhe, and ME-Arg-Gly-Leu were weak compared to those signals obtained when the peptides were analyzed individually; these data confirm the well-known signal suppression phenomenon of peptides when present in a mixture during FAB MS analysis (37). Because we had prepared a mixture of known peptides, we could easily confirm the sequence of each of the four components, even though the individual amino acid sequence signals were not very strong. However, this mutual suppression of ionization may be a severe limitation of FAB when applied to the sequencing of a mixture of unknown peptides, even following HPLC purification, which does not necessarily always separate all peptides in a biologic extract. The positive ion FAB produced even more inferior data. CONCLUSIONS
In this study, positive and negative ion FAB MS methods were evaluated for rationalization of peptide bond fragmentation of endogenous opiates of M, up to approximately 1250 amu. The fragmentation behavior of the opioid peptides studied here is in accordance with the known fragmentation characteristics of other studied peptides (8,13b,23,24). However, the patterns of the relative abundance of various types of ion series differ. These differences are related to the hydrophobicity of the amino acid residues and perhaps to a certain extent to the conformational features of these peptides. FAB ionization produces abundant (M + H)+ and (M - H)- ions, from which molecular weight information is readily deduced. Sequence information is obtained by considering the intense Z-type C-terminus ion series in the positive ion mode. For sub-
FAST
ATOM
BOMBARDMENT
stantiating these data, the use of C-type Nterminus and Y- and Z-type C-terminus ion series in the negative ion mode is also recommended, especially when characterizing an unknown compound-if sufficient material is available. It is necessary to understand the behavior of each ion in the mass spectrum of each peptide before quantification of that peptide can be undertaken. Toward that goal of quantification of specific peptides, this manuscript has provided data rationalizing the fragmentation of several neuropeptides and the ion genesis of fragment ions. ACKNOWLEDGMENTS The authors gratefully acknowledge the financial assistance of NIH (GM 26666) and the typing assistance of Dianne Cubbins.
REFERENCES 1. Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B. A., and Morris, H. R. (1975) Nature (London) 258,577-579. 2. Kosterlitz, H. W., and Paterson, S. J. (1985) Philos. Trans. R. Sot. London Ser. B 308,291-297. 3. Leclercq, P. A., Smith, L. C., and Desiderio, D. M. (197 1) Biochem. Biophys. Res. Commun. 45, 937-944. 4. Morris, H. R. (1980) Nature (London) 286, 447-452. 5.
6.
7. 8.
9. 10.
(a) Frank, H., and Desiderio, D. M. (1978) Anal. Biochem. 90, 413-419; (b) Burgus, R., Dunn, T. F., Desiderio, D. M., Ward, D. N., Vale, W., and Guillemin, R. (1970) Nature (London) 226, 321-325. Barber, M., Bordoli, R. S., Sedgwick, R. D., and Tyler, A. N. (1981) J. Chem. Sot. Chem. Commun., 325-327; Barber, M., Bordoli, R. S., Sedgwick, R. D., Tyler, A. N., and Whally, E. T. (198 1) Biomed. Mass Spectrom. 8, 337-342. Surman, D. J., and Vickerman, J. C. (1981) J. Chem. Sot. Chem. Commun.. 324-325, (a) Williams, D. H., Bradley, C., Bojesen, G., Santikam, S., and Taylor, L. C. E. (1981) .I. Amer. Chem. Sot. 103,5700-5704; (b) Williams, D. H., Bradley, C., Santikam, S., and Bojesen, G. (198 1) Biochem. .I. 201, 105-l 17. Barber, M., Bordoli, R. S., Garner, G. V., Gordon, D. B., Sedgwick, R. D., Tyler, L. W., and Tyler, A. N. (1981) Biochem. J. 197,401-404. Morris, H. R., Panico, M., Barber, M., Bordoli,
MASS
SPECTROMETRY
65
R. S., Sedgwick, R. D., and Tyler, A. N. (1981) Biochem. Biophys. Res. Commun. 101,623-63 1. 11. Smith, L. A., and Caprioli, R. M. (1983) Biomed. Mass Spectrom. 10, 98-102; Self, R., and Parente, A. (1983) Biomed. Mass Spectrom. 10, 78-82; Hong, Y. M., Takao, T., Aimoto, S., and Shimonishi, Y. (1983) Biomed. Mass Spectrom. 10,450-457.
12. Katakuse, I., Matsuo, T., Matsuda, H., Shimonishi, Y., Hong, Y-M., and Izumi, Y. (1982) Biomed. Mass Spectrom. 9, 64-68; Bradley, C., Williams, D. H., and Hanley, M. R. (1982) Biochem. Biophys. Res. Commun. 104, 1223-1230. 13. (a) Benninghoven, A., and Sichtermann, W. (1978) Org. Mass Spectrom. 50, 1180-l 184; (b) Seki, S., Kambara, H., and Naoki, H. (1985) Org. Mass Spectrom. 2, 18-24. 14. Beckey, H. D. (1972) in Biochemical Applications of Mass Spectrometry (Waller, G., Ed.), pp. 795-815, Wiley, New York; Winkler, H. U., and Beckey, H. D. (1972) Biochem. Biophys. Res. Commun. 46, 391-398; Desiderio, D. M., and Sabbatini, J. Z. (198 1) Biomed. Mass Spectrom. 8, 565-568; Matsuo, T., Matsuda, H., Katakuse, I., Wada, Y., Fuji@ T., and Hayashi, A. (1981) Biomed. Mass Spectrom. 8,2S-30. 15. MacFarlane, R. D. (1982) Act. Gem. Res. 15, 268-275; MacFarlane, R. D., and Torgeson, T. F. (1976) Science 191, 920-925; Hakansson, P., Kamensky, I., Sundquist, B., Fohlman, J., Peterson, P., McNeal, C. J., and MacFarlane, R. D. (1982) J. Amer. Chem. Sot. 104,2948-2949. 16. Cotter, R. J. (1984) Anal. Chem. 56, 485a-504a; Wilkins, C. L., Akil, D. A., Yang, C. L. C., and Ijames, C. F. (1985) Anal. Chem. 57,520-524. 17. Kim, H. Y., Pilosof, D., Dyckes, D. F., and Vestal, M. L. (1984) J. Amer. Chem. Sot. 106, 7304-7309; Blakley, C. R., Carmody, J. J., and Vestal, M. L. (1980) J. Amer. Chem. Sot. 102, 5931-5933; Blakley, C. R., and Vestal, M. L. (1983) Anal. Chem. 55, 750-754; Pilosof, D., Kim, H. Y., Dyckes, D. F., and Vestal, M. L. (1984) Anal. Chem. 56, 1236-1240. 18. Lippstreu-Fisher, D. L., and Gross, M. L. (1985) Anal. Chem. 57, 1174-l 180. 19. Desiderio, D. M. (1984) Analysis of Neuropeptides by Liquid Chromatography and Mass Spectrometry, Elsevier, Amsterdam. 20. Katakuse, I., and Desiderio, D. M. (1983) Int. J. Mass Spectrom. Ion Phys. 54, l-15. 2 1. Katakuse, I., and Desiderio, D. M. (1985) Shitsuryo Bunseki, 33, 35 l-370. 22. (a) Yu, T. J., Schwartz, M. A., Cohen, S. A., Vouros, P., and Karger, B. L. (1984) .I. Chromatogr. 301, 425-440; (b) Hunt, D. F., Buko, A. M., Ballard, J. M., Shabanowitz, J., and Giordani, A. B. (198 1) Biomed. Mass Spectrom. 8,397-408.
66
DASS AND DESIDERIO
23. Schafer, W. (1983) in Modem Methods in Protein Chemistry (Tschesche, H., Ed.), pp. 337-359, de Gruyter, New York. 24. Roepstorlf, P., and Fohlman, J. (1984) Biomed. Mass Spectrom. 11,60 1. 25. Rapaka, R. S., Renugopalakrisyhnan, V., and Bhatnagar, R. S. (1985) Amer. Biotech. Lab., 8-21. 26. Renugopalkrishnan, V., Rapaka, R. S., Collette, T. W., Carreija, L. A., and Bhatnagar, R. S. (1985) Biochem. Biophys. Rex Commun. 126, 1029-1035. 27. Roepstorff, P., Hojrup, P., and Moller, J. (1985) Biomed. Muss Spectrom. 12, 181-189. 28. Pelzer, G., and De Pauw, E. (1985) in 33rd Annual Conference on Mass Spectrometry and Allied Topics, Extended Abstract, pp. 247-249. 29. Bull, H. B., and Breese, K. (1974) Arch. Biochem. Biophys. 161,665-670. 30. Heurling, M. E., Yu, R. K., Sedgwick, R. D., and Rinehart, K. L., Jr. (1984) Biochemistry 23, 5706-5713; Busch, K. L., Hsu, B. H., Xie, Y-X,
31. 32.
33. 34. 35. 36. 37.
and Cooks, R. G. (1983) Anal. Chem. 55, 1157-l 160. Bowie, J. H. (1984) Muss Spectrom. Rev. 3, 161-207. Pang, H., Costello, C. E., and Biemann, K. ( 1984) in 32nd Annual Conference on Mass Spectrometry and Allied Topics, Extended Abstract, pp. 751-752. Desiderio, D. M. (1987) ht. J Mass Spectrom. Zen Phys., in press. Desiderio, D. M., and Dass, C. ( 1986) Anal. Lett. 19, 1963-1971. Udenfriend, S., and Kilpatrick, D. L. (1983) Arch. Biochem. &ophys. 221, 309-333; Hughes, B. J. (1983) Brit. Med. Bull. 39, 17-24. Tang, J., Yang, Hy.-T, and Costa, E. (1982) Neuropharmacology 21,595-600. Clench, M. R., Gamer, G. V., Gordon, D. B., and Barber, M. (1985) Biomed. Mass Spectrom. 12, 355-357; Naylor, S., Findeis, A. F., Gibson, B. W., and William, D. H. (1986) J. Amer. Chem. Sot. 108,6359-6363.
BIOCHEMISTRY
163,52-66
Fast Atom Bombardment CHHABIL
(1987)
Mass Spectrometry
Analysis of Opioid Peptides
DASS* AND DOMINIC M. DESIDERIO*,?
Charles B. Stout Neuroscience Mass Spectrometry Laboratory, and Departments of *Neurology and tBiochemistry, University of Tennessee, 800 Madison Avenue, Memphis, Tennessee 38163 Received October 13, 1986 Positive and negative ion fast atom bombardment mass spectrometries have been used to determine the amino acid sequence-determining fragment ion information of opioid peptides containing from 5 to 10 amino acid residues. The opioids investigated include several enkephalins, dynorphin A fragments l-7 through 1- 10, and cc-and &neoendorphins. Data obtained in the two ionization polarities provide complementary information and exhibit the C-terminaland the N-terminal-containing amino acid sequence-determining fragment ions that are formed by cleavage of (a) the bond between the carbonyl group and the a-carbon (-CHR-CO-), (b) the peptide amide bond (-CO-NH-), and (c) the amino-alkyl (-NH-CHR-) bond. The C-terminal sequence ions are dominant in the positive ion mode, whereas the C-terminal and N-terminal ions are equally important in the negative ion mode. Detection limits for full mass scans extend down to the picomole range. The apparent role of hydrophobicity of the amino acid residues on the fragmentation characteristics of the peptide is discussed. o 1987 Academic Ress, Inc. KEY WORDS: opioid peptides; mass spectroscopy; fast atom bombardment; enkephalins; dynorphins.
Since the discovery in 1975 of endorphins as endogenous opiates (l), an increasing number of polypeptides with opioid activity has been identified in the central nervous system and the periphery. These endogenous substances bind to opiate receptors and mimic the biological effects of morphine; hence they were termed endorphins (endogenous morphinelike factors). Three classes of endogenous opioid peptides-enkephalins, dynorphins, and endorphins-are known, along with their corresponding precursors, preproenkephalins A and B and proopiomelanocortin (POMC),’ respectively. Furthermore, several types of opioid receptors (mu, sigma, delta, epsilon, kappa) in brain and gut tissue have been pharmacologically defined
(2). However, our understanding of the molecular events that occur in the endogenous peptidergic pathways is still not complete, and toward that end, quantitative measurements must be made in biologic extracts of selected opioid peptides. Before those analytical measurements are made, however, a firm understanding of the detailed mass spectrometric behavior of those peptides must be provided from model studies. Mass spectrometry (MS) has been used for nearly three decades to determine the amino acid sequence of peptides. Chemical derivatization is necessary whenever electron ionization (EI) and chemical ionization (CI) are used for analyzing this very polar class of compounds (3-5, but see 5a). However, underivatized peptides are now analyzed with ,newer desorption methods such as fast atom bombardment (FAB; 6-12), secondary ion mass spectrometry (SIMS; 13), field desorption (FD; 14), Califomum-252 plasma desorption (15), and laser desorption (16). Of
’ Abbreviations used: POMC, proopiomelanocortin; EI, electron ionization; CI, chemical ionization; FAB, fast atom bombardment; SIMS, secondary ion mass spectrometry; FD, field desorption; RIA, radioimmunoassay; RRA, radioreceptorassay; SRM, selected reaction monitoring. 0003-2697187 $3.00 Copyright 0 1987 by Academic Press, Inc. AII rights of reproduction in any form reserved.
sequencing;
52
FAST ATOM
BOMBARDMENT
these desorption techniques, FAB is an ap propriate technique for the analysis of peptides due to its simplicity, reproducibility, and extended mass range. Recently, another soft ionization technique, thermospray liquid chromatography/MS, has been employed for sequencing peptides ( 17). MS sequencing methodology is useful, and in certain cases necessary, as an adjunct to the more common wet chemical (Edman) and gas phase methods. In certain cases for sequence determination of biological peptides, MS offers the only alternative method; for example, when the target peptide has blocked termini, especially the N-terminus, and/or when very little biologic material is available (5b). In this paper, we present the application of FAB MS for the characterization of several selected opioid peptides (listed in Table 1) by acquiring positive and negative ion FAB mass spectra. The peptides investigated are important in our continuing studies on the basic and clinical neurochemistry of neuropeptides, and the data in this paper are important first to identify a particular peptide in a biologic extract and second to quantify that peptide. TABLE THEOPIOID FWTIDESSTUDIEDBY Peptide Group A Leucine enkephalin (LE) Dynorphin A Fragment l-7 Fragment l-8 Fragment l-9 Fragment l-10 8-Neoendorphin a-Neoendorphin Group B Methionine enkephalin (ME) ME-enkephalin-Lys ME-enkephalin-Lys-Lys ME-enkephalin-Lys-Arg ME-enkephalin-Arg-Phe ME-enkephatin-Arg-Gly-Lcu
M, 555 867 980 1136 1233 1099 1227 573 701 829 857 876 899
MASS SPECTROMETRY
53
A few scattered reports of MS studies have appeared in the literature for some of these opioid peptides, such as for leutine enkephalin (LE=YGGFL) (1,9,18-20), methionine enkephalin (ME=YGGFM) ( 1,9,10,19,2 1,22), and cy- and P-neoendorphins (13b). However, to our knowledge, no systematic investigation has yet been carried out to rationalize the fragmentation mechanisms of all of the peptides contained in this present set of biologically important peptides. Moreover, the commonly used methods for quantification of the neuropeptides rely on radioreceptorassay (RRA) and radioimmunoassay (RIA) measurements. These techniques lack the maximum molecular specificity that is offered by MS methods. Because of the continuing interest in neuropeptides in general and the rapid expansion of neurosciences in particular, the need to continue the development of measuring other peptides that derive from the four gene product precursors protachykinin, preproenkephalins A and B, and proopiomelanocortin exists. Our need for measuring these peptides derives directly from our HPLC-RRA and HPLC-RIA methodologies, where those data consistently indicate 1 FAB MASS~PECTROMETRY Amino acid sequence Tyr-Gly-Gly-Phe-Leu-OH TyrGly-Gly-Phe-Leu-Arg-Arg-OH Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Be-OH Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Be-Arg-OH Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Be-Am-Pro-OH Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-OH Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys-OH Tyr-Gly-Gly-Phe-Met-OH Tyr-Gly-Gly-Phe-Met-Lys-OH Tyr-Gly-Gly-Phe-Met-Lys-Lys-OH Tyr-Gly-Gly-Phe-Met-Lys-Arg-OH Tyr-Gly-Gly-Phe-Met-Arg-Phe-OH Tyr-Gly-Gly-Phe-Met-Arg-Gly-Lcu-OH
54
DASS AND DESIDERIO
the presence of other C-terminally extended ME and LE peptides, which must be quantified ( 19).
(St. Louis, MO) and were used without any further purification. RESULTS AND DISCUSSION
EXPERIMENTAL
SECTION
Fragmentation Mechanisms
The positive and negative ion FAB mass For ease in data tabulation and rationalspectra were obtained with a VG 7070E-HF ization of their positive and negative ion fragmentation mechanisms, the peptides double-focusing forward geometry (E,B, are divided into two where E = electric and B = magnetic sector) under investigation mass spectrometer equipped with a standard groups (Table 1) based upon the amino acid VG FAB ion source and an Ion Tech B 11NF sequence of the N-terminus of each peptide. saddle-field primary atom gun. Xe atoms of The peptides containing the amino acid seapproximately 7 keV impact energy and a quence of LE at the N-terminus are classified beam flux of 1 mA were used as the ionizing as Group A, and those with ME at the N-terbeam. The gas flow of Xe was regulated to ,minus are collected into Group B. produce a source pressure of 1 X 1Oe6 Torr as Positive ion FAB mass spectra. The posiindicated by an ion gauge connected to the tive ion FAB mass spectra of Group A and ion source housing. The FAB target con- Group B peptides are listed in Tables 2 and sisted of a stainless-steel probe tip of approxi3, respectively, which include only the amino mately 8 mm* surface area beveled at an ap- acid sequence-determining fragment ions propriate angle of incidence and fitted onto (hereafter called sequence ions). With the the tip of the direct insertion probe. The ions exception of a few low-mass (<200) fragment were accelerated to 6 keV from the source ions, the (M + H)+ ions are the most intense region. The mass spectra were acquired ions in the spectra. Their preponderance faunder computer (Digital PDP 1 l/24) con- cilitates the determination of the molecular trolled magnet scans at a resolution of ap- weight, one of the most important experiproximately 2000 (10% valley definition) mental parameters used in characterizing an and scan rates of 10 s per decade by using VG unknown peptide or corroborating the pres1 l-250 M+ software. CsI was used as the ence of a known peptide in a biologic extract. Similar to the earlier studies (8a,13b,23) on mass calibrant in the positive and negative other oligopeptides, the genesis of the imporionization modes. Glycerol served as the liquid matrix. tant sequence ions from the neuropeptides The test samples were dissolved in under investigation is rationalized in terms H20:CH30H (1: 1) at a concentration of 0.5 of cleavage of (a) the bond between the carpg $‘. An amount of l-2 pg, corresponding bony1 group and a-carbon (-CHR-CO-), (b) and (c) to 2-4 nmol of the peptide, was applied to the peptide amide bond (-CO-NH-), bond (-NH-CHR-). The the FAB probe tip that already contained a the amino-alkyl layer of 0.2 ~1 of glycerol. The methanol was cleavage of these bonds, in many instances, is evaporated by heating under an infrared associated with a transfer of hydrogen(s) lamp, and the probe was introduced into the from the neutral fragment to the charged ion source via a vacuum lock. For the data in species. In designating the sequence ions, the Fig. 3, a stock solution of dynorphin A 1-7 of nomenclature proposed recently by Roepstoti and Fohlman is utilized (24). For ex0.5 nmol ~1~’ concentration was prepared, ample, the N-terminus-containing ions and an appropriate amount (0.25 to 1 nmol) was mixed with 0.2 ~1 of glycerol on the formed according to the aforementioned probe tip. All the peptides listed in Table 1 mechanism are designated A, B, and C and were purchased from Sigma Chemical Co. the corresponding C-terminal ions are de-
FAST
ATOM
BOMBARDMENT
MASS
TABLE AMINO
r
ACID SEQUENCE-DETERMINING
:,
136 A la0 c 117 Z" 132 y" 158 x 136 159 175 201 136
c
at,
02
193 221 236c 264 279 307
7 16 42 5
A 166 z' loo y" 137 x 53 A 215 -
115 y' 141 'X
d-NO0 endorphin
A a Z" y" X"
193 A 221 B 315 2 331 y" 357 x 221 B
a
i
c
a
:
9 11 3 8 25 3
251 279 295 321 336 361,x
A 8' c" Z" y"
5397A 25 425 5 442 4 378 IO 393 2419x
B C" z" y"
26 23
278 B 429 z" 444 y" 470 x 278 B
24 16 IO 33
23 72 14 6 37
397 A 427 8" 576-Y 591 y" 617X 397 A -
c
a 5 3 2 3 5 1
MASS SPECTRA OF GROUP
221 B
480 146
136 A 222 115 Y' 153 141 'x 58
136 A -
-
196
(47 y" 107 174 x' 25
501 35 278 1 B
34
y-j 427
B
:
c
a
5 76 19 7
666A 696 711 690 705 733
:
5-14 555 c" lo6 633 T 21 648 y" IO 674 X IO -
“y;;,539
12
8" c" z" y" x" -
4 4 12 50 11 4
/
B
‘;I-;: 14 696
z' Y
14 II
B
795 F al0 Y"
ig 14 a
193 223
z y" X'
II 7 7
519 z 533Y 561 X
7 11 7
noted as X, Y, and 2 ions, respectively. The number of hydrogens transferred to or lost from the charged moiety is indicated by the corresponding number of apostrophes to the right or left of the letter designation, respectively. Peptides are very complex molecules consisting of the 20 naturally occurring amino acid residues. For n known amino acids, n! sequences are possible; for an unknown with m amino acids, 20” possible peptides exist. Due to the presence of several noncovalent molecular forces, the enkephalins in solution are known to fold to a state of lowest free energy and to exist in several conformations, the more common one being the P-sheet or
c
a
8 b
cla
I
9
b
cl
II0
675 691 719
Z y" x"
7 789 z" 5ao4v 3830x
i
a68 (M+H)
-
263 278 307
391 407 434
b
867 c" 803z" 818~" 644x
193 221
33 46 24
a
556
255 272 298
228 z" 244 y" 272 X-
7
c
A PEPTIDES
(M+H)+
II_‘yy/i
@-NW endorphin
ION FAB
Amino Acid Reaidua a
LE
2
IONS IN THE POSITIVE
T
55
SPECTROMETRY
a 4 3
5 30 a 2
867 C902z" 917 y" 943x 622 A a67 c" 942 Z" 957 y" 985 X"
8 63 15 10
794A a24 8" 839 cm a65z" aao Y" 908x" 794 A a24 B a39 c" 936 z" 951 y" 978 x'
3 5 5 7 4 2 5 13 19 29 11 8
6 33 IO 5 5
extended structure involving intermolecular H bonds and several types of &turns-a lomember intramolecular H-bonded ring involving the ith and (i + 3)th residues (25,26). A few conformations may pedominate, depending upon the particular chemical microenvironment (solvent, ionic strength) in which that peptide is found and the nature (hydrophobicity, charge, size) of the unique amino acid residues in the peptide chain. For example, it was shown recently that the pturn structure is less prevalent in an aqueous solution of ME than in that of LE (26). A specific conformation of a peptide dissolved in the FAB matrix will affect the overall energy content of the molecule, which in turn
56
DASS AND DESIDERIO TABLE 3 AMINO
ACID
SEQUENCE-DETERMINING IONS IN THE POSITIVE OF GROUP B PE~TIDES
/ 1
ME
ME-Lys
2
ME-Lys-Arg
c IME-Arg-Phe
ME-Arg Gly-Lsu
3
I
AminoAcidResidues 4 I
MASS SPECTRA
5
a b c 136 A 173 18Oc' 30 133 2 61 15oy" 92 178 x" 31 136A 201 131 z' 284 146 y' 312 136A 165B'
ME-Lys-Lys
I
ION FAB
64 91
193 A 223 B"
97 89
131 z' 147 y" -
66 46
259 F
73
136 A 165B'
114 54
301 x 193 A 223 B" 287 Z' 303 y" 329 x 193 A 221 B 305 z 322 y" 348 X 193 A
20 23 80
159 175 203 136 165 180 151 166 192 136 18oc' 117 132 158
r 81 y" 77 X" 29 A 281 B' 102 c' 114 z" 90 y" 128 x 75 A 205 66 z" 245 y" 163 x 70
29 22 14 66 54 79 52 46 40
391 406
279 419 434 460x 251 276 293c 438 453 479 251
Z" y" 8' Z" y" A B Z" y" X A'
399A"
24 529A' 442 c" 15 573 39 538 Z" 40 595 13 553 Y" 20 610 581 X" 9638X" 399A"8 20 556 28 566 z" 55 623 12 581 y" 12 636 5609x" 76&4x 53 399 A" 74 52 29 571 80 585 z" 112 642 28 600 y" 23 657 17 685 19 628 x" 31 399 A" 83
C" z" y" S z" y" c z" y" x"
6 9 24 16 6 5 33 16 5
12 52 27 15
721X 684A 712 6 729c" 699z" 714 y"
4 14 13 12 50 27
-
a, b, c. - See Talbls? II
may influence the ease of formation of the (M + H)+ ion and its subsequent fmgmentation. The energy content of an ion is also controlled by the lattice forces associated with solute-solvent interaction. Furthermore, the charge distribution in a peptide may also influence the fragmentation behavior. In view of these complexities and the diverse number and nature of the amino acid residues present in peptides, it is a nearly impossible task to predict a priori the fragmentation behavior of a peptide and the occurrence of all specific sequence ions in their spectra. Nevertheless, from the data in Tables 2 and 3, the following salient features can be summarized for the particular peptides studied here. In general, the C-terminal-containing sequence ions are more abundant than the N-
terminal sequence ions. A likely explanation for this observation is that the charge site in the molecular ion generally influences the fragmentation reactions. Because peptides exist as zwitterions, the initial site of protonation is assumed to be the negatively charged terminal carboxyl group, leaving the amino group at the N-terminal as positively charged during the generation of (M + H)+ ions of the peptides by FAB ionization. The formation of the positively charged sequence ions occurs by sequential migration of the positive charge in the peptide chain from the terminal HJN+ group as depicted in Scheme 1, which rationalizes the production only of the series of C-terminus-containing ions. Alternately, the fragment ions may be formed by transfer of a proton from the terminal HJN+ group to a specific basic site within the pep-
FAST
+,PQ
ATOM
BQ
H,N-CH-C-NH-CH-C-OH
BOMBARDMENT
BQ
MASS
90
---* +C$C-NH-CH-C-OH SCHEME
tide chain, resulting in cleavage of the bond adjacent to the newly created charge site. This mechanism will favor both the N-terminus and C-terminus ions. The charge site within a peptide is affected by the nature of the amino acid side chains constituting that peptide. The basic amino acid residues Arg and Lys play a role in controlling the charge site prior to and/or after dissociation of the positively charged protonated molecular ion. The majority of the opioid peptides studied here contains Arg and Lys at or near the carboxylic group of the peptide, suggesting that formation of the positively charged C-tenninus ions via Scheme 1 is aided by the presence of Arg and Lys at the C-terminus of these peptides. This rationale is further supported by the fact that in the positive ion FAB MS of the opioid peptides ME and LE, in which Arg and Lys are absent, the N-terminus ions are equally important, consistent with the observation of Roepstorff et al. (27) in their study of other peptides of a more hydrophobic nature. The role of Arg and Lys in controlling the type of fragmentation has also been discussed by Schafer (23). The sequence ions containing only the single N- or C-terminus amino acid residue are the most abundant ions in the spectra and even more so when the amino acid residue is hydrophobic in nature (vide infra); compare the Z, , Y, , and X1 ions with the remaining C-terminus ions of a particular peptide in Tables 2 and 3. Cleavage of the -CHR-CObond, with few exceptions (e.g., Ai ions), yields A-type N-terminal sequence ions, whereas the corresponding C-terminus sequence ions are formed with or without H transfer (Tables 2 and 3). In other words, both X and X” ions are observed. However, the ions formed by cleavage of the -CHR-CObond are relatively less important in the spectra cornoared
57
SPECTROMETRY
Q
7 0
--* +C,-NH-CH-C-OH
-+ -+
1
to other sequence ions, except for some Atype ions, which are discussed below. The cleavage of the peptide bond is almost always followed by a transfer of a hydrogen atom from the neutral fragment to form a Y”-type C-terminal ion (quatemary ammonium ion). The N-terminal sequence ions, on the other hand, are formed with or without H transfer following the peptide bond cleavage to yield B” and B ions, respectively. These N-terminal sequence ions are normally weak and in some instances are not observed in the spectrum (see Tables 2 and 3). The data of Tables 2 and 3 reveal that the principal process of ion formation in the positive ion FAB MS of the peptides under study is cleavage of the amino-alkyl bonds of the (M + H)+ ion of the peptides accompanied by H transfer from the neutral moieties to the charged species (the N-terminal or the C-terminal) leading to the formation of amino-amide (C”) and amino-carboxyl (Z”) ions, respectively. This observation is consistent with the other FAB MS studies @a), but is in contrast to the results of EI and CI studies of chemically derivatized peptides (22a). In the latter studies, it was shown that the prominent fragmentation process of the peptide molecular ion is the cleavage of the peptide bond. The discussion so far is consistent with the formation of the six known types of amino acid sequence-determining fragment ions when these opioid peptides are subjected to positive ion FAB. However, not all of the ions are formed with equal facility, and in some instances a few of the fragments are even absent (Tables 2 and 3). On the other hand, some fragments are unusually dominant in the mass spectra. For example, the C-terminal ions that contain three or more amino acid residues are intense in the spec-
58
DASS
AND
n-urn of dynorphin A 1-7 (Fig. 1). Likewise, the fragments that terminate with the hydrophobic amino acids Leu, Pro, and Phe residues are of significant abundance in the spectra of the other neuropeptides being considered here (Tables 2 and 3). Although it is not easy to assign a single factor, from these results it appears that, at least to a certain extent, the hydrophobicity of the amino acid side chain influences the fragmentation pattern of the peptides. Recently, Pelzer and DePauw also attributed the increase in the ion fragment intensity to the hydrophobicity of the solute (28). Because of the complex structural nature of the peptide molecules, it is not easy to rationalize the manner in which the hydrophobic residues act in controlling fragmentation of the peptides. However, the following explanation may account, at least partly, for the role of hydrophobic residues. It is well known that charged side chains such as Lys, Arg, Gly, and Asp are usually located on the outer surface of the three-dimensional conformation of a peptide molecule dissolved in a polar solvent, whereas the nonpolar residues are found within the hydrophobic volume. Therefore, the polar side chains are predominantly involved via H bonds in interaction with the surrounding matrix molecules and also participate in the molecular folding of the peptide itself. Consequently, it is logical to rationalize that the
se
--Arg
-
----
e
FIG.
l-7.
1. Positive
ion FAB
mass spectra
of dynorphin
A
DESIDERIO
energy requirements for cleavage of the bonds connected to the polar versus nonpolar residues are different. We believe that this phenomenon could affect the intensity of the various peptide fragments found in the mass spectra. This hypothesis, indeed, is corroborated by the present results (Tables 2 and 3). For instance, the C-terminus ions having more than two amino acid residues found at the site of Lys or Arg are commensurately weaker than the ions that are formed at the sites of more hydrophobic residues such as Leu, Be, Met, Phe, and Tyr in the positive ion FAB mass spectra of the opioids under study (for example, compare 25 ion intensity with those of Z$ and Z5 and Zg ions in the spectra of Dynorphin 1-8; Table 2). The decreasing order of hydrophobicity based on the Bull and Breese index (29) for the amino acid residues present in these peptides is Leu > Phe > Ile > Tyr % Met % Lys > Arg. Other factors such as charge delocalization of the carbonium ion and the size of the fragment ion also influence the fmgmentation yield. For example, cleavage of the bond between Tyr and Gly residues (m/z 136) and between Phe and either a Leu or a Met residue (m/z 397 or 399) yields intense A-type N-terminus ions. Similarly, the Z-type ions (see Table 2 for Zi, Z’; , ZI; , and Z’; ions from dynorphin A fractions 1-7, l-8, 1-9, and 1- 10, respectively, and Zl; and Z$ from /3- and cr-neoendorphins, respectively; see Table 3 for Z; from ME-Lys, Z$ from ME-Lys-Lys, ME-Lys-Arg, and ME-Arg-Phe, and Z? from ME-Arg-Gly-Leu) generated at the site of cleavage between Phe and Gly are always the dominant C-terminus ions. The dominance of these ions in these spectra may result from charge delocalization by the phenyl ring by H-atom migration as depicted in Scheme 2. The effect of fragmentation size, i.e., the number of amino acid residues present in that fragment, is clearly evident when we compare the relative abundance of Z; ions with the Z’; and Zg ions in the spectra of ME-Lys-Lys, ME-Lys-Arg, and ME-ArgPhe (see Table 3).
FAST ATOM
BOMBARDMENT
Considering all these aspects, we suggest that it is not prudent to assign a single specific factor responsible for the formation of a fragment. We feel that the relative abundance of a fragment ion will depend upon a combination of several factors such as hydrophobicity, charge delocalization, and size. In addition to the sequence ions discussed so far, the presence of other ions characteristic of particular amino acid residues are noted. Most important are the immonium ions, which are produced only from the hydrophobic amino acids. For example, ions of significant abundance occur at nominal masses 104 (H*N=CH-CH2--CHz-SCH3) and 120 (H2N=CH-CH2-Ph). In fact, m/z 120 is the most abundant ion found in the spectra of several of these peptides. The corresponding ions characteristic of the basic amino acid residues (Lys and Arg) were either absent or present in very low abundance. The ions at m/z 107, 116, 131, and 147, presumably having structures a, b, c, and d, respectively (Scheme 3), also occurred in significant abundance. The c’ and d’ ions are also likely candidates for the structures of
r!o
I
P
II
270 (-HC-C-NH-&H,
![(A,Z&],
0 R 0 II II HZN-CH-C-NH-CH-C-[(B6Y$],or
Ill
0
298 (H,C-C-NH-CH-C-
the ions found at m/z 131 and 147, respectively. However, the exact nature of the distonic ions b-d is not known and is the subject of further investigation. The intense signals due to the aforementioned ions are further evidence of the influence of hydrophobicity of the amino acid side chain. The occurrence of these ions in the spectra aid in confirming the presence of the suspected, indicated amino acid residues in the peptides and will probably provide clues to their presence in the spectra of unknown peptides. The ions arising from the loss of the side chain of specific amino acids are also seen at the high mass end of the spectrum and can be helpful in identifying the presence of an amino acid residue. The losses of 15 (CH,), 44 (CO&, 45 (HCOOH), 61 (CH2-S-CH3), and 107 (CH,-Ph-OH) mass units are often noticed (see Fig. 1). However, some ions of undefined origin are also present in the positive ion FAB spectra of the opioid peptides studied here. For example, the ions of ambiguous character (with the probable structure shown in parentheses) seen in the spectra of dynorphin A 1-7 are m/z 229 (A, + G + H, where G = glycerol), 0
R
Q
-H&NH-&H-&-NH,
L
R
59
MASS SPECTROMETRY
[(cl,Z,),],
0 L 0 II II -C-NH-CH-C-NH-CH2
P
[(&X&l),
0
P
II
[(B,Z$] 0 or
0
II
F
L
II
0
R
II
0
II
-C-NH-CH-C-NH-AH-C-H 0
II
L
385 (C, + G), 459 (-C-NH-CH-C-NH-CH-C-NH-CH-C-NH-
0
II
R
[(BbX,),]),
0
II
[(C,X,),]),
5 17 (B4 + G), and 533 (C, + G). However, further work is needed to unambiguously assign structures to these ions. Finally, it was also observed that fragment
ion intensities increase with the time of FAB irradiation, as shown in Fig. 2 for dynorphin
60
DASS AND DESIDERIO 140. 120lOO-
SCHEME
2
A 1-7. The increase in fragmentation is attributed to an increase in the internal energy of the molecular ion caused by changes in the relative\ concentration of the peptides, because with time the solvent is depleted from the probe tip. As a result, fewer solvent molecules are associated with the desorbed clusters, a phenomenon that causes the molecular ions to be preferentially energy enriched because now less energy is dissipated in desolvating the remaining decreased number of solvent molecules. This rationale was confirmed by loading different concentrations of dynorphin A l-7 on the probe tip (see Fig. 3); increased fragmentation was observed with
2%
0I I ;jH +
+Ii)+-CHJ 20-
I , 6
1
10
15
FIG. 2. Increase in the fragmentation of (M + H)+ of dynorphin A l-7 with respect to the time of irradiation.
the more concentrated solution because clusters with fewer solvent molecules were formed. The increase in the fragment ion intensity with concentration was also observed by other workers (30). Negative ion FAB mass spectra. To gain additional structural information and possibly to increase the molecular specificity of quantitative MS measurements, negative ion FAB mass spectra of these opioid peptides were acquired. Because the structural features that stabilize a negative charge are not usually the same as those that stabilize a positive charge (see below), the negative ion
a
180
0 Hi-CH-Cd
&-t-tiH
160 3
LH I *
{H2 140
-
F+ $ a3
c-+ 0 H-&JH Y
b
0.2
:I
0.4
0.6
0.8
1 .o
1.2
Cont. (nM) SCHEME
3
FIG. 3. Increase in the fragment ions intensities with concentration of dynorphin A 1-7.
FAST
ATOM
BOMBARDMENT
MASS
TABLE AMINO
ACID SEQUENCE-DETERMINING
61
SPECTROMETRY
4
IONS IN THE NEGATIVE
ION FAB
SPECTRA OF GROUP
A WITIDES
LE
364 “X Dynorphin A l-7
135 163 179
'A 'B c
70 10 7
191 "A 219 "6 236 C
11 4 8
249 277 293 427 442 470
'A 'B c Z Y x
277 'B 293 c 426 'Z 441 'Y 468 "X 249 A 293c4644oc 426 'Z 441 'Y 468 "X
Dynorphin A 1-a
Dynorphin A l-10
417 "X
4 I
1 3 12 2 3 1
395 "A 425 B 441 c 575 z 590 Y' 617 x
1 1 8 4 3 1
6 14 4 4 2 18
395 "A 425 E 440 c 539 'Z 554 'Y 581 "X 395 -A 425 B
14 11 9
582 598 626
c - Relative abundanceofthe
'A B c Z Y 'X -
2 2538B a 553 c 5 687 z 12 702 Y 2 730 x 9 508 “A IO 538 B 20553c 11 695 Z 4 710 'Y 4 -
2 665'A 1 694 B 8709C 368EZ 3704y' 1 732 x' 3 7 709 c 5 744 z 3 759 Y 1 787 x 8 665 'A 7 694 B 28709C 7 a43 z 7 858 Y -
'B c Z Y x
9 33 IO 8 7
395 "A 425 B 440 c 523 'Z 538 'Y 565 "Z
2 2 IO 6 5 2
509 'A 538 B 553 c 679 'Z 694 'Y 721 "X
3 665'A 5694B 15 709 6 792 2 3
277 293 389 405 433
'B C 'Z Y x
38 38 9 5 5
397 425 440 545 5EQ 587
A B C 'Z 'Y x
12 4 17 8 4 2
395 -A 425 B 440 c 532 'Y 559 "X
30 2 7
508 "A 538 E 554 c' 658 'Z 674 Y 701 'X 508 "A 553 c 673 'Z 715 "X
6 5 26 6 4 3 2
'A 240 'B 12 389 'Z IO 431 "X 2
d-Neo endorphin
'Z Y X
509 538 554 632 647 673
277 293 368 383 411
249 277
a. b - see Tablell,
3
sequence
ions considemg
2 1
3 1 3 4 4 1
866 (M-H)-
4a65c 6 801 Z 4816Y la44X 4 5 13a65c 10 !3N z 4915Y -
c 'Z -
3 3 6 a65 C 39402 955 Y 983 x
B C 'Z Y 'x 'A 7 709 c 2 786 'Z 802 Y 2 -
2 792 "A 3 a22 B 12 a37 C 5 862 ‘Z 2 078 Y 2 5 822 B 8 a37 c 4 934 z 2 950 y' 977 x
665'A 694 709 805 821 848 665
2 4 4 2
4 12 6
4 6 4 1 3 4 7 6 4 3 9 4 6 2
(M-H)- asthe base peak
spectra may provide information complementary to that obtained from the positive ion mode. Tables 4 and 5 list the sequence ions from the negative ion FAB mass spectra of Groups A and B peptides, respectively. The negative ion mass spectra of dynorphin A 1-7 is shown in Fig. 4 and may be compared to the corresponding positive ion spectra in Fig. 1. By comparing the negative ion data in Tables 4 and 5 with the positive ion data listed in Tables 2 and 3, we find that the mechanism of fragmentation of the quasimolecular (M - H)- anions of the peptides generated by negative FAB ionization is comparable to the pattern of the (M + H)+
ions of these peptides. However, by comparing the two ionization modes, some striking differences are found in the data. The most significant difference is seen in the relative fragment ion yields. In the negative ion spectra, the bulk of the total ion current consists of the quasimolecular (M - H)- anion. This difference suggests that (M - H)- anions formed by the FAB desorption process are energetically more stable than the corresponding (M + H)+ cations, consistent with the general observation that the negative ions formed by electron capture are of low internal energy (3 1). Another important difference is that the N-terminal ions, which were
62
DASS
AND
DESIDERIO
TABLE AMINO
5
ACID SEQUENCE-DETERMINING IONS IN THE NEGATIVE OF GROUP B PEPTIDES
ION FAB M.us
SPECTRA
1
Amino AC
1 a
ME
ME-Lys
ME-Lys-Lys
ME-Lyo-Arg
ME-Arg-Phe
b
163 ‘6 7 179c 5 146 Y 12 175 ‘X 5 __135 ‘A 15 163 ‘6 7 130 z 15 145 Y 15 173x 7 135 ‘A 501 163 ‘B 113 13Oz 38 145Y 46 173 X 24 135 ‘A 163 ‘B 173 Y -
2 a
b “A B c z Y X “A
E 4 4 4 7 7 3 7
23s; 260 ‘Z 276 Y 303 ‘X 221 6 236 C 273 Y 301 x
a 6 8 3
T 191
221 236 280 295 323 191
13 19
191 “A 221 B 236 c
20
301 327 191 221 236 303 319 34.8 191
135 ‘A 29 163 ‘B 73 151 z” 130 164 Y 108 191 ‘X 17
135 ‘A 163 ‘6 179C 115 z 13OY 158x a. b. c. - See Talblc? IV
ME-ArgGly-LSIJ
c
16 9 6 18 60 4
236 172 187 213
572 (M-H)277 ‘B 293c 4082 424 Y 451 x
21 26 75 8
293 389 405
C z Y
395 “A 5 1344Oc 4465z 446OY 2 1 506 “X 397 A 425 B 27 440 C 6536Z 4 551 Y
13 14 11
Y 4 “X 3 “A 17 6 190 C 19 “Z 12 ‘Y 9 “X 13 “A 27 C 6 2 21 Y 35 “X 18
ion FAB mass spectra
5 4 4 1 9 5 20 7 16
554 “B 572 c’ 522 z 537 Y 563 “X 554 “El 571 c 593 z @Y3Y 63’X 554 “6 572 c’ 620 ‘Z 636Y 663’X 554 “6 571 c 6402 655Y 661 “X 530
A”
572 ;’ 607z 622 y’ 648,x
rare in the positive ion spectra, are now quite conspicuous in the negative ion spectra (compare C-type ions in Tables 4 and 5).
FIG. 4. Negative l-7.
2
of dynorphin
A
8 12 2 4 2
700 (M-H)-
11 12 7 6 3 13 6 5 5 2
677 ‘Z 693Y 720 ‘X
7 5 2
(M-H)-
26 9 7 9 3 2 4 5 7 2
898 (M-H)-
This observation is explained by the fact that one of the major processes of generation of negatively charged sequence ions of the opioid peptides of Table 1 is the initial charge localization at the terminal carboxy group yielding an (M - H)- anion by donation of the proton from the H3N+ terminal group and subsequent sequential migration of the negative charge in the peptide chain from the carboxyl group in a process that is the reverse of the formation of the positive ions discussed earlier in Scheme 1. Furthermore, contrary to the positive ion data, the Z-type negative ions are not always the dominant ions, and in some cases, cleavage of the peptide bond also gains importance in forming the Y-type C-terminal sequence ions (Tables 4 and 5). The cleavage of
FAST
ATOM
BOMBARDMENT
the -CHR-CObond produces two types of carbonyl-containing fragment ions (X and “X).
Formation of Adduct Ions Previous studies have reported the occurrence of ionic adducts between glycerol and solute molecules containing primary and secondary amines (32). A similar phenomenon was observed in the current study of these opioid peptides. The positive ion spectra include several ions having mass numbers greater than the protonated molecular ion of the peptides. Most prominent is the addition of I2 mass units. Other adduct ions corresponded to the addition to the (M + H)+ ion of 24,29,44, 54,66, 74, and 104 mass units. On the other hand, the formation of adduct ions in the negative ionization mode is not as facile. However, an addition of 98 mass units to the (M - H)- anion was frequently observed. A detailed mechanistic study concerning the adduct formation, presumably between the glycerol and these opioid peptides, is underway and will be reported elsewhere.
SequenceDetermination In the course of describing peptide fragmentation processes, with our specific goal of quantification, some general observations on features relating to amino acid sequence determination were made. For sequence determination of a peptide, identification of the terminal amino acid residues is quite important. In the case of the peptides studied here, the N-terminal amino acid Tyr is readily recognized by considering the intense C-terminal sequence ions of the (n - 1)th amino acid residue, which is formed by the loss of the Tyr residue from the peptide. Another piece of evidence for the presence of Tyr at the N-terminus is the presence of an ion of m/z 136 (A,-type ion). On the other hand, recognition of the Cterminal amino acid is somewhat ambiguous
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when the C-terminal sequence ions such as X1, Y, , and Z1 are considered. This limitation is due to the excessive chemical noise at the lower end of the FAB spectra that arises due to the matrix ions and to the fragment ions from the peptide itself. Furthermore, it is sometimes not possible to unambiguously assign a signal to the specific amino acid residue (33). For example, the ion at m/z 13 1 in the spectra of ME-Lys and ME-Arg-GlyLeu can be due to either Lys (Z’) and Leu (Y’) residues, respectively, or Met (structure b). However, these limitations can be overcome effectively by the careful choice of the solvent matrix and by searching for N-terminal sequence ions formed due to the (n - 1)th amino acid residue. These ions are generally of significant abundance in the FAB MS of the opioid peptides studied here. The remaining amino acid residues can be recognized by considering the most intense C-terminal ion series. Figure 1 demonstrates the efficacy of FAB positive ion mass spectrometry in sequencing these known opioid peptides. The data reported in this study were obtained using l-2 nmol of the peptides. However, a better approach for sequencing the peptides reported here is to use negative ion FAB MS. This polarity advantage arises because (a) the ions belonging to the C-type N-terminal sequence ion series are of significant abundance in the negative ion spectra; (b) compared to the Z-type C-terminal ion series, the Y-type series is equally dominant in the negative ion spectra and, therefore, may be used for sequencing the peptides; and (c) in comparison to positive ion spectra, the negative ion spectra are relatively clean at the upper mass end. Therefore, in the negative ion mode, it is easier to recognize a specific sequence ion and it is possible to use the N-terminus ion series to substantiate the conclusion drawn based on the C-terminus ion series, thereby lending considerable confidence to the structure elucidation of an unknown peptide. The example of the mass spectrum of dynorphin A l-7
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(Fig. 4) demonstrates the usefulness of negative ion FAB MS for sequencing peptides.
Detection Limits Although we normally quantify a peptide in a biological extract by MS/MS selected reaction monitoring SRM (19,34), on occasion we obtain a full mass spectrum of an HPLC fraction to survey for the possible presence of a peptide. Toward that latter end, we also investigated the minimum amount of a peptide needed to obtain the meaningful sequence data from a full mass spectrum. This experiment was accomplished by acquiring the spectra of the pentapeptides LE and ME in the positive and negative ionization modes. It was noticed that, in the negative ion mode, the (M - H)- anion of LE could be seen in a single scan with as little as 10 ng (18 pmol) of that peptide. However, the interpretable amino acid sequence-determining data could only be obtained with amounts greater than 25 ng (ca. 50 pmol). The corresponding detection limits in the positive ionization mode were 20 and 40 ng, respectively. These detection limits may be considerably improved with signal averaging techniques. ME, on the other hand, cannot be detected with the same ease. The lowest amounts necessary to obtain useful amino acid sequence data of ME in the negative and positive ion modes were 100 and 200 ng, respectively.
Mixture Analysis In order to test the utility of FAB MS as a tool for the characterization of a specific peptide in the presence of other peptides, which is a situation that could be experienced with biologic extracts even, in our experience, after HPLC separation, a synthetic mixture of four peptides consisting of 120, 350, 150, and 150 pmol of LE, ME, MEArg-Phe, and ME-Arg-Gly-Leu, respectively, was prepared. The choice of this mixture is important from a practical point of
view because these opioids have a common precursor, the preproenkephalin A (35) and have been found together in body tissues (36). It was observed that the (M + H)+ and sequence ion signals due to ME, ME-ArgPhe, and ME-Arg-Gly-Leu were weak compared to those signals obtained when the peptides were analyzed individually; these data confirm the well-known signal suppression phenomenon of peptides when present in a mixture during FAB MS analysis (37). Because we had prepared a mixture of known peptides, we could easily confirm the sequence of each of the four components, even though the individual amino acid sequence signals were not very strong. However, this mutual suppression of ionization may be a severe limitation of FAB when applied to the sequencing of a mixture of unknown peptides, even following HPLC purification, which does not necessarily always separate all peptides in a biologic extract. The positive ion FAB produced even more inferior data. CONCLUSIONS
In this study, positive and negative ion FAB MS methods were evaluated for rationalization of peptide bond fragmentation of endogenous opiates of M, up to approximately 1250 amu. The fragmentation behavior of the opioid peptides studied here is in accordance with the known fragmentation characteristics of other studied peptides (8,13b,23,24). However, the patterns of the relative abundance of various types of ion series differ. These differences are related to the hydrophobicity of the amino acid residues and perhaps to a certain extent to the conformational features of these peptides. FAB ionization produces abundant (M + H)+ and (M - H)- ions, from which molecular weight information is readily deduced. Sequence information is obtained by considering the intense Z-type C-terminus ion series in the positive ion mode. For sub-
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stantiating these data, the use of C-type Nterminus and Y- and Z-type C-terminus ion series in the negative ion mode is also recommended, especially when characterizing an unknown compound-if sufficient material is available. It is necessary to understand the behavior of each ion in the mass spectrum of each peptide before quantification of that peptide can be undertaken. Toward that goal of quantification of specific peptides, this manuscript has provided data rationalizing the fragmentation of several neuropeptides and the ion genesis of fragment ions. ACKNOWLEDGMENTS The authors gratefully acknowledge the financial assistance of NIH (GM 26666) and the typing assistance of Dianne Cubbins.
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