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A comparison of keV atom bombardment mass spectra of peptides obtained with a two-sector mass spectrometer with those from a four-sector tandem mass spectrometer
International Journal of Mass Spectrometry and Zon Processes, 78 (1987) 213-228 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
213
A COMPARISON OF keV ATOM BOMBARDMENT MASS SPECTRA OF PEJYIIDESOBTAINED WITH A TWO-SECTOR MASS SPECTROMETER WITH THOSE FROM A FOUR-SECTOR TANDEM MASS SPEC~OME~R
STEPHEN A. MARTIN
* and KLAUS BIEMANN
**
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 (U.S.A.) (Received 26 November 1987)
ABSTRACT The information content of the conventional fast (keV) atom bombardment (FAB) mass spectra of six peptides, substance P, des-Arg’substance P, Fibrinop~tide A, desArgy6fibrinopeptide A, oxidized somatostatin, and reduced somatostatin, ranging in molecular weight from 1190.6 to 1638.7 is compared with that of the corresponding spectra measured by a high-performance (four-sector) tandem mass spectrometer after collision-induced decomposition (CID). The CID spectra exhibit distinct peaks corresponding to extended series of sequence characteristic ions free of the chemical background and contribution of the matrix which makes the weak fragment ion signals in conventional FAB mass spectra so difficult to interpret. The presence of arginine, a strongly. basic amino acid, in either the N-terminal or C-terminal position is reflected in the dominance of characteristic N-terminal and C-terminal fragment ions. The resolving power of the first of the two mass spectrometers is clearly sufficient to permit the recording of separate CID spectra of oxidized and reduced somatostatin in a mixture.
INTRODUCTION
Mass spectral data of relatively large, polar, thermally labile, non-volatile compounds can now be obtained by sputtering them from a solution in a polar matrix of low vapor pressure, such as glycerol. The sputte~ng agent is a beam of neutral atoms (argon or xenon) in the case of fast (keV) atom bombardment (FAB) ionization [l] or ions (Cs’) in a variant thereof, referred to as “liquid SIMS” (secondary ion mass spectrometry) [2]. While * On leave of absence from the Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC 29425, U.S.A. * * To whom correspondence should be addressed. 0168-1176/87/$03.50
Q 1987 Elsevier Science Publishers B.V.
214
these “soft ionization” techniques generate abundant protonated molecular ions, (M + H)+, or their cationated analogs, such as (M + Na)+, these ions are very stable and do not fragment extensively. Therefore, the resulting spectra exhibit little structural information. This can be remedied, however, by collisional-induced decomposition (CID) of the (M + H)+ ion (precursor ion) while passing through a collision cell filled with a neutral gas, such as helium. The resulting product ions, when mass analyzed in a second spectrometer, provide a clue to the structure of the precursor. Since this method requires two mass analyzers, it is known as tandem mass spectrometry (MS/MS). The principles and some applications of MS/MS have been summarized in a recent book [3]. One area of current interest in our laboratory is the determination of the amino acid sequence of peptides, ranging in length from two to at least twenty-five amino acids, derived by chemical or enzymatic cleavage of proteins for the purpose of determining their primary structure. The molecular weights of these peptides may be as high as 3000 u but the fragment ions are distributed over the entire mass range from m/z - 60 to the precursor ion and their mass has to be determined to better than k 0.5 u to allow the unambiguous assignment of the sequence. The performance requirements with respect to mass range, resolution, and sensitivity of the mass spectrometer are, therefore, very stringent and far exceed those of the instruments previously available [3]. For this reason, we use a high-performance tandem mass spectrometer (JEOL HXllO/HXllO) which consists of two consecutive double-focussing mass spectrometers arranged in the optimal C-configuration of an E,B,E,B, system. This instrument has been described previously [4-61. In this paper, we discuss the information which can be
TABLE I Structure
and molecular
weight of the peptides
used in this work
Substance P(l), M, 1346.73 Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH, des-Arg’ Substance P(2), M, 1190.63 Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH, Fibrinopeptide A (human)(3), M, 1535.68 Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg A(4), M, 1379.58 des-Arg ” Fibrinopeptide Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val Somatostatin, oxidized(S), M, 1636.72 Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys Somatostatin, reduced(6), M, 1638.73 Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys
215
derived from the product ion spectra obtained by CID of the precursor ion in such a ~~-perfo~an~ tandem mass spectrometer and compare them with the more limited information of the conventional FAB mass spectra. Of the large number of CID spectra of peptides which we have determined over a period of more than one year, we selected the set listed in Table 1. The conventional FAB spectra of 1 and 3 have been reported previously [7]. EXPERIMENTAL
A JEOL HXllO/HXllO high-performance tandem mass spectrometer of E,B,E,B, geometry with a mass range of 14500 u at 10 kV accelerating potential was used in these experiments f4-6]. The samples were dissolved in glycerol acidified with acetic acid and 0.5 ~1, containing approximately l-2 nmol of peptide, was applied to a stainless steel probe tip. The probe was inserted into the ion source via a vacuum lock and the sample was bombarded with a beam of xenon atoms from a JEOL neutral atom gun (6 kV, 2 A cathode current, 10 mA emission). The desorbed ions were accelerated, energy and mass selected, and detected after the first mass spectrometer, MS-l, using the combination of an off-axis post-acceleration device ( k 20 kV) and a 16-stage secondary electron multiplier. The post-acceleration unit is normally operated at - 18 kV in the positive ion mode. In the MS/MS mode, the precursor ion is selected in MS-l by setting the resolution such as to transmit only the “‘C component of the (M -t H) + isotope cluster into the collision region. Helium is introduced into the collision cell at such a pressure that the precursor ion is attenuated to lo-30% of its original abundance as measured by comparison of the precursor ion signal before and after introduction of helium and recorded at the detector located after B, of MS-2. Wit~n this attenuation range, the relative abundance distribution of the product ions does not change significantly. The resolution of MS-2 for the B/E scans reported was 1 : 1000 for peptides 1 - 4 and 1: 500 for peptide 5 and 6. It should be noted that lower than unit resolution suffices for reliable mass assignment because the 13C contributions have been eliminated by MS-l. An ion source almost identical to that in MS-1 is located in the interface region of the JEOL HXllO/HXllO and is used to calibrate MS-Z for normal and B/E scans. This ion source contains an integrated collision cell, which was employed in these experiments. A mixture of LiI, NaI, KI, RbI, and Csl was used to calibrate MS-2 from 0 to 3600 for the recording of the product ion spectra. A single ca~bration file is applicable to all precursor ions of any mass lower than the upper limit of the calibration file. The B/E scans are acquired in the profile mode (raw data) scanning from low mass to the precursor ion at scan rates which range from 30 s to 3 min for m/z
216 TABLE
2
Mass assignment of fragment A (human) (3) (see Fig. 4).
ions observed
A
upon CID of the (M + H)+ ion of fibrinopeptide
Ion type
m/z
Ys
Yll
758.58 784.52 905.69 931.47 1020.84 1046.52 1077.58
+0.16 +0.13 + 0.21 + $01 + 0.33 + 0.03 + 0.05
x11
1103.52
+0.01
+0.01
Yl2
1206.78
+0.21
445.13
-0.12
Xl2
1232.64
+0.09
x5
471.14
-0.09
Y13
1263.80
+0.20
Y6
574.24
-0.05
X13
1289.77
+0.19
X6
600.03
-0.24
Y14
1350.81
+0.18
Y7
645.34
+ 0.01
X14
1377.07
+0.46
x7
671.35
+0.04
Ion type
m/z
obs.
V L E F Y1 Y2 Y3
71.59 85.70 101.74 119.73 174.91 274.10 331.07
- 0.49 -0.39 - 0.31 - 0.35 - 0.21 - 0.09 -0.14
Y4
388.11
-0.12
x4
414.22
Y5
‘8
Y9 x9
YlO x10
obs.
A
30-2000. Individual or summed scans are then processed in the data system using the peak detection software. The accuracy of the mass assignments is always k 0.3 u or better. As an example, the m/z values determined by the data system for some of the product ions obtained upon CID of the (M + H)+ ion (m/z 1535.68) of fibrinopeptide A (3) are listed in Table 2 along with their deviation from the theoretical values. The peptides used in these experiments were purchased from Bachem [16], checked for homogeneity by reversed-phase HPLC, and used without further purification. Partial reduction of somatostatin was carried out by adding 0.2 ~1 of dithiothreitol/dithioerythritol to the probe tip which contained the sample in a glycerol matrix. The sample was re-inserted into the mass spectrometer and the reduced protonated molecular ion was selected for the B/E scan.
RESULTS
AND DISCUSSION
The fragmentation processes generally observed in FAB mass spectra of peptides are illustrated in Scheme 1, which represents a summary of proposals put forward specifically or implicitly in the early 1980s. chiefly by Barber et al. [8,9], Williams et al. [7,10], and Morris et al. [II] (for a review see ref. 12). In addition, we have observed, in many CID spectra, fragmentation leading to ions denoted as the w, series. The FAB spectrum of the
217
$4
Y+ $3
Ps
NH-CH-CO-NE-CR-CO-NH-CH-COoE ii/ Y3
R
R2
R4
R3
RS
Ha-VIA-CO-NB-~A-CO-~-OH-CO-MI-AH-CO-MI-~A-COOB +
A transfer
\,
R
R
R2
E$N-&-CO-NE-&R-CrO+
R2
E&t&-co-NR-&I-co-ki, c2
b2
1 F1
5
E$wi-co-ili=cH a2
Rx
k2
R&&i immonium fon (labelled
F
H$?-CH-CO-NE-CH-co+ internal with single
letter
cleavage
ion
code)
Scheme 1. Schematic presentation of the major fragmentations observed in normal positive ion FAB mass spectra and/or in collision spectra. Cleavage of the bonds between amino acids 2 and 3 of a pen~peptide is used as a general example. The notation is a variant of that proposed by Roepstorff and Fohhnan 1171. a, Protonation at 1st or 2nd amino acid. b, Protonation at 4th or 5th ammo acid.
218
*
I *
* I
( M+~H) 2+
*
a,-57
X5.0
(b)
,/
u4
a5
a,-57
x4.0
~ ,
(a)
x2.0
7
a:,
Fig. 1. Comparison (top two panels low mass, lower two panels high mass) of (a) the conventional FAB mass spectrum of the undecapeptide substance P (1) and (b) the product ion spectrum obtained upon CID of the (M+H)+ ion of this peptide. For notation, see Scheme 1. Peaks due to the matrix in (a) are indicated by an asterisk.
219
undecapeptide substance P (1) of M, = 1346.7 is shown in Fig. l(a). The lower mass range (m/z < 500) is dominated by the cluster ions of glycerol, the matrix. At higher mass, N-terminal ions of type c, and C-terminal ions of type yn are observable above the background ions (usually referred to as chemical noise). In contrast, the CID spectrum of the product ions formed upon collision of the precursor (M + H)+ with He in the collision cell consists mainly of a continuous series of ions of the type a, [Fig. l(b)]. They are distinct and abundant (compared with background) single peaks because they are derived from the ‘*C component of the (M + H)+ isotope cluster by transmitting only m/z 1347.7 + 0.5 from MS-1 to MS-2. The mass difference between consecutive peaks of this ion series clearly defines the sequence from the second to the tenth amino acid in this peptide. The eleventh amino acid is recognized to be methionine (in the form of the carboxy-terminal amide) by the difference in mass (176 u) between the a,, and (M + H)+ ion. The only ambiguity that remains is at the N-terminus, which could be Arg-Pro or Pro-Arg. This question can be easily resolved upon removal of the N-terminal amino acid by a one-step Edman degradation involving treatment of the peptide with phenylisothiocyanate (PITC) resulting in the formation of the decapeptide 2. The difference (157.1 u) in molecular weight of this product (M, = 1190.6) [Fig. 2(a)] and the original peptide reveals the nature of the N-terminal amino acid, in this case arginine. However, the removal of this strongly basic amino acid dramatically changes the relative abundance and distribution of the sequence ions in the CID spectrum [Fig. 2(b)]. Whereas the presence of arginine in 1 favors the appearance of a continuous series of ions of type a,, after its removal the acyl ions b, become equally dominant. The only C-terminal ion of note is ys, which represents the fragment formed by cleavage of the N-terminal peptide bond of the proline in position 4. Its predominance may be the reason for the relatively low abundance of the N-terminal fragments, especially b,, which involve cleavage of the C-terminal amide bond of this proline. Another consequence of the proline is the appearance of an internal fragment ion, PQQ, the formation of which also involves cleavage of the N-terminal amide bond of Pro. Such internal fragments are often present as a series of extended length. The most significant difference between the conventional FAB spectrum [Fig. 2(a)] and the CID spectrum [Fig. 2(b)] is the appearance of one or two continuous ion series in the latter, whereas the former exhibits some fragments due to cleavage at certain peptide bonds, but often without a general trend. While this allows for a correlation of peaks in the conventional FAB spectra with known structures of peptides, it is not very helpful for the unambiguous determination of the sequence of peptides of unknown structure. Another effect of the absence of N-terminal a&nine is the much clearer
(a)
T,‘I
700
aoc
900
1000
1100
1200
Fig. 2. Comparison (top two panels low mass, lower two panels high mass) of conventional FAB mass spectrum of the decapeptide (2) obtained by removal N-terminal arginine from 1 and (b) the product ion spectrum obtained upon CID (M + H)+ ion of this peptide. For notation, see Scheme 1. Peaks due to the matrix in indicated by an asterisk.
(a) of of (a)
the the the are
221
m/z
60
70
Fig. 3. Region from m/z
80
90
100
110
120
130
50 to m/z 125 of the CID spectrum shown in Fig. 2(b).
appearance of the immonium ions indicative of the presence (but independent of their position) of certain amino acids. As an illustration, the lower mass range (m/z 50-125) of Fig. 2(b) is shown expanded in Fig. 3, which reveals the presence of Pro, Lys, Leu or Ile, Gln, Met, and Phe. Although the immonium ions of Gln and Lys have the same nominal mass (m/z lOl), we have made the observation that only the latter preferentially cyclizes by elimination of NH, to form m/z 84. Finally, ions of the a,, series that have lost 42 or 57 u indicated in Figs. 1 and 2 need to be mentioned. They are due to loss of part of the side chain of leucine and glutamic acid, respectively, (with transfer of a hydrogen) whenever this amino acid represents the C-terminal position of an ion of type a,, [13]. This fragmentation permits the differentiation of the isomers leucine and isoleucine (Ile gives rise to an a, - 28 ion). Asparagine, glutamine, aspartic acid, and glutamic acid behave in a similar way (loss of 43, 57, 44, and 58 u from the a, ion). These fragment ions are absent in the conventional FAB spectra, which thus do not allow these important differentiations to be made. At the high mass end of the CID spectra, ions due to the loss of the side chain of some of the amino acids are discernible and these are labeled with the single letter code of the corresponding amino acid, preceded by a minus sign. While not as distinct as the immonium ions at low mass, they are also helpful indicators of the presence of certain amino acids. The influence of the presence of a strongly bzsic amino acid, such as arginine, at the C-terminus of a peptide is also noticeable. As an example of such a case, the mass spectra (Fig. 4) of fibrinopeptide A (human) (3) are compared with those of the analog 4, lacking the C-terminal arginine (Fig. 5). The much higher information content of the CID spectra is again evident
I
x3.2 ',-
:
(a)-
1’
I
i I f
“>
) ?
q I_! 82
I,
,,,,
1 1L
‘_
,>
_
! .‘I‘L
I 7-t_
121,
Fig. 4. Comparison (top two panels low mass, lower two panels high mass) of (a) the conventional FAB mass spectrum of fibrinopeptide A (human) (3) and (b) the product ion spectrum obtained upon CID of the (M+ H)+ ion of this peptide. For notation, see Scheme 1. Peaks due to the matrix in (a) are indicated by an asterisk.
223
t
(a)
b,
(b) YS
b,
GEGD
Y7
SGEGD
700
600
500
x 1.5
(a)
YS
m/z
800
900
1000
1100
12bo
1300
? d b; -L
-L
b,
-D ! I
YlO
m/z
800
900
LA
1000
,100
1200
1300
-!
tcIO
Fig. 5. Comparison (top two panels low mass, lower two panels high mass) of (a) the conventional FAB mass spectrum of des-Arg 16-fibrinopeptide A (4) and (b) the product ion spectrum obtained upon CID of the (M+H)+ ion of this peptide. For notation, see Scheme 1. Peaks due to the matrix in (a) are indicated by an asterisk.
224
from Fig. 4, which compares the conventional FAB spectrum of 3 with its CID spectrum. Although, in Fig. 4(a), the fragment ions at higher mass can be correlated with the structure as indicated by the labeling of the corresponding peaks, which are mainly due to the C-terminal ions of the w,,, y,, and z, series, the lower mass range is dominated by matrix ions and one needs to compare carefully the spectrum with that of glycerol alone to recognize the peaks due to the peptide. Even that becomes impossible below m/z - 300, where those N- and C-terminal ions would be found that are indicative of the first and last two or three amino acids. In contrast, the CID spectrum [Fig. 4(b)] very distinctly exhibits a complete y,, series, with the exception of the last one (y,,), and other C-terminal ions, chiefly of the type x, and w,,. That the dominance of these is due to the strongly basic arginine at the C-terminus is demonstrated by the CID spectrum of peptide 4 [Fig. 5(b)]. In this case, the most complete series are the N-terminal ions, b,-bi5. The low abundance of b, is due to the fact that it requires cleavage of the amide bond of glycine which sometimes appears to be relatively unfavorable. In the normal FAB spectrum of 4, the b, series is of minor significance and much less extensive [b,_,, Fig. 5(a)]. As an illustration of the peaks due to the loss from the (M + H)+ ion of the side chains of various amino acids mentioned earlier and evident in Figs. l(b), 2(b), 4(b), and 5(b), the upper end of Fig. 5(b) is expanded in Fig. 6. For a number of reasons, cystine-containing peptides fragment poorly, even after collisional activation. In part, this may be so because a -S-Sbond converts a linear peptide to a cyclic one and two bonds have to be
_E”
b-15
(M+H)+
Fig. 6. Expansion of the upper end of Fig. 5(b) covering the mass region of peaks due to the loss of the side chains of various amino acids (labelled with single letter code).
225
m nA.nn
h
A
100
200
300
400
500
600
700
800
X30
(M+H)
m/z
900
1000
1100
1200
1300
Fig. 7. Conventional FAB spectrum of oxidized somatostatin indicated by an asterisk.
1400
1500
1600
(5). Peaks due to the matrix are
cleaved to produce fragments from the cyclic portion of the peptide. The normal FAB spectrum of the oxidized form of somatostatin (5), shown in Fig. 7 exhibits almost no fragmentation. When the 12C component of the (M + H)+ ion of m/z 1637.7 [Fig. 8(a)] was transmitted by MS-l into the collision region and the product ions analyzed by MS-2, very few peaks were observed [Fig. 9(a)], in contrast to the CID spectra shown in Figs. l(b), 2(b), 4(b), and 5(b). Treatment of the glycerol solution used in this experiment with dithiothreitol/dithioerythritol converted about half of the material to the reduced form 6, as evidenced by the molecular ion pattern before and after the reduction [Fig. 8(b)]. When the ion of m/z 1639.7 was transmitted by MS-l, a much more detailed CID spectrum corresponding to the reduced linear peptide was observed [Fig. 9(b)] which is dominated by N-terminal fragments, particularly the b, series. Although the precursor ion also contains some of the i3C2 component of (M + H)+ of the oxidized form 5, it
226
I
r.
&+H);
;
162:
(M,.,+H)
(a)
1743
hg’
1 b53
rr ;
'635
'nLiJ
+
-~-F
1045
(b)
1 t 5,3
Fig. 8. (a) Molecular ion region of the sample of somatostatin used to generate the spectra shown in Figs. 7 and 9(a). (b) After partial reduction of the disulfide bond to generate the spectrum shown in Figure 9(b).
contributes little to the product ion spectrum because it undergoes very little fragmentation (see above). In general, this lack of fragmentation of peptides which are cyclic by virtue of the disulfide bond is not a problem in the sequencing of peptides produced by proteolytic digestion of a protein because, in that situation, cystine is always first reduced to cysteine and then converted to a stable thioether (by reaction with iodoacetic acid or iodoacetamide). The situation is different when one needs to determine the location of -S-Sbridges which requires first that this bond is kept intact and then reduced [14,15]. However, this question always arises after the primary structure of the reduced polypeptide has been established and the amino acid sequence itself is not the issue.
CONCLUSION
The data discussed in this paper demonstrate that the CID spectra of (M + H)+ ions of peptides not only exhibit much more distinct fragment ions than the normal FAB spectrum but also that the predominant fragmentations may be quite different. The CID spectra contain more complete series of a few sequence ion types, whereas the normal FAB spectra often exhibit more sporadic fragment ions of many different ion types. The latter may be an artifact of the general appearance of FAB spectra which are characterized by a periodicity of peak clusters that may accidentally fall at the mass of a possible sequence ion. Comparison of the two types of spectra clearly indicates that the CID spectra permit the sequencing of a peptide with much greater ease and confidence than the normal FAB spectra.
221
W
100
NFF
200
300
400
500
600
700
800
(b)
T/Z IO0
200
300
400
500
600
700
800
m/z
1000
1100
1200
1300
1400
1500
1600
1000
1100
1200
1300
1400
1500
1600
900
X 2.5
m/z
900
Fig. 9. CID spectra of (a) the oxidized form of somatostatin (5) and (b) of the reduced form (6).
228 ACKNOWLEDGEMENTS
The authors Johnson, and aspects of this display of the Health (Grants
express their appreciations to Hubert A. Scoble, Richard S. Catherine E. Costello for stimulating discussions of various work and to James E. Biller for help with the processing and data. This work was supported by the National Institutes of RR00317 and GM05472).
REFERENCES 1 M. Barber, R.S. Bordoli, R.D. Sedgwick and A.N. Tyler, J. Chem. Sot. Chem. Commun., (1981) 325. 2 W. Aberth, K.M. Straub and A.L. Burlingame, Anal. Chem., 54 (1982) 2029. 3 F.W. McLafferty (Ed,), Tandem Mass Spectrometry, Wiley, New York, 1983. 4 Y. Kammei, Y. Itagaki, E. Kubota, H. Kunihiro and M. Ishihara, 33rd Annu. Conf. Mass Spectrom. Allied Topics, ASMS, San Diego, CA, 1985, p. 855. 5 K. Biemann, S.A. Martin, H.A. Scoble, R.S. Johnson, LA. Papayannopoulos, J.E. Biller and C.E. Costello, in C.J. McNeal (Ed.), Mass Spectrometry in the Analysis of Large Molecules, Wiley, Chichester, 1986, p. 131. 6 K. Biemann, Anal. Chem., 58 (1986) 1289A. 7 D.H. Williams, C.V. Bradley, S. Santikarn and G. Bojesen, Biochem. J., 201 (1982) 105. 8 M. Barber, R.S. Bordoli, G.V. Garner, D.B. Gordon, R.D. Sedgwick, L.W. Tetler and A.N. Tyler, Biochem. J., 197 (1981) 401. 9 M. Barber, R.S. Bordoli, R.D. Sedgwick, A.N. Tyler and E.T. Whalley, Biomed. Mass Spectrom., 8 (1981) 337. 10 D.H. Williams, C. Bradley, G. Bojesen, S. Santikarn and L.C.E. Taylor, J. Am. Chem. Sot., 103 (1981) 5700. 11 H.R. Morris, M. Panico, M. Barber, R.S. Bordoli, R.D. Sedgwick and A. Tyler, Biochem. Biophys. Res. Commun., 101 (1981) 623. 12 K. Biemann and S.A. Martin, Mass Spectrom. Rev., 6 (1987) 1. 13 S.A. Martin and K. Biemann, 34th Annu. Conf. Mass Spectrom. Allied Topics, ASMS, Cincinnati, OH, 1986, p. 854. 14 H.R. Morris and P. Pucci, Biochem. Biophys. Res. Commun., 126 (1985) 1122. 15 R. Yazdanparast, P.C. Andrews, D.L. Smith and J.E. Dixon, Anal. Biochem., 153 (1986) 348. 16 Bachem Inc., Torrance, CA, U.S.A. 17 P. Roepstorff and J. Fohlman, Biomed. Mass Spectrom., 11 (1984) 601.
213
A COMPARISON OF keV ATOM BOMBARDMENT MASS SPECTRA OF PEJYIIDESOBTAINED WITH A TWO-SECTOR MASS SPECTROMETER WITH THOSE FROM A FOUR-SECTOR TANDEM MASS SPEC~OME~R
STEPHEN A. MARTIN
* and KLAUS BIEMANN
**
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 (U.S.A.) (Received 26 November 1987)
ABSTRACT The information content of the conventional fast (keV) atom bombardment (FAB) mass spectra of six peptides, substance P, des-Arg’substance P, Fibrinop~tide A, desArgy6fibrinopeptide A, oxidized somatostatin, and reduced somatostatin, ranging in molecular weight from 1190.6 to 1638.7 is compared with that of the corresponding spectra measured by a high-performance (four-sector) tandem mass spectrometer after collision-induced decomposition (CID). The CID spectra exhibit distinct peaks corresponding to extended series of sequence characteristic ions free of the chemical background and contribution of the matrix which makes the weak fragment ion signals in conventional FAB mass spectra so difficult to interpret. The presence of arginine, a strongly. basic amino acid, in either the N-terminal or C-terminal position is reflected in the dominance of characteristic N-terminal and C-terminal fragment ions. The resolving power of the first of the two mass spectrometers is clearly sufficient to permit the recording of separate CID spectra of oxidized and reduced somatostatin in a mixture.
INTRODUCTION
Mass spectral data of relatively large, polar, thermally labile, non-volatile compounds can now be obtained by sputtering them from a solution in a polar matrix of low vapor pressure, such as glycerol. The sputte~ng agent is a beam of neutral atoms (argon or xenon) in the case of fast (keV) atom bombardment (FAB) ionization [l] or ions (Cs’) in a variant thereof, referred to as “liquid SIMS” (secondary ion mass spectrometry) [2]. While * On leave of absence from the Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC 29425, U.S.A. * * To whom correspondence should be addressed. 0168-1176/87/$03.50
Q 1987 Elsevier Science Publishers B.V.
214
these “soft ionization” techniques generate abundant protonated molecular ions, (M + H)+, or their cationated analogs, such as (M + Na)+, these ions are very stable and do not fragment extensively. Therefore, the resulting spectra exhibit little structural information. This can be remedied, however, by collisional-induced decomposition (CID) of the (M + H)+ ion (precursor ion) while passing through a collision cell filled with a neutral gas, such as helium. The resulting product ions, when mass analyzed in a second spectrometer, provide a clue to the structure of the precursor. Since this method requires two mass analyzers, it is known as tandem mass spectrometry (MS/MS). The principles and some applications of MS/MS have been summarized in a recent book [3]. One area of current interest in our laboratory is the determination of the amino acid sequence of peptides, ranging in length from two to at least twenty-five amino acids, derived by chemical or enzymatic cleavage of proteins for the purpose of determining their primary structure. The molecular weights of these peptides may be as high as 3000 u but the fragment ions are distributed over the entire mass range from m/z - 60 to the precursor ion and their mass has to be determined to better than k 0.5 u to allow the unambiguous assignment of the sequence. The performance requirements with respect to mass range, resolution, and sensitivity of the mass spectrometer are, therefore, very stringent and far exceed those of the instruments previously available [3]. For this reason, we use a high-performance tandem mass spectrometer (JEOL HXllO/HXllO) which consists of two consecutive double-focussing mass spectrometers arranged in the optimal C-configuration of an E,B,E,B, system. This instrument has been described previously [4-61. In this paper, we discuss the information which can be
TABLE I Structure
and molecular
weight of the peptides
used in this work
Substance P(l), M, 1346.73 Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH, des-Arg’ Substance P(2), M, 1190.63 Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH, Fibrinopeptide A (human)(3), M, 1535.68 Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg A(4), M, 1379.58 des-Arg ” Fibrinopeptide Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val Somatostatin, oxidized(S), M, 1636.72 Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys Somatostatin, reduced(6), M, 1638.73 Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys
215
derived from the product ion spectra obtained by CID of the precursor ion in such a ~~-perfo~an~ tandem mass spectrometer and compare them with the more limited information of the conventional FAB mass spectra. Of the large number of CID spectra of peptides which we have determined over a period of more than one year, we selected the set listed in Table 1. The conventional FAB spectra of 1 and 3 have been reported previously [7]. EXPERIMENTAL
A JEOL HXllO/HXllO high-performance tandem mass spectrometer of E,B,E,B, geometry with a mass range of 14500 u at 10 kV accelerating potential was used in these experiments f4-6]. The samples were dissolved in glycerol acidified with acetic acid and 0.5 ~1, containing approximately l-2 nmol of peptide, was applied to a stainless steel probe tip. The probe was inserted into the ion source via a vacuum lock and the sample was bombarded with a beam of xenon atoms from a JEOL neutral atom gun (6 kV, 2 A cathode current, 10 mA emission). The desorbed ions were accelerated, energy and mass selected, and detected after the first mass spectrometer, MS-l, using the combination of an off-axis post-acceleration device ( k 20 kV) and a 16-stage secondary electron multiplier. The post-acceleration unit is normally operated at - 18 kV in the positive ion mode. In the MS/MS mode, the precursor ion is selected in MS-l by setting the resolution such as to transmit only the “‘C component of the (M -t H) + isotope cluster into the collision region. Helium is introduced into the collision cell at such a pressure that the precursor ion is attenuated to lo-30% of its original abundance as measured by comparison of the precursor ion signal before and after introduction of helium and recorded at the detector located after B, of MS-2. Wit~n this attenuation range, the relative abundance distribution of the product ions does not change significantly. The resolution of MS-2 for the B/E scans reported was 1 : 1000 for peptides 1 - 4 and 1: 500 for peptide 5 and 6. It should be noted that lower than unit resolution suffices for reliable mass assignment because the 13C contributions have been eliminated by MS-l. An ion source almost identical to that in MS-1 is located in the interface region of the JEOL HXllO/HXllO and is used to calibrate MS-Z for normal and B/E scans. This ion source contains an integrated collision cell, which was employed in these experiments. A mixture of LiI, NaI, KI, RbI, and Csl was used to calibrate MS-2 from 0 to 3600 for the recording of the product ion spectra. A single ca~bration file is applicable to all precursor ions of any mass lower than the upper limit of the calibration file. The B/E scans are acquired in the profile mode (raw data) scanning from low mass to the precursor ion at scan rates which range from 30 s to 3 min for m/z
216 TABLE
2
Mass assignment of fragment A (human) (3) (see Fig. 4).
ions observed
A
upon CID of the (M + H)+ ion of fibrinopeptide
Ion type
m/z
Ys
Yll
758.58 784.52 905.69 931.47 1020.84 1046.52 1077.58
+0.16 +0.13 + 0.21 + $01 + 0.33 + 0.03 + 0.05
x11
1103.52
+0.01
+0.01
Yl2
1206.78
+0.21
445.13
-0.12
Xl2
1232.64
+0.09
x5
471.14
-0.09
Y13
1263.80
+0.20
Y6
574.24
-0.05
X13
1289.77
+0.19
X6
600.03
-0.24
Y14
1350.81
+0.18
Y7
645.34
+ 0.01
X14
1377.07
+0.46
x7
671.35
+0.04
Ion type
m/z
obs.
V L E F Y1 Y2 Y3
71.59 85.70 101.74 119.73 174.91 274.10 331.07
- 0.49 -0.39 - 0.31 - 0.35 - 0.21 - 0.09 -0.14
Y4
388.11
-0.12
x4
414.22
Y5
‘8
Y9 x9
YlO x10
obs.
A
30-2000. Individual or summed scans are then processed in the data system using the peak detection software. The accuracy of the mass assignments is always k 0.3 u or better. As an example, the m/z values determined by the data system for some of the product ions obtained upon CID of the (M + H)+ ion (m/z 1535.68) of fibrinopeptide A (3) are listed in Table 2 along with their deviation from the theoretical values. The peptides used in these experiments were purchased from Bachem [16], checked for homogeneity by reversed-phase HPLC, and used without further purification. Partial reduction of somatostatin was carried out by adding 0.2 ~1 of dithiothreitol/dithioerythritol to the probe tip which contained the sample in a glycerol matrix. The sample was re-inserted into the mass spectrometer and the reduced protonated molecular ion was selected for the B/E scan.
RESULTS
AND DISCUSSION
The fragmentation processes generally observed in FAB mass spectra of peptides are illustrated in Scheme 1, which represents a summary of proposals put forward specifically or implicitly in the early 1980s. chiefly by Barber et al. [8,9], Williams et al. [7,10], and Morris et al. [II] (for a review see ref. 12). In addition, we have observed, in many CID spectra, fragmentation leading to ions denoted as the w, series. The FAB spectrum of the
217
$4
Y+ $3
Ps
NH-CH-CO-NE-CR-CO-NH-CH-COoE ii/ Y3
R
R2
R4
R3
RS
Ha-VIA-CO-NB-~A-CO-~-OH-CO-MI-AH-CO-MI-~A-COOB +
A transfer
\,
R
R
R2
E$N-&-CO-NE-&R-CrO+
R2
E&t&-co-NR-&I-co-ki, c2
b2
1 F1
5
E$wi-co-ili=cH a2
Rx
k2
R&&i immonium fon (labelled
F
H$?-CH-CO-NE-CH-co+ internal with single
letter
cleavage
ion
code)
Scheme 1. Schematic presentation of the major fragmentations observed in normal positive ion FAB mass spectra and/or in collision spectra. Cleavage of the bonds between amino acids 2 and 3 of a pen~peptide is used as a general example. The notation is a variant of that proposed by Roepstorff and Fohhnan 1171. a, Protonation at 1st or 2nd amino acid. b, Protonation at 4th or 5th ammo acid.
218
*
I *
* I
( M+~H) 2+
*
a,-57
X5.0
(b)
,/
u4
a5
a,-57
x4.0
~ ,
(a)
x2.0
7
a:,
Fig. 1. Comparison (top two panels low mass, lower two panels high mass) of (a) the conventional FAB mass spectrum of the undecapeptide substance P (1) and (b) the product ion spectrum obtained upon CID of the (M+H)+ ion of this peptide. For notation, see Scheme 1. Peaks due to the matrix in (a) are indicated by an asterisk.
219
undecapeptide substance P (1) of M, = 1346.7 is shown in Fig. l(a). The lower mass range (m/z < 500) is dominated by the cluster ions of glycerol, the matrix. At higher mass, N-terminal ions of type c, and C-terminal ions of type yn are observable above the background ions (usually referred to as chemical noise). In contrast, the CID spectrum of the product ions formed upon collision of the precursor (M + H)+ with He in the collision cell consists mainly of a continuous series of ions of the type a, [Fig. l(b)]. They are distinct and abundant (compared with background) single peaks because they are derived from the ‘*C component of the (M + H)+ isotope cluster by transmitting only m/z 1347.7 + 0.5 from MS-1 to MS-2. The mass difference between consecutive peaks of this ion series clearly defines the sequence from the second to the tenth amino acid in this peptide. The eleventh amino acid is recognized to be methionine (in the form of the carboxy-terminal amide) by the difference in mass (176 u) between the a,, and (M + H)+ ion. The only ambiguity that remains is at the N-terminus, which could be Arg-Pro or Pro-Arg. This question can be easily resolved upon removal of the N-terminal amino acid by a one-step Edman degradation involving treatment of the peptide with phenylisothiocyanate (PITC) resulting in the formation of the decapeptide 2. The difference (157.1 u) in molecular weight of this product (M, = 1190.6) [Fig. 2(a)] and the original peptide reveals the nature of the N-terminal amino acid, in this case arginine. However, the removal of this strongly basic amino acid dramatically changes the relative abundance and distribution of the sequence ions in the CID spectrum [Fig. 2(b)]. Whereas the presence of arginine in 1 favors the appearance of a continuous series of ions of type a,, after its removal the acyl ions b, become equally dominant. The only C-terminal ion of note is ys, which represents the fragment formed by cleavage of the N-terminal peptide bond of the proline in position 4. Its predominance may be the reason for the relatively low abundance of the N-terminal fragments, especially b,, which involve cleavage of the C-terminal amide bond of this proline. Another consequence of the proline is the appearance of an internal fragment ion, PQQ, the formation of which also involves cleavage of the N-terminal amide bond of Pro. Such internal fragments are often present as a series of extended length. The most significant difference between the conventional FAB spectrum [Fig. 2(a)] and the CID spectrum [Fig. 2(b)] is the appearance of one or two continuous ion series in the latter, whereas the former exhibits some fragments due to cleavage at certain peptide bonds, but often without a general trend. While this allows for a correlation of peaks in the conventional FAB spectra with known structures of peptides, it is not very helpful for the unambiguous determination of the sequence of peptides of unknown structure. Another effect of the absence of N-terminal a&nine is the much clearer
(a)
T,‘I
700
aoc
900
1000
1100
1200
Fig. 2. Comparison (top two panels low mass, lower two panels high mass) of conventional FAB mass spectrum of the decapeptide (2) obtained by removal N-terminal arginine from 1 and (b) the product ion spectrum obtained upon CID (M + H)+ ion of this peptide. For notation, see Scheme 1. Peaks due to the matrix in indicated by an asterisk.
(a) of of (a)
the the the are
221
m/z
60
70
Fig. 3. Region from m/z
80
90
100
110
120
130
50 to m/z 125 of the CID spectrum shown in Fig. 2(b).
appearance of the immonium ions indicative of the presence (but independent of their position) of certain amino acids. As an illustration, the lower mass range (m/z 50-125) of Fig. 2(b) is shown expanded in Fig. 3, which reveals the presence of Pro, Lys, Leu or Ile, Gln, Met, and Phe. Although the immonium ions of Gln and Lys have the same nominal mass (m/z lOl), we have made the observation that only the latter preferentially cyclizes by elimination of NH, to form m/z 84. Finally, ions of the a,, series that have lost 42 or 57 u indicated in Figs. 1 and 2 need to be mentioned. They are due to loss of part of the side chain of leucine and glutamic acid, respectively, (with transfer of a hydrogen) whenever this amino acid represents the C-terminal position of an ion of type a,, [13]. This fragmentation permits the differentiation of the isomers leucine and isoleucine (Ile gives rise to an a, - 28 ion). Asparagine, glutamine, aspartic acid, and glutamic acid behave in a similar way (loss of 43, 57, 44, and 58 u from the a, ion). These fragment ions are absent in the conventional FAB spectra, which thus do not allow these important differentiations to be made. At the high mass end of the CID spectra, ions due to the loss of the side chain of some of the amino acids are discernible and these are labeled with the single letter code of the corresponding amino acid, preceded by a minus sign. While not as distinct as the immonium ions at low mass, they are also helpful indicators of the presence of certain amino acids. The influence of the presence of a strongly bzsic amino acid, such as arginine, at the C-terminus of a peptide is also noticeable. As an example of such a case, the mass spectra (Fig. 4) of fibrinopeptide A (human) (3) are compared with those of the analog 4, lacking the C-terminal arginine (Fig. 5). The much higher information content of the CID spectra is again evident
I
x3.2 ',-
:
(a)-
1’
I
i I f
“>
) ?
q I_! 82
I,
,,,,
1 1L
‘_
,>
_
! .‘I‘L
I 7-t_
121,
Fig. 4. Comparison (top two panels low mass, lower two panels high mass) of (a) the conventional FAB mass spectrum of fibrinopeptide A (human) (3) and (b) the product ion spectrum obtained upon CID of the (M+ H)+ ion of this peptide. For notation, see Scheme 1. Peaks due to the matrix in (a) are indicated by an asterisk.
223
t
(a)
b,
(b) YS
b,
GEGD
Y7
SGEGD
700
600
500
x 1.5
(a)
YS
m/z
800
900
1000
1100
12bo
1300
? d b; -L
-L
b,
-D ! I
YlO
m/z
800
900
LA
1000
,100
1200
1300
-!
tcIO
Fig. 5. Comparison (top two panels low mass, lower two panels high mass) of (a) the conventional FAB mass spectrum of des-Arg 16-fibrinopeptide A (4) and (b) the product ion spectrum obtained upon CID of the (M+H)+ ion of this peptide. For notation, see Scheme 1. Peaks due to the matrix in (a) are indicated by an asterisk.
224
from Fig. 4, which compares the conventional FAB spectrum of 3 with its CID spectrum. Although, in Fig. 4(a), the fragment ions at higher mass can be correlated with the structure as indicated by the labeling of the corresponding peaks, which are mainly due to the C-terminal ions of the w,,, y,, and z, series, the lower mass range is dominated by matrix ions and one needs to compare carefully the spectrum with that of glycerol alone to recognize the peaks due to the peptide. Even that becomes impossible below m/z - 300, where those N- and C-terminal ions would be found that are indicative of the first and last two or three amino acids. In contrast, the CID spectrum [Fig. 4(b)] very distinctly exhibits a complete y,, series, with the exception of the last one (y,,), and other C-terminal ions, chiefly of the type x, and w,,. That the dominance of these is due to the strongly basic arginine at the C-terminus is demonstrated by the CID spectrum of peptide 4 [Fig. 5(b)]. In this case, the most complete series are the N-terminal ions, b,-bi5. The low abundance of b, is due to the fact that it requires cleavage of the amide bond of glycine which sometimes appears to be relatively unfavorable. In the normal FAB spectrum of 4, the b, series is of minor significance and much less extensive [b,_,, Fig. 5(a)]. As an illustration of the peaks due to the loss from the (M + H)+ ion of the side chains of various amino acids mentioned earlier and evident in Figs. l(b), 2(b), 4(b), and 5(b), the upper end of Fig. 5(b) is expanded in Fig. 6. For a number of reasons, cystine-containing peptides fragment poorly, even after collisional activation. In part, this may be so because a -S-Sbond converts a linear peptide to a cyclic one and two bonds have to be
_E”
b-15
(M+H)+
Fig. 6. Expansion of the upper end of Fig. 5(b) covering the mass region of peaks due to the loss of the side chains of various amino acids (labelled with single letter code).
225
m nA.nn
h
A
100
200
300
400
500
600
700
800
X30
(M+H)
m/z
900
1000
1100
1200
1300
Fig. 7. Conventional FAB spectrum of oxidized somatostatin indicated by an asterisk.
1400
1500
1600
(5). Peaks due to the matrix are
cleaved to produce fragments from the cyclic portion of the peptide. The normal FAB spectrum of the oxidized form of somatostatin (5), shown in Fig. 7 exhibits almost no fragmentation. When the 12C component of the (M + H)+ ion of m/z 1637.7 [Fig. 8(a)] was transmitted by MS-l into the collision region and the product ions analyzed by MS-2, very few peaks were observed [Fig. 9(a)], in contrast to the CID spectra shown in Figs. l(b), 2(b), 4(b), and 5(b). Treatment of the glycerol solution used in this experiment with dithiothreitol/dithioerythritol converted about half of the material to the reduced form 6, as evidenced by the molecular ion pattern before and after the reduction [Fig. 8(b)]. When the ion of m/z 1639.7 was transmitted by MS-l, a much more detailed CID spectrum corresponding to the reduced linear peptide was observed [Fig. 9(b)] which is dominated by N-terminal fragments, particularly the b, series. Although the precursor ion also contains some of the i3C2 component of (M + H)+ of the oxidized form 5, it
226
I
r.
&+H);
;
162:
(M,.,+H)
(a)
1743
hg’
1 b53
rr ;
'635
'nLiJ
+
-~-F
1045
(b)
1 t 5,3
Fig. 8. (a) Molecular ion region of the sample of somatostatin used to generate the spectra shown in Figs. 7 and 9(a). (b) After partial reduction of the disulfide bond to generate the spectrum shown in Figure 9(b).
contributes little to the product ion spectrum because it undergoes very little fragmentation (see above). In general, this lack of fragmentation of peptides which are cyclic by virtue of the disulfide bond is not a problem in the sequencing of peptides produced by proteolytic digestion of a protein because, in that situation, cystine is always first reduced to cysteine and then converted to a stable thioether (by reaction with iodoacetic acid or iodoacetamide). The situation is different when one needs to determine the location of -S-Sbridges which requires first that this bond is kept intact and then reduced [14,15]. However, this question always arises after the primary structure of the reduced polypeptide has been established and the amino acid sequence itself is not the issue.
CONCLUSION
The data discussed in this paper demonstrate that the CID spectra of (M + H)+ ions of peptides not only exhibit much more distinct fragment ions than the normal FAB spectrum but also that the predominant fragmentations may be quite different. The CID spectra contain more complete series of a few sequence ion types, whereas the normal FAB spectra often exhibit more sporadic fragment ions of many different ion types. The latter may be an artifact of the general appearance of FAB spectra which are characterized by a periodicity of peak clusters that may accidentally fall at the mass of a possible sequence ion. Comparison of the two types of spectra clearly indicates that the CID spectra permit the sequencing of a peptide with much greater ease and confidence than the normal FAB spectra.
221
W
100
NFF
200
300
400
500
600
700
800
(b)
T/Z IO0
200
300
400
500
600
700
800
m/z
1000
1100
1200
1300
1400
1500
1600
1000
1100
1200
1300
1400
1500
1600
900
X 2.5
m/z
900
Fig. 9. CID spectra of (a) the oxidized form of somatostatin (5) and (b) of the reduced form (6).
228 ACKNOWLEDGEMENTS
The authors Johnson, and aspects of this display of the Health (Grants
express their appreciations to Hubert A. Scoble, Richard S. Catherine E. Costello for stimulating discussions of various work and to James E. Biller for help with the processing and data. This work was supported by the National Institutes of RR00317 and GM05472).
REFERENCES 1 M. Barber, R.S. Bordoli, R.D. Sedgwick and A.N. Tyler, J. Chem. Sot. Chem. Commun., (1981) 325. 2 W. Aberth, K.M. Straub and A.L. Burlingame, Anal. Chem., 54 (1982) 2029. 3 F.W. McLafferty (Ed,), Tandem Mass Spectrometry, Wiley, New York, 1983. 4 Y. Kammei, Y. Itagaki, E. Kubota, H. Kunihiro and M. Ishihara, 33rd Annu. Conf. Mass Spectrom. Allied Topics, ASMS, San Diego, CA, 1985, p. 855. 5 K. Biemann, S.A. Martin, H.A. Scoble, R.S. Johnson, LA. Papayannopoulos, J.E. Biller and C.E. Costello, in C.J. McNeal (Ed.), Mass Spectrometry in the Analysis of Large Molecules, Wiley, Chichester, 1986, p. 131. 6 K. Biemann, Anal. Chem., 58 (1986) 1289A. 7 D.H. Williams, C.V. Bradley, S. Santikarn and G. Bojesen, Biochem. J., 201 (1982) 105. 8 M. Barber, R.S. Bordoli, G.V. Garner, D.B. Gordon, R.D. Sedgwick, L.W. Tetler and A.N. Tyler, Biochem. J., 197 (1981) 401. 9 M. Barber, R.S. Bordoli, R.D. Sedgwick, A.N. Tyler and E.T. Whalley, Biomed. Mass Spectrom., 8 (1981) 337. 10 D.H. Williams, C. Bradley, G. Bojesen, S. Santikarn and L.C.E. Taylor, J. Am. Chem. Sot., 103 (1981) 5700. 11 H.R. Morris, M. Panico, M. Barber, R.S. Bordoli, R.D. Sedgwick and A. Tyler, Biochem. Biophys. Res. Commun., 101 (1981) 623. 12 K. Biemann and S.A. Martin, Mass Spectrom. Rev., 6 (1987) 1. 13 S.A. Martin and K. Biemann, 34th Annu. Conf. Mass Spectrom. Allied Topics, ASMS, Cincinnati, OH, 1986, p. 854. 14 H.R. Morris and P. Pucci, Biochem. Biophys. Res. Commun., 126 (1985) 1122. 15 R. Yazdanparast, P.C. Andrews, D.L. Smith and J.E. Dixon, Anal. Biochem., 153 (1986) 348. 16 Bachem Inc., Torrance, CA, U.S.A. 17 P. Roepstorff and J. Fohlman, Biomed. Mass Spectrom., 11 (1984) 601.