Anal. Chem. I Q W ,57, 1174-1180
1174
Fast Atom Bombardment and Tandem Mass Spectrometry for Sequencing Peptides and Polyamino Alcohols Dixie L. Lippstreu-Fisher and Michael L. Gross*
Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588 Fast atom bombardment combined wlth colilslonal activation decomposltlon (CAD) spectrometry has been used to investigate eight peptides contalnlng from three to SIXamlno aclds In order to derive some general concluslons about the CAD of the (M H)' of peptldes. A scheme for lnterpretlng the CAD spectra is developed and exempllfied uslng a pentapeptide. The primary peptide backbone fragmentatlons give rlse to N- and C-termlnus-containing lons, resulting from peptide bond cleavage, and N-terminal-containing Ions minus CO. I n addition, the (M 4- H)' Ions of polyamlno alcohols, formed by chemical reductlon of these peptides, were submitted to collisional activation. Complementary amlno acid sequencedetermlnlng lnformatlon is obtalned. Moreover, the NH-CH, cleavage, wlth charge retentlon on the C termlnus, is now prominent for the reduced peptldes. The potential of CAD spectrometry for studles of mlxtures of peptldes and mixtures of poiyamino alcohols Is demonstrated by analyzlng an equimolar mixture of four peptides.
+
The development of mass spectrometric methods for determining and confirming the primary structure of proteins has become a major effort in the last several years. Reasons for the high activity are that mass spectrometryoffers not only the capability for accurate mass measurements at extremely high sensitivity but also the potential for degrading the peptide into constituent amino acid residues. It also has potential for the analysis of mixtures by utilizing direct probe sample ini troduction, GCIMS, and recently tandem mass spectrometry
(MSJMS).
Early mass spectrometric studies of peptides were often limited by thermal lability and the lack of sample volatility. One approach involved the development of chemical modifications to decrease these limitations. Morris et al. (1) employed the method of N-acetyl-N,O,S-permethyl derivatization in sequencing the enzyme, dihydrofolatereductase (2,3). This mass spectrometric protein determination was the first to be done independent of classical sequentor methods. Biemann and co-workers have developed another approach based on chemical reduction of peptides to their corresponding polyamino alcohols (4). They have carefully evaluated the reduction and coupled it with GC/MS for analysis of the product mixtures which result from chemical reduction of a mixture of peptides from a digest (5-13). Both methods are limited to small (di- to deca-) peptides, and overlap techniques must be used to determine the sequence of larger peptides. One must often rely on typical nonspecific fragment ions such as (M - H20)+.and (M - CH# in order to determine the molecular mass. In addition, the predicted fragment ions are not always observed, and thus sequencing may be incomplete. Desorption ionization methods have greatly enhanced the potential for mass spectrometric determinations of peptides. The limitations of involatility and thermal sensitivity can now be avoided and relatively large, nonderivatized peptides can be studied. The choice of a desorption technique depends on 0003-2700/85/0357-1174$01 S O / O
the strengths of the individual method and the type of information needed. For example, field desorption mass spectrometry (FD-MS) yields primarily molecular ion information and has been shown to be a good technique for the quantification of peptides at the nonogram level (14). Fragmentation information has been obtained by using collision activated dissociation (CAD) (15,16) and by submitting the sample to a digestion process ( I 7). A static SIMS method (18) makes use of the excellent sensitivity observed for quaternary ammonium salt derivatives of small peptides to obtain sequence information with improved S I N . The liquid matrix desorption techniques,such as FAB, have been used in studies of both model peptides and unknown proteins and have shown that molecular weight and sequence information can be obtained, often without derivatization (19-37). These studies range from small peptides of less than 1000 to intact proinsulin, (M H)+ 9390 (37). Sequence information from FAB mass spectra i s often limited and ambiguous, and additional techniques are often needed to minimize this limitation. For example, tryptic digests of large peptides have been analyzed without extensive purification, and the ions observed were compared with the anticipated (M + H)+ ions. This technique has been used to verify peptide sequences (35) as well as to locate incorrectly sequenced portions of peptides, such as abnormal hemoglobin (38) and neocarzinostatin (34). Additional digestion with enzymes such as a carboxypeptidase or an aminopeptidase followed by FAB studies has been useful in supplementing information from peptide bond cleavages, resulting in further sequence information (31,32). N-terminus isotopic acetylation (1:lCH3CO:CD3CO)has been successfully used by Morris et al. (36) to identify and sequence the N-terminus fragment ions of cardioactive peptide. Another promising source of sequence-containing information is collisionally activated decomposition (CAD). By coupling CAD with MS/MS, one is able to focus on the ion of interest and observe enhanced fragmentation with minimal interferences from the sample matrix or impurities (39). Hunt et al. (40) have used a triple quadrupole system to demonstrate the advantages of MS/MS for studying mixtures of small isotopically labeled peptides. Other peptides studied with MSIMS techniques include a heptapeptide (41))angiotensins (23), and cyclic peptides (42). Leu-enkephalin (43) and Substance P (44) are examples studied by using linked scan techniques. The only CAD study of a set of peptides to date is by Neumann and Derrick (45),who used field desorption as the ionization method. The major emphasis of the paper was translational energy loss in relation to mass, although there was some discussion of the type of fragmentation observed. The major fragment ions described were N- and C-terminal-containing sequence ions, resulting from peptide bond cleavage, and N-terminal-containingsequence ions minus CO. The usefulness of FAB-MS coupled with CAD for studying peptide sequences is demonstrated in this paper for the pentapeptide Tyr-Ala-Gly-Phe-Leu (YAGFL). Generalizations about typical fragment ions, drawn from a study of spectra of seven additional peptides, are applied toward
+
0 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
YAGFL as if it had an unknown sequence. Chemical modification of structure followed by reinvestigation has often been effective in structure determination. Chemical reduction was chosen as the means for peptide modification because it has proved useful in GC/MS studies of small quantities (50-100 nmol) of unknown peptides ( 4 , 5 ) . Accordingly, YAGFL was converted to its coresponding polyamino alcohol and reinvestigated by using FAB and MS/ MS. Observations about fragment ion types, from an investigation of a series of reduced peptides, are applied to interpreting the spectrum of the (M + H)+ of reduced YAGFL. These additional peptides are used for illustrating further the advantages of studying CAD spectra of both peptides and the corresponding polyamino alcohols. The last portion of this paper deals with the potential of the method for mixture analysis, as that would be desirable when studying a protein digest which contained various small peptides. Four peptides were combined in equimolar amounts and were studied by using FAB-MS and CAD spectrometry for analyzing the original mixture and the mixture of the reduced peptides.
EXPERIMENTAL SECTION Reduction with Diborane. Peptides were reduced by using the diborane method described previously by Biemann et al. (4). The reaction was done in a Teflon-sealed Reactivial,using 0.1-5.0 mg of peptide and approximately 200 p L of 1 F BzHB/THF per mg of peptide (or a minimum of 200 pL). The mixture was sonicated so that dissolution of the peptide was expedited and heated for at least 30 min at 90 "C. Excess diborane was quenched with anhydrous methanol and the solvents were evaporated under a stream of Nz. The borane/polyamino alcohol complexes were hydrolyzed with 1 F HCl/anhydrous methanol for 30 min at 90 OC. Solvent and trimethoxyborane were removed under a stream of dry N2, and the residue was dried briefly "in vacuo". The acid hydrolysis and drying steps were repeated. The polyamino alcohol residue was taken up in 200 pL of 25% KzC03and the pH was adjusted to between 10.5 and 11.5 with 1 F KOH. The aqueous phase was extracted three times with 300 pL of methylene chloride, and the mixture was centrifuged to obtain clean separation. The ionic strength was then increased to enhance the extraction of the more polar residues from the aqueous phase. This was done by adding 50-100 mg of solid KzC03and 200 pL of a solution containing 12 g of K2C03per 10 mL of water. Again, the aqueous solution was extracted with methylene chloride. The high ionic strength was used to enhance the extraction of the more polar residues from the aqueous phase. The two extracts were kept separate and dried under a stream of N2 Prior to mass spectrometric analysis, the residues were taken up in ca. 15 pL of methylene chloride. Mass Spectrometric Analysis. The peptides and polyamino alcohols were analyzed with a Kratos MS-50 triple sector mass spectrometer, which has been previously described (46). It consists of a high-resolution MS-I of Nier-Johnson geometry, followed by an electrostatic analyzer used as MS-11. The fast atom bombardment (FAB) ion source, of standard Kratos design, was equipped with an ION TECH atom gun. Solutions of the peptides (1pL containing ca. 1-10 pg) were dissolved in a drop of glycerol containing 1% formic acid on the copper target of a direct insertion probe, and the solution was bombarded with 7 kV xenon atoms. Ions were accelerated from the source region at 8 kV and mass analyzed at a resolution of approximately 3000 (10% valley definition) and a scan rate of 30 s per decade. CAD spectra were obtained by selecting an ion with MS-I, activating the ion by collisions with an inert gas in the third field-freeregion, and scanning MS-11. A Kratos DS-55 data system and software written in this laboratory were used to acquire the spectra at 40 s per scan; 20 to 40 scans were averaged per spectrum. Helium was used as the collision gas at a pressure resulting in 50% main beam suppression. The polyamino alcohols were studied in a similar manner, using acidified glycerol as the liquid matrix. Application to the copper target was accomplished by first dissolving the reduced peptides
1175
'0
'0
c 2. ;;SO
z r
550 Y
> -IO r
-
-
75
57
-
< _1 =wl o ,
/I11120
I
I/ &
I PO
200
300
k00
500
500
W Z
Figure 1. FAB full scan mass spectrum of Tyr-Ala-Gly-Phe-Leu, (M 4- H)+ 570.
in CH2Clzand transferring 10-20% (1-2 pL) of the solution to a droplet of glycerol on the probe. The minimum amount of polyamino alcohol that can be determined by using this method was not evaluated. Preparation and Analysis of a Mixture. A solution containing 1.2 pmol each of Gly-Phe-Ala, Gly-Leu-Tyr, Tyr-GlyGly-Phe-Leu,and Tyr-Ile-His-Pro-Phe was split, and 20% of the original solution was retained. The FAB and CAD mass spectra were obtained as described above by using an aliquot corresponding to 2 nmol of each peptide per experiment. The remaining 80% of this mixture was reduced by using the BzH6/THFmethod described, and the low ionic strength and high ionic strength extracts were studied independently. CAD spectra were obtained by using aliquots representing 10-15% of the extract. Reagents. The 1 M diborane/THF was obtained from Ventron/Alfa. Methanol and methylene chloride, reagent grade, were obtained from Fischer Chemical Co. Potassium carbonate was from Baker Chemical Co., also reagent grade. The peptides Gly-Leu-Tyr, Gly-Phe-Ala, and N-Cbz-GlyPro-Gly-Gly-Pro-Ala were obtained from Sigma Chemical Co. Angiotensin I1 pentapeptide (Tyr-Ile-His-Pro-Phe) and leu-5enkephalin (Tyr-Gly-Gly-Phe-Leu)were from Chemical Dynamics Corp. Tyr-Ala-Gly-Phe-Leu was synthesized by John Pelton at the University of Nebraska and was provided as a generous gift.
RESULTS AND DISCUSSION Study of a Pentapeptide. The FAB mass spectrum of Tyr-Ala-Gly-Phe-Leu,YAGFL (Figure l),demonstrates the problems often observed with liquid matrix spectra. The (M + H)+(m/z570) is one of the most prominent nonmatrix ions in the spectrum and is easily identified. However, fragment ions are of relatively low abundance, and many are observed a t approximately the same abundance as the matrix ions, particularly those with masses below 300 amu. On the basis of this spectrum, it would be difficult to obtain unambiguously the amino acid sequence of this peptide. In order to eliminate matrix ions and to obtain an unambiguous fragmentation pattern, the (M + H)+ of the peptide YAGFL was submitted to collisional activation (Figure 2). Nearly all of the abundant product ions can be assigned structures which result directly from cleavage of the peptide backbone of (M + H)+ (see Table I). Note that approximately 75-80% of the total ion current, excluding the (M + H)+, is borne by three types of fragment ions: N,, Nj - 28, and C,, where N, and C, result from peptide bond cleavage with charge retention on the N-terminal portion and the C-terminal portion, respectively, Nj - 28 corresponds to the loss of CO from fragment Nj and j is the number of amino acid residues in the fragment ion (Scheme I). Ions resulting from the cleavage of side chains of (M + H)+ are observed in the region
1176
ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985 Scheme I. Principal Decomposition Pathways Observed in CAD Spectra of Peptides: Nj, Cj, and Nj - 28
T I R- AL A-GLI-PHE-LEU 439
I
R
I
200
IO0
400
300
cJ
SO0
P
91
hi/ 2
Flgure 2. CAD spectrum of the (M 570.
+ H)+ of Tyr-Ala-Gly-Pheleu, m l r
I
*2
Table I. Fragment Iona Observed in the CAD Spectrum of the Peptide Tyr-Ala-Gly-Phe-Leu,m / z 570 (M €I)+
+
fragment
% re1 mass abundance
fragment
% re1 mass abundance
amino acid sequence-determining information. The first major ion observed, working from high to low mass, is mlz 552, clearly (M + H - HzO)+. There are ions observed in the mass region of 445-525 amu, at 10-20% relative abundance; however, the next abundant ion is mlz 439 (80% relative abundance). Note that there is a prominent ion observed at mlz 411,28 amu below mlz 439. This pattern is suggestive of N, and N, - 28 fragment ions. The difference between 552 and 439 corresponds to the -HNCH(R)CO- moieties of leucine, isoleucine, or hydroxyproline. Other possible N,/(N, - 28) pairs are observed at m/z 2921264 (40%/6%relative abundance) and mlz 2341206 (25%130% relative abundance). These probably correspond to the subsequent loss of phenylalanine and glycine units from the carboxylic acid terminus. Thus, the sequence at the C-terminal end is likely ...GlyPhe-Leu, ...Gly-Phe-Ile, or ...Gly-Phe-ProOH. Judging from the molecular weight, 569, and the presence of the low mass glycine, it may be deduced that the peptide probably contains five amino acid units. If this is correct, then C3and Cz should be observed at mlz 336 and 279, respectively. Ions of 40% relative abundance each are observed at these masses. Furthermore, low abundance ions are observed which could correspond to ammonia loss from C3 and Cz. Information about the remainder of the sequence could be obtained from the fragments N1 or C,; however, there are no obvious choices for either ion. The mass of the molecule yet to be accounted for corresponds to the amino acid pairs serinelphenylelanine, prolinelhistidine, tyrosinelalanine, and cysteinelmethionine. The most likely choice would by tyrosinelalanine, since ions at mlz 136 and 462 could be explained by the presence of tyrosine. However, neither of these ions can be interpreted to show conclusively whether tyrosine or alanine is the N-terminal residue. Study of Reduced Tyr+Ala-Gly-Phe-Leu(YAGFL). As a strategy to obtain more information on a peptide we have chosen to reduce chemically the peptide to a polyamino al-
Cleavage of CO-NH Bond MH-HzO N4 N3 NZ N1
552 439 292 235 164
MH-COZ Nd-28 N3-28 Nz-28 N1-28
524 411 264 207 136
100 79 38 24
C4 c3
Cz
c1
407 336 279 132
18 39 37 5
Cleavage of CH-CO Bond 20 42 6 29 49
C4 + 26 Cs 26 Cz 26 C1 + 26
+ +
433 362 305 158
Cleavage of NH-CH Bond N4 + 17 N3 + 17 Nz 17 N1 + 17
+
456 309 252 181
MH-NH3 C4-17 C3-17 Cz-17 c1- 17
553 390 319 262 115
6 2 6
mlz 455 to mlz 512, at 10-20% relative abundance. The losses of HzO and CHzOzaccount for ions mlz 552 and 524, respectively. The mlz 120 ion probably originates from the tyrosyl side chain. The application of CAD to structural studies of unknown peptides can be generalized if the three fragment ion types observed for YAGFL are found to be as abundant for other peptides. As a test, CAD data obtained for six additional peptides studied in our laboratory and one from the literature (41)are compared with YAGFL (see Table 11). It is clear from the evidence in Table I1 that YAGFL may be considered typical of these peptides; fragment types N,, N, - 28, and C, account for an average of 70-75% of the total product ion current observed. Let us now consider the CAD spectrum of fragments from protonated YAGFL (Figure 2) in terms of ease of extracting
Table 11. Competition between Decomposition Pathways of Peptides
peptide:
CO-NH N, c,
70 re1 abundance CH-CO NH-CH cleavage N, - 28 C, + 26 N, + 17 C, - 17
Tyr-Ala-Gly-Phe-Leu Tyr-Gly-Gly-Phe-Leu Tyr-Ile-His-Pro-Phe N-Cbz-GIy-Pro-Gly-Gly-Pro-Ala Ala-Leu-Trp(F0R)-Asn-Arg-Ala Trp-Gly-Gly Gly-Leu-Tyr Gly-Gly-Leu
39 44 36 35 26 6 28 25
16 6 17 19 14 9 28 36
23 20 12 26 34
1 0 1 2 7
12
10
9 13
3 5
mean re1 abund std dev
33 7
19 10
20 9
3 2
2 3
other
2
19
6 5 1 11 54 4 11
23 28 15 NA 8 25
6
20 7
4
10
ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
1177
100-
Table 111. Fragment Ions Observed in the CAD Spectrum of the Polyamino Alcohol Form of Tyr-Ala-Gly-Phe-Leu, m/z500 (M H)+
93 SO
+
x5
-
80
% re1
fragment
mass abundance
% re1
fragment
mas8 abundance
Cleavage of CH2-NH Bond MH-HzO "4
"3 "2 "1
482 383 250 207 150
100 30 36
C'4 C'3 C'2 C'l
351 294 251 118
28 31 30 12
363 306 263 130
5 8 12 6
Cleavage of CH-CH2 Bond 14
N'4-
"3-14 14
"2-
"1-14
38 6 43 4
369 236 193 136
C'4 C'3 C'z C'l
+ 12 + 12 + 12
+ 12
+ 17 + 17 + 17 + 17
400 267 224 167
13 16 8
MH-NH3 C'd-17 C'3- 17 C'z-17 C'1 - 1 7
110
I00
250
200
wz
IS0
300
400
Flgure 3. FAB full mass spectrum of the (M Ala-Gly-Phe-Leu, (M HI+ 500.
+
Cleavage of NH-CH Bond N'4 N'3 N'2 N'1
50
483 334 217 234 101
44 24 19 41
150
IS0
500
+ H)+ of reduced Tyr-
REDUCED TYR-ALA-CLY-PHE-LEU
i
cohol. All amide groups were reduced to amines and the carboxylic acid was reduced to the alcohol (eq 1). B2H6
200
IO0
300
4 00
500
-HNCHRCQNH-CHRCOOH THF~ -HNCHRCH2NH-CHRCH20H (1)
M/ 2 Figure 4. CAD spectrum of the (M Phe-Leu, m lz 500.
FAB and CAD spectra were obtained in the same manner as for the original peptide. The FAB spectrum of reduced YAGFL (Figure 3) shows an abundant (M H)+ at m / z 500, a decrease of 70 amu due to the reduction of five CO groups to CH2. Several sequence ions are observed, again a t the same relative abundance as matrix ions. In addition, lower abundance ion clusters are found at m/z 514 and 528, possibly the result of incomplete reduction. The CAD spectrum of the (M H)+ of reduced YAGFL (Figure 4) can be interpreted to show that four types of fragment ions predominate (see Table 111). The four ion types represent 70% of the total ion current and are shown in Scheme 11. Three of the ion types, N'], N'] - 14,and C',, are the same as for nonreduced peptides, but C', - 17, a low abundance ion type in the decompositions of nonreduced peptides, is now quite abundant. The six additional peptides were also reduced. None of the full scan FAB mass spectra of the reduced peptides showed abundant (M + K)+ ions even though a high concentration of potassium chloride was used in the workup of the redudion
Scheme 11. Principal Decomposition Pathways Observed in CAD Spectra of Reduced Peptides: N'j, C'j, N'j - 14, and C'j - 17
+
+ H)+ of reduced Tyr-Ala-Gly-
81
NJ
\HAcH2!%w/%\ -.-D \,,/Cn\& + HzN\,/MF.
I
R2
A2
+
reaction. CAD spectra of the (M + H)+ions of the reduced peptides were acquired so that the generality of the frag-
Table IV. Competition between Decomposition Pathways of Reduced Peptides %
polyamino alcohol of Tyr-Ala-Gly-Phe-Leu Tyr-Gly-Gly-Phe-Leu Tyr-Ile-His-Pro-Phe N-Cbz-Gly-Pro-Gly-Gly-Pro-Ala Trp-Gly-Gly Gly-Leu-Tyr Gly-Gly-Leu mean re1 abund std dev
"1
23 22 22 35 16 24 26
CHZ-NH C', 14 11
5 or 23" 2 5 5 4
CH-CHZ C',
N f 1- 14 12 11 22 or 4" 21 10 18 9
24 7 or 9" 15 or 12" 6 4-8 6 " Mass of fragment coincides with mass of another fragment type.
+ 12
NH-CH cleavage N'j + 17 C'j- 17
4 3 5 4 6 5 7
5 5 3 4 9 2
5 1
other 24
1
18 20 14 9 17 24 44
4 3
21 11
25 8
28
29 25 37 22 9
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
mentations could be checked. The CAD results (Table IV) show that the same four ion types represent approximately 70% of the total ion current. The decompositionsof reduced YAGFL appear to be typical. The next issue that must be addressed is whether the CAD spectrum of the (M + H)+ of the polyamino alcohol is useful for completing the sequence and verifying it. Interpretation will be based on: (1)mass shifts from the N,, Nl - 28, and C, ions and (2) the appearance of C, - 17 ions in the reduced YAGFL spectrum. For example, N4, N3, Nz, and N1 would be expected to shift downward by 56, 42, 28, and 14 amu, respectively, due to the conversion of CO to CH2. N’] - 14 ions would be observed 14 amu below N’j ions. C4,C3,C2,and C1 would also decrease by 56,42,28, and 14 amu to form C’, series of ions. In addition, a complement to the C’] ions at 17 amu lower due to loss of ammonia would be expected. Those ions which are observed at the same mass in both CAD spectra (and cannot be accounted for by mass shifts) may be assumed to originate from side chains and not from a reducible portion of the peptide backbone. If the sequence is UNK1-UNK2-Gly-Phe-Leu (Leu being either leucine, isoleucine, or hydroxyproline) then N’4, N’3, and N’2 ions should be observed for the reduced peptide at m / z 383, 250, and 207. They are indeed abundant ions, at loo%, 30%, and 30%, respectively (Figure 4). Members of the complementary N’3 - 14 series are expected at m/z 369, 236, and 193. N’4 - 14 and W2 - 14 are observed at 40% relative abundance each, but the peak for N’3 - 14, m / z 236, is obscured by the intense peak at mlz 234. Two sets of mass-shifted ions are observed which indicate C’] and C’] - 17 fragment types, at m / z 3511334 and m / z 2941277. By subtracting the masses of N’] ions directly from the (M + H)+ at m / z 500, it can be seen that these ions correspond to subsequent losses of tyrosine and alanine from the N-terminus, which is suggestive of a sequence of TyrAla-Gly-Phe-Leu. The C’2 and C’2- 17 ions m / z 251 and 234, observed at 30% and 40% relative abundance, respectively, are indicative that the third amino acid is glycine. An ion representing C’l is observed at mlz 118, at 10% relative abundance. The CAD spectrum of the (M + H)’ of nonreduced YAGFL can now be reexamined to look for the ions C4 and N1. C4, m/z 407, is obscured by the intense peak at m / z 411. C4 17 may be present at m/z 390, but is part of a broad multiplet. N1 is not observed above SIN. By reducing the pentapeptide, these ambiguities have been circumvented and the sequence can be given as Tyr-Ala-Gly-Phe-Leu, with “Leu” indicating leucine or isoleucine. Higher mass resolution for MS-I1would be advantageous because m / z 407 and 411 would be cleanly separated. However, it is likely that mlz 411 and 407 are part of a multiplet and, even at higher mass resolution, it is doubtful that m / z 407 would be identified as a significant ion. Discussion of Other Peptides. The peptide reduction technique coupled with FAB-MS and CAD provided useful information for all of the peptides reported here. In some cases the additional information obtained by studying the reduced peptide was critical in order to assign a sequence with considerable certainty. The usefulness of this method and the types of problems it can be used to solve will be further demonstrated with the examples Tyr-Ile-His-Pro-Phe (YIHPF), Trp-Gly-Gly (WGG), and N-Cbz-Gly-Pro-Gly-Gly-ProAla (N-Cbz-GPGGPA). The CAD spectrum of the (M H)+ of YIHPF (Figure 5A) does not contain enough information to assign a sequence, primarily due to the lack of abundant ions in the high mass region. Fragments N3,C3,and Cz can be identified at masses 414,400, and 263, respectively. If we assume a pentapeptide
+
A.
IYR-It€-HIS-PRO-PHE
67
I 400
I10
100
E,
400
300
200
500
600
REDUCED T Y R - I L E - H I S-PRO-PHE
M/Z Flgure 5. (A) CAD spectrum of the (M 4- H)’ of Tyr-Ile-His-Pro-Phe, m / r 676. (B) CAD spectrum of the (M H)’ of reduced Tyr-IleHis-Pro-Phe, m l z 606.
+
50
6.
I bo
I
io
200
2i0
360
REDUCED T R P - C L Y - C L T
I
50
100
M/Z
150
200
250
+
Flgure 6. (A) CAD spectrum of the (M H)+ of Trp-Gly-Gly, m l r 319. (B)CAD spectrum of the (M H)’ of reduced Trp-Gly-Gly, m l z 277.
+
based on the molecular mass, histidine must be the third amino acid (400 - 263 = mass of a histidine residue). The presence of a tyrosine residue is indicated by the ions m / z 568 and 136. An ion at m / z 513 or 164 would indicate an N-terminal tyrosine, but the meaning of the weak, broad peaks at m / z 511-513 and 164-166 is not clear. The problem of determining sequence based on the CAD spectrum of the original peptide (M + H)”is resolved by studying the spectrum of the (M + H)+ of reduced YIHPF (Figure 5B). Abundant ions are observed at the appropriate shifted masses for N’3, N’3 - 14, N’2, C3,C’3 - 17, and C’2 and confirm the third amino acid as histidine. In addition, abundant ions N’4 and N’4 - 14 are indicators of a C-terminal sequence of ...Pro-Phe. Cr4is not observed; however, C’4 - 17 is confirmation of the N-terminal tyrosine and a sequence of Tyr-Leu-His-Pro-Phecan be assigned. As before, “Leu” must be considered to be generic for leucine or isoleucine. A study of the tripeptide WGG serves to demonstrate that those ions having the same masses in both spectra of the (M + H)’ ions of reduced and the nonreduced peptide may be as important as those which show mass shifts. The only amino acid readily assigned from the CAD spectrum of the (M + H)’ of WGG (Figure 6A) is the C-terminal glycine, identified from ions N2 and N2 - 28. Most of the prominent ions in the
ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
A.
N-CEZ-GLY-PRO-CLI-CLY-PRO-ALA MH-441
1.-111
7'
1179
sponding polyamino alcohols was tested by analyzing mixtures of three and four of the peptides previously described. A study of an equimolar mixture of the peptides Gly-Phe-Ala (GFA), Gly-Leu-Tyr (GLY), Tyr-Ile-His-Pro-Phe (YIHPF), and Tyr-Gly-Gly-Phe-Leu (YGGFL) is presented here. The (M + H)+ ions were seen easily in the FAB spectrum: m / z 676 corresponds to (M + H)+ YIHPF, m / z 556 to (M H)+ YGGFL, m / z 352 to (M + H)+ of GLY, and m / z 294 to the peptide (M + H)+ of GFA. These masses were observed with relative abundances of 2:610:4, respectively, in the spectrum of the mixture even though the peptides were present in equimolar amounts. The CAD spectra acquired for mixture components were nearly identical with those obtained from individual peptides. A split of this mixture, which contained 960 nmol of each peptide, was reduced by using the diborane method described. The two resultant extracts, obtained at different ionic strengths, were studied separately. The FAB spectrum of the low ionic strength extract contained all four of the anticipated (M H)+ ions; however, only the (M + H)+ ions of reduced YIHPF ( m / z 606) and reduced YGGFL (mlz 486) were abundant enough to be recognized as major ions. The (M + H)+of reduced GLY, m / z 310, was of much lower abundance than the nearby ions m / z 294 and 320. The (M + H)+ of reduced GFA, m / z 252, was dwarfed by a large peak at m / z 251. Furthermore, an intense peak was found at m / z 294 which looks like it could be nonreduced GFA. The FAB spectum of the high ionic strength extract was strikingly different. Ions m/z 310 and 252 were now the major non-glycerol ions in the spectrum. The (M + H)' ions of reduced YIHPF and reduced YGGFL were observed, but at very low abundance. In addition, the ions m / z 251 and 294 (fragment ions of reduced YGGFL) were much less abundant than in the spectrum of the first extract. The dramatic difference in the spectra of these two extracts demonstrates that polyamino alcohols are differentially extracted at a given ionic strength (14) and that both extraction steps are essential to recover sufficient amounts of all the polyamino alcohols present. Those extracts in which a given ion was more abundant were used for further CAD studies. In each case, spectra were obtained by using aliquots corresponding to 10-15% of the extract, the equivalent of 100-150 nmol of the original peptide, assuming 100% yield in the reduction (the actual yield was not measured, but it certainly is less than 100%). CAD spectra obtained for the (M + H)+ ions of the polyamino alcohols in admixture were identical with those obtained of (M + H)+ of individual peptides. We also studied a mixture of three peptides: Gly-Leu-Tyr, Gly-Gly-Leu, and N-Cbz-Gly-Pro-Gly-Gly-Pro-Ala. All of the expected (M + H)+ ions were observed for both the original and the reduced mixtures. The CAD spectra were identical with those obtained for (M + H)+ ions of the pure peptides and of the corresponding polyamino alcohols. Evaluation of the Detection Limit. CAD spectra of the hexapeptide N-Cbz-GPGGPA were acquired, using amounts varying from 16 ng to 2.7 pg, in order to evaluate the amount necessary to obtain meaningful sequence data. Twenty scans were averaged for each peptide dissolved in acidified glycerol. Only 15 scans were acquired for the 16 ng sample because the beam was essentially undetectable after 15 scans. Additional acquisitions only reduced the SIN ratio. Spectra obtained by using 2.7 pg, 880 ng, and 440 ng were essentially the same. A 10-V main beam was obtained at a multiplier gain of approximately lo6. Spectra were also acquired for 160 ng, 80 ng, 32 ng, and 16 ng. The ion beam and SIN ratio progressively dropped with lower amounts of peptide; however, 80 ng of the peptide still provided an in-
+
B.
REDUCED N-CEZ-CLY-PRO-GLI-CLI-PRO-I
M/Z Flgure 7. (A) CAD spectrum of the (M H)+ of N-Cbz-Gly-Pro-GlyGly-Pro-Ala, m Ir 589. (B)CAD spectrum of the (M H )' of reduced N-Cbz-Gly-Pro-Gly-GIy-Pro-Ala, m / r 385.
+
+
spectrum are related to the indole ring of tryptophan and do not arise via cleavages of peptide bonds. The N1 or Nz ions, which would allow one to determine the presence of tryptophan and the second glycine, cannot be distinguished. However, by comparing this spectrum to that of the (M + H)+ of reduced WGG (Figure6B), it is observed that ions m/z 130, 144, 159, and 170 (structures 1,2,3, and 4,respectively) are
2 -
-I
WNH3 t
I
O+== - J -Q
-3
A
4 -
k
present in both spectra and, therefore, cannot be the result of CO-NH (or CH,-NH) bond cleavage. This similarity of spectra coupled with the abundant loss of ammonia observed in both spectra would probably lead to the conclusions that an N-terminal tryptophan is present and that the sequence must be Trp-Gly-Gly. The CAD spectrum of the (M + H)+ of the amino-blocked hexapeptide N-Cbz-Gly-Pro-Gly-Gly-Pro-Ala (N-CbzGPGGPA) is another example where there is insufficient sequencing information (Figure 7A). Ions that can be clearly identified are N5,N5 - 28, N4,NB, C3, and C2 In addition one might guess a t Nz (4% relative abundance) and C4 (4% relative abundance). C5 is in the tail of N4 and probably could not be identified. This indicates a C-terminal sequence of ...Gly-Gly-Pro-Ala and leaves 289 not accounted for. Reduction of the peptide results in a dramatic mass shift, from (M H)+ 589 to (M H)+ 385 (Figure 7B). That mass shift would indicate the cleavage of a rather large blocking group. The CAD spectrum of the (M + H)+ of the reduced peptide contains the entire N'j series and its complementary N'j - 14 series. The C'j series is not as prominent, but the abundant C15 and C16 ions are indicative of N-terminal CH3-Gly-Pro. Again, the sequence can be confidently and correctly assigned. Application to Mixtures. The capability of MS/MS for studying mixtures of peptides and mixtures of their corre-
+
+
+
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
terpretable CAD spectrum of (M + H)' at a multiplier gain of 107. As the concentration of peptide was decreased, changes in the CAD spectra occurred. The ion resulting by loss of water increased in relative abundance, as did the ion mlz 497, (M + H - 92)'. These ions probably result from the losses of water and glycerol from a glycerol matrix ion. A likely candidate for the interference is (glycerol, + K - 2H)+. As the concentration of the peptide was decreased, the relative proportion of the matrix ion in the main beam increased. No attempt was made to evaluate the detection limit for the polyamino alcohols because no appropriate standards were readily available. Registry No. Tyr-Ala-Gly-Phe-Leu, 60284-47-1;Tyr-GlyGly-Phe-Leu, 58822-25-6;Tyr-Ile-His-Pro-Phe, 52530-60-6; N Cbz-Gly-Pro-Gly-Gly-Pro-Ala, 13075-38-2; Ala-Leu-Trp(F0R)Asn-Arg-Ala, 95935-87-8; Trp-Gly-Gly,20762-31-6; Gly-Leu-Tyr, 4306-24-5;Gly-Gly-Leu, 14857-82-0;Tyr-Ala-Gly-Phe-Leupolyamino alcohol, 95935-88-9;Tyr-Gly-Gly-Phe-Leupolyamino alcohol, 95935-89-0; Tyr-Ile-His-Pro-Phe polyamino alcohol, 95935-90-3;N-Cbz-Gly-Pro-Gly-Gly-Pro-Ala polyamino alcohol, 95935-91-4; Trp-Gly-Gly polyamino alcohol, 95935-92-5; GlyLeu-Tyr polyamino alcohol, 95935-93-6;Gly-Gly-Leupolyamino alcohol, 95935-94-7.
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(19) Morrls, H. R.; Panico, M.; Barber, M.; Bordoli, R. S.;Sedgwick, R. C.; Tyler, A. Biochem. Biophys. Res. Commun. 1981, 107, 623-631. (20) Morris, H. R. Int. J. Mass Spectrom. Ion Phys. 1982, 4 5 , 331-342. (21) Williams, D. H.; Bradley, C. V.; Santikarn, S.; Bojesen, G. Biochem. J. 1982, 201, 105-117. (22) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N.; Garner, G. V.; Gordon, D. B.; Tetler, L. W.; Hider, R. Biomed. Mass Spectrom. 1982, 9 , 265-268. (23) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. Biomed. Mass Spectrom. 1982, 9 , 208-214. (24) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N.; Whalley, E. T. Biomed. Mass Spectrom. 1981, 8 , 337-342. (25) Barber, M.; Bordoli, R. S.; Garner, G. V.; Gordon, D. B.; Sedgwick, R . D.; Tetler, L. W.; Tyler, A. N. Biochem. J. 1981, 797, 401. (26) Desiderio, D. M.; Katakuse, I. Biomed. Mass Spectrom. 1984, 11. 55-59. (27) Buko, A. M.; Phillips, L. R.; Fraser, 8. A. Biomed. Mass Spectrom. 1983, 10, 324-333. (28) Buko, A. M.; Philllps, L. R. and Fraser, B. A. Biomed. Mass Spectrom. 1983, IO, 407-419. (29) Garner, G. V.; Gordon, D. B.; Tetler, L. W.; Sedgwick, R. D. Org. Mass Spectrom. 1983, 18, 486-488. (30) Rose, M. E.; Prescott, M. C.; Wilby, A. H.; Galpin, I. J. Biomed. Mass Spectrom. 1984, 1 1 , 10-23. (31) Bradley, C. V.; Williams, D. H.; Hanley, M. R. Biochem. Biophys. Res. Commun. 1982, 104, 1223-1230. (32) Self, R.; Parente, A. Biomed. Mass Spectrom. 1983, IO, 78-82. (33) Williams, D. H.; Santikarn, S.; Oelrlchs, P. B.; DeAngelis, F.; MacLeod, J. K.; Smith, R. J. J. Chem. SOC., Chem. Commun. 1982, 1394-1396. (34) Blemann, K. Int. J. Mass Spectrom. Ion Phys. 1983, 4 5 , 183-194. (35) Takao, T.; Hitouji, T.; Shimonishi, Y.; Tanabe, T.; Inuye, S.; Inouye, M. J . Bioi. Chem. 1984, 259, 6105-6109. (36) Morris, H. R.; Panico, M.; Karplus, A,; Lloyd, P. E.;Rinlker, B. Nature (London)1982, 300, 643-645. (37) Barber, M.; Bordoli, R. S.; Elliott, G. J.; Horoch, N. J.; Green, B. N. Biochem. Biophys. Res. Commun. 1983, 110, 753-757. (38) Wada, Y.; Hayashl, A.; Masanori, F.; Katakause, I.; Ichihara, T.; Nakabushi, H.; Matsuo, T.; Sakurai, T.; Matsuda, H. Biochim. Biophys. Acta 1983, 749, 244-248. (39) Crow, F. W.; Tomer, K. B.; Gross, M. L. Mass Spectrom. Rev. 1983, 2 , 47-76. (40) Hunt, D. F.; Bone, W. M.; Shabanowitz, J.; Rhodes, J.; Ballard, J. M. Anal. Chem. 1981, 5 3 , 1704-1706. (41) Heerma, W.; Kamerling, J. P.; Slotboom, A. J.; van Scharrenburg, G. J. M.; Green, B. M.; Lewis, I. A. A. Biomed. Mass Spectrom. 1983, IO, 13-16. (42) Tomer, K. 8.; Crow, F. W.; Gross, M. L.; Kopple, K. D. Anal. Chem. 1984, 5 6 , 880-886. (43) Katakuse, I.; Desiderio, D. M. Int. J. Mass Spectrom. Ion Proc. 1983, 5 4 , 1-15. (44) Desiderio, D. M.; Katakusei, I. Anal. Biochem. 1983, 129, 425-429. (45) Neumann, G. M.; Derrick, P. J. Org. Mass Spectrom. 1984, 19, 165-1 70. (46) Gross, M. L.; Chess, E. K.; Lyon, P. A.; Crow, F. W.; Evans, S.; Tudge, H. Int. J. Mass Spectrom. Ion Phys. 1982, 4 2 , 243.
RECEIVED for review December 26,1984. Accepted February 25,1985. This research was supported by the Midwest Center for Mass Spectrometry, an NSF regional instrumentation facility (Grant No. CHE 8211164).
