Field desorption-collisional activation mass spectrometry with

416

Anal. Chem. 1981, 53, 416-421

Field Desorption-Collisional Activation Mass Spectrometry with Accumulated Linked-Scan Technique for Peptide Structure Elucidation Takekiyo Matsuo' and Hlsashi Matsuda Institute of Physics, College of General Education, Osaka University, Toyonaka 560, Japan

Itsuo Katakuse Department of Physics, faculty of Science, Osaka University, Toyonaka 560, Japan

Yasutsugu Shimonishi Institute for Protein Research, Osaka University, Suita 565, Japan

Yusuke Maruyama, Tetsuo Higuchi, and Eiji Kubota JEOL Ltd., Nakagami Akishirna, Tokyo 197, Japan

Collisional activation (CA) mass spectra of field desorbed peptides were Investigated. Protonated molecular ions (M 4H)' and sodium cluster Ions (M Na)' of a hexapeptide amido, dipeptide hydrazide, pentapeptide hydrazide, and hexapeptlde were examined. Since prominent N-terminal sequence peaks were observed, R was posdbie to elucidate peptide sequences from these CA mass spectra.

+

Metastable ion peaks are important for the structural analysis of complex compounds. Since the technique called MIKES (mass analyzed ion kinetic energy spectrometry) (1) or CA (collisional activation) (2)was introduced, it has been effectively used for structure elucidation. It may be helpful to utilize this technique in sequencing peptides, e.g., N-termind sequencing peaks have been clearly observed in electron impact (E1)-MIKES experiments performed on derivatized peptides by using a double focusing mass spectrometer with inverse geometry ( 3 , 4 ) . There are two technical limitations on this method using free and rather large peptides; one is low resolving power (200-300) and the other is the difficulty involved in producing molecular or protonated molecular ions of free peptides. However, these difficulties may be avoided by using the following techniques: Considerable improvement in the resolving power may be expected by applying the linked-scan technique (5), and the protonated molecular ions of large free peptides up to m/z 3000 can be obtained (6)with field desorption ionization (7). In the present work, we have studied the possibility of amino acid sequencing of free peptides, peptide amides, and peptide hydrazides by using the techniques described above. Furthermore, we have recently developed a computer system which accumulates the CA mass spectra of a linked-scan mode. Since the signals of the CA mass spectra are normally very weak, even if molecular or protonated molecular ions are produced in a FD ion source, a satisfactory signal/noise (S/N) ratio cannot be obtained by a single scanning. The computer-accumulated system with a reversed-geometry double focusing spectrometer has already been reported (8). EXPERIMENTAL SECTION Mass Spectrometer. Mass spectra with electric and magnetic linked scan and the normal magnetic scan were obtained with 0003-2700/81/0353-0416$01.00/0

a JMS-DBOO (JEOL) double focusing mass spectrometer and a Matsuda-type double focusing mass spectrometer (9). Both carbon (IO) and silicon (11) emitters were used. The JMA-2000 data analysis system (JEOL) was used for data acquisition, accumulation, and treatment of the CA mass spectra. The linked-scan speed was 10,20, or 60 s. During a measurement, the linked scan was repeated approximately 50-100 times. Helium gas was used as the collision gas. Its pressure was controlled so that the intensity of the precursor ion was decreased by the introduction of gas into the collision chamber to one-third the initial intensity. The pressure in the collision chamber was estimated to be approximately 10-2Pa under such conditions, The collision chamber (18 mm length) was located between the source slit and the ion source block. Accumulated Linked-Scan System. When a Hall element was used as a sensor, the output signal of the sensor was not linearly proportional to the magnetic field strength due to its S-type characteristic curve. In a normal scan, the mass calibrating procedure is done by the software of the computer. In this study, when using the linked-scan mode, the output signal of the Hall probe was corrected by means of the hardware (magnetic field linearizer), so that the output was always directly proportional to the magnetic field strength. The configuration of a linked-scan system is shown in Figure 1. The following abbreviations are used to identify two special terms: B/E scan, the type of linked scan in which magnetic field B and electric field E are scanned simultaneously such that B/E is constant (5);B2/E scan, the type of linked scan in which B and E are scannec! simultaneously such that B2/E is constant (5). The output signal of digital to analogue converter (DAC), 1,was used as a reference voltage for both the electric field, 2, and magnetic field, 3, power supply. The system can be operated in either the B/E = constant mode or the B2/E = constant mode with switch 5. When it is set in position a, the system works as B2/E mode and when in position b, the system runs as a B/E mode. The potentiometer marked 7 is provided in order to set the initial m / z value of the scan. The precursor ion (for a B/E scan) or daughter ion (for a B2/E scan) can be set by adjusting the potentiometer appropriately. The magnetic field linearizer consists of an analogue to digital converter (ADC), a read only memory (ROM), and a digital to analogue converter (DAC). After measurement of the characteristic curve of a Hall probe, it linearizes the effective magnetic field strength. The electric field strength is controlled in proportion to the scan signal from DAC, 1. This scan signal is inverted to the abscissa (mass scale) of the spectrum. The EECA mass spectrum of perfluorokercwene (pK)was used for mass calibration of the linked scan system. Since the predominant peaks can be interpreted as CA fragments of C,F,+, 0 1981 American Chemlcal Society

ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981 JMA2000 DATA ANALYSIS SYSTEM

Research, Osaka University. The products, except for H-AlaLeu-Leu-Ser-Ser-NzH3,were confirmed to be homogeneous on a thin-layer chromatogram (Merck silica-gel G Art. 7731) and a paper electropherogram (pH 4.8, pyridine/acetate buffer), and also by an amino acid analysis of the acid hydrolysates. H-AlaLeu-Leu-Ser-Ser-NzH3was used without purification for mass measurements.

COMPUTER ( T I 9808)

16 B I T D/A CONVERTER I

1 POWER SUPPLY

I ON

RESULTS AND DISCUSSION Hexapeptide Amide (H-Lys-Phe-IleGly-Leu-Met-NH2). A normal FD mass spectrum is shown in Figure 2. From this spectrum we can deduce that the mass peak a t m/z = 707 is the protonated molecular peak, the peak at m/z = 1413 is a protonated dimolecular peak, and m / z = 463 is a fragment peak. Sodium and potassium cluster peaks (M + Na)+ and (M + K)+ are observed at m / z = 729 and 1435 and m / z = 745 and 1451, respectively. We took the field desorption-collisional activation (FD-CA) mass spectra in the B/E scan mode selecting the protonated molecular peak (M + H+ = 707) as a precursor ion. The CA spectrum is shown in Figure 3. We can clearly recognize the predominant N-terminal sequence peaks. C-terminal sequence peaks were not clearly observed. Two fragment ions (m/z = 632,650) were produced by the loss of a side chain of amino acids due to simple cleavage accompanied by a proton transfer reaction. I t is possible to deduce the partial sequence from this CA linked-scan mass spectrum as Gly-Leu-Met-NH2. If the mass range is between m/z = 100 and 707, all N-termind sequence peaks may be clearly observed. Secondly, we chose the intense peak a t m/z = 463 as a precursor in order to determine the origin of this peak. It is difficult to distinguish between a fragment peak or another impurity peptide in the normal FD spectrum in Figure 2. The CA mass spectrum of this peak is shown in Figure 4. Since the predominant peaks in this figure can be interpreted as N-terminal sequence peaks, the intense peak which was observed in the normal FD spectrum a t m / z = 463 is clearly a fragment peak indicating cleavage between glycine and leucine. The intensity of amine and aminoacyl fragments is rather strong. C-terminal sequence peaks were also observed at m / z = 333 and 361. The partial sequence can be deduced from the observed N- and C-terminal sequence peaks as H-LysPhe-Ile-Gly-NH,. To determine the origin of this peak, we

l6

POWER SUPPLY

(linked scan)

(normal scan)

- ELECTRIC

SOURCE

1

LINYARIZER

MAGNETIC DETECTOR

FIELD

...

FIELD

JMS D 3 0 0 MASS SPECTROMETER

Figure 1. Configuration of the accumulated linked-scan system.

and it is easy to determine the mass numbers of these intense peaks, the EI-CA linked-scan mass spectrum of PFK is a very effective mass reference. Sample Preparation. The peptide (H-Val-Tyr-Ile-His-ProPhe-OH) and peptide derivatives (H-Lys-Phe-Ile-Gly-Leu-MetNHz and H-Ala-Leu-Leu-Ser-Ser-NzH~ were prepared from their protected intermediates (p-methoxybenzyloxycarbonyl-valTyr-Ile-His-Pro-Phe-OBzl, di-tert-butoxycarbonyl-Lys-Phe-IleGly-Leu-Met-NHz,and tert-butoxycarbonyl-Ala-Leu-Leu-SerSer-NzH3,respectively) by conventional methods (12) and purified by ion-exchange chromatography in the Institute for Protein H-Lys-

P h e - l le- G l y - L e u - M e t - N H 2

M+ H 707 463

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418

ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981 I le

Phe

LYS

:'N 2

C H 61

cy 2

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Flgure 3. CA linked-scan mass spectrum of a peptide amMe. The protonated molecular Ion ( m l z = 707)was chosen as a precusor ion. Mass range of linked scan was from m l z = 707 to m l z = 354. LY.

11.

Pha

0 lY

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Figure 4. CA linked-scan mass spectrum of a peptWe amide (KLys-Phe-Ile-Gly-Leu-Met-NH2). The ions at m l z = 463 were chosen as precursor ions. The mass range of a linked scan was from m l z = 463 to m l z = 230. used a B2/E linked scan. The broad peak was observed near m / z = 940. The origin of this peak is not clear. Hardly any peak was observed near at m / z = 707. Thirdly, the linked-scan CA mass spectrum was then taken from a sodium cluster (M + Na)+ as shown in Figure 5. A very intense molecular peak (M+. = 706) was observed. The intensity ratio of (M+.)/(M Na)+ was 0.5-0.7. In spite of a low intensity, N-terminal sequence peaks were observed. It was interesting to note that no distinguishable N-terminal sequence peaks retaining sodium were observed. In addition, it was reported that the alkali ion is predominantly retained in the fragment ions of oligosaccharides (13,14). The binding between a peptide amide and a sodium is weak enough to be

+

broken by helium gas collision. Pentapeptide Hydrazide (H-Ala-Leu-Leu-Ser-SerNH-NH2). In the normal FD mass spectrum of this compound, a protonated molecular ion is observed at m / z 504. Cationated peaks for (M + Na)+ and (M K)+ are observed at m / z = 526 and m/z = 542, respectively. The CA mass spectrum from the protonated molecular ion is shown in Figure 6. In this case, intense N-terminal sequence peaks were observed. C-terminal sequence peaks were &o observed. Amino fragments of m / z = 357 and 444 were not observed. Some peaks at m/z = 227,241, and 447 were not interpreted. The partial sequence ...Leu-Ser-Ser-NH-NH2may be deduced from the spectrum.

+

ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981 LY.

Ph.

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600

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Flgure 5. CA linked-scan mass spectrum of a peptide amide. The sodium cluster ion ( m l z = 729) was chosen as a precursor ion. The mass range of linked scan was from m l z = 729 to m / z = 365. AI.

Lau

Leu

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OH

OH

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185

Flgure 6. CA linked-scan mass spectrum of a pep* scan was from m l z = 504 to m / z = 100.

/

298

385

4 1 2 487

503

hydrazide. The precursor ion was the protonated molecular ion. The mass range of linked

The CA mass spectrum from the sodium cluster ( m / z = 526) was recorded. Very intense molecular peaks were also observed a t m / z = 503. N-terminal sequence peaks (aminoacyl fragments) without sodium attachment were clearly observed. Dipeptide Hydrazide (H-Val-Tyr-N2HJ). For highly polarized compounds, multiple cluster peaks (M H, 2M + H, 3M + H, ...) are often observed in the normal FD mass spectrum. It was considered interesting to note what kind of CA spectrum was obtained when a multiple cluster ion was selected as a precursor ion. For this purpose a small hydrazide was used. In its normal FD mass spectrum, the following peaks were observed: M+' = 294, (M + H)+ = 295, (2M + H)+ = 589, and (3M + H)+= 883. The (2M + HI+ion was selected

+

as a precursor ion. The CA maas spectrum is shown in Figure 7. The predominant peak represented only (M + H)+ a expected. Small peaks at mlz = 196,235, and 263 supplied information concerning the structure of a monomer. There were no peaks between m / z = 295 and 589 except at m / z = 514 which was interpreted as being due to (2M + H - CHB)+.Next, the (3M + H)+ion was used as a precursor ion. The CA mass spectra showed two prominent peaks a t m/z = 295 (M + H)+ and m/z = 589 (2M + H)+.The intensity of these two peaks was greater than that of the precursor ion (3M + H)+. Hexapeptide (H-Val-Tyr-Ile-His-Pro-Phe-OH). We studied a free peptide to see if it also produced N- or C-terminal sequence peaks in the CA mass spectrum. The most

420

ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981

H-VOl-Tyt

-N,H,

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prominent peaks in the normal FD spectrum were at m / z = 775 (M + H)+and m/z = 797 (M+ Na)'. The CA mass spectrum from the protonated molecular ion is shown in Figure 8. N-terminal sequence peaks were clearly observed at m / z = 235, 263, 485, 513, and 582. Next, the CA mass spectrum from the sodium cluster (M + Na)+ was studied. The raw spectrum shown on the UV recorder is also shown in Figure 9. An intense molecular peak was observed at m/z = 774. The daughter ion resolution of the B/E scan was estimated at more than loo0 in Figure 9. N-terminal sequence peaks were also observed in this case.

CONCLUSIONS Owing to the capability of data accumulation by the computer, we can add each CA mass spectrum in the linked-scan mode as many times as we like. When the accumulated data from a single sample are not sufficient, the data from another loading can also be added over and over again. We can thus

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obtain good CA mass spectra even from weak FD signals in the linked-scan mode. For a free hexapeptide, a hexapeptide amide, and a pentapeptide hydrazide, prominent N-terminal sequence peaks were observed. From these peaks it is possible to determine peptide sequence. If the precursor ions are protonated molecular ions or sodium cluster ions, an intense peak for the molecular ion M+*may be observed in the CA spectrum. If the precursor is a nth multiple cluster ion, then the CA mass spectra also contain multiple cluster ions (M H, 2M + H, -, (n l ) M + H). When any intense peak is observed in a normal FD spectrum, the character of this peak can be interpreted as (M H)+, (2M + HI+, (3M + H)+, or (M + Na)+ by simply taking a CA linked-scan mass spectrum. Many skillful methods of peptide sequencing using mass spectrometry have been reported (15-17) and recently we proposed still another method of peptide sequencing by combining Edman degradation and FD mass spectrometry

+

-

+

Anal. Chem. 1981, 53, 421-427 M+Na

M

v 760

770

790

780

Flgure 9. Raw CA linked-scan mass spectrum of a free peptide (H-Val-Tyr-Iie-His-Pro-Phe-OH). The precursor ion was the sodium cluster ion ( m l z = 797). An intense molecular peak was observed.

421

(3) Schuiunegger, U. P. Angew. Chem. 1075, 14, 679-688. (4) Levsen, K.; Wipf, H.-K.; Mclafferty,F. W. Org. Mess Spectrom.1074, 8 , 117-128. (5) Bruins, A. P.; Jennings, K. R.; Evans, S. Int. J. Mess Specfrom. Ion P h y ~1078, . 26, 395-404. (6) Matsuo, T.; Mats&, H.; Katakuse, I.; Wada, Y.; Fujita, T.; Hayashi, A. 6 b M . Mass Specfrom., in press. (7) Beckey, H. D. “Principle of Field Desorption Mass Spectrometry”; Pergamon Press: New Yo&, 1977. (8) Wachs. T.; Van de Sande, C. C.; Bente, P. F., III; Dymerski, P. P.; McLafferty. F. W. Int. J. Mess Spectrom. Ion Phys. 1077, 23, 21-27. (9) Matsuda, H. Inf. J . Mess Specfrom. Ion. Phys. 1074, 14, 219-233. (IO) Schuiten, H. R.; Beckey, H. D. Org. Mess Spectrom. 1072, 6 , 885-895. (11) Matsuo, T.; Matsuda, H.; Katakuse. I. Anal. Chem. 1070, 51, 69-72. (12) Bodanszky, M.; Klausner, Y. S.; Ondetti, M. A. “Peptide Synthesis”, 2nd ed.;Wiley: New York, 1976;pp 18-84. (13) Roilgen, F. W.; Giessmann, U.; Borchers, F.; Levsen, K. Org. Msss Specfrom. 1078, 13, 459-461. (14) Kambara, H.; Burlingame. A. L. Proceedings of the 27th Annual Conference on Mass Spectromeby and Allied Topics, Seattle, WA, 1979; on 897-69s. rr

(18-20). The FD-CA method using metastable ions and the

method introduced above complement each other and provide valuable information in the structural analysis of peptides. ACKNOWLEDGMENT The authors acknowledge helpful discussions with Y. Izumi, Institute of Protein Research, Osaka University. LITERATURE CITED (1) Cooks, R. G.; Beynon, J. H.; Caprioii, R. M.; Lester, G. R. “Metastable Ions”, Elsevier: Amsterdam, 1973;Chapter 3. (2) Mclafferty, F. W.; Bente, P. F., 111; Kornfekl, R. T.; Sal, S . C . ; Howe, I. J . Am. Chem. SOC.1073, 95, 2120-2129.

(15) Biemann, K.; Cone. C.; Webster, B. R.; Arsenauk, 0. P. J . Am. Chem. Sm. 1068, 88, 5598-5606. (16) Monis, H. R.; Williams. D. H.; Ambler, R. P. Bbchem. J. 1071, 125, 189-201. (17) Wipf, H.-K.; Irving, P.; McCamish, M.; Venkataraghavan, R.; McLafferty, F. W. J. Am. Chem. Soc. 1073, 95, 3389-3375. (18) Shimonishi, Y.; Hong, Y. M.; Matsuo, T.; Katakuse, I.; Matsuda, H. Chem. Left. 1070, 1369-1372. (19) Matsuo, T.; Matsuda, H.; Katakuse, I. A&. Mess Spectrom. 1080, BA, 990-996. (20) Matsuo, T.; Matsuda, H.; Katakuse, I. Shltsutyo Bunseki 1080, 28, 169-174.

RECEIVED for review September 26,1980. Accepted December 10, 1980.

Scans for Preselected Neutral Fragment Loss in Double-Focusing Mass Spectrometry Bori Shushan and Robert K. Boyd’ Guelph-Waterloo Centre for Graduate Work in Chemistty, University of Guelph, Guelph, Ontario, Canada N7G 2W7

The theory of linked scans of the fields of a double-focusing mass spectrometer, for a preselected mass of the neutral fragment from decompositions of ions In the first field-free region, is presented. One of the scan laws thus derived has been described previously, but the other Is new. The problem of artifact peaks in such scans, and the theory and practice of the method of ac modulation in identifying them, is described with reference to a model system, 1,ldiphenylethane. Application of these techniques to identification of polychlorinated biphenyls, present at levels of order 1 % in compkx matrices of other compounds, illustrates both the power and llmltations of the method.

Interpretation of the mass spectrum of an unknown substance is usually based upon reasonable assumptions concerning the nature of the fragments produced in ionic decompositions of the type ml+

-

m2+

+ m,

(1) A conventional electron impact (EI) mass spectrum reflects all those ions formed in the ion source and thus corresponds to a superposition of several fragmentation reactions described 0003-2700/81/0353-0421$01.00/0

by eq 1 with varying ml+ and/or m2+. It is possible to study specific reactions of the general type (eq 1)by investigating only those fragmentations which occur in the field-free regions in a mass spectrometer. Such techniques available up to about 1973 have been fully discussed in a recent monograph (1).The well-established value of “metastable peaks”, which underlie the conventional mass spectrum, is limited (1) by ambiguity in the interpretation of the apparent mass mt = (mZ2/ml). Consequently, various unorthodox methods of scanning double-focusing mass spectrometers have been developed, in order to study specified reactions of the type (I). For many purposes, it is most convenient to specify the parent ion ml+ and to generate a spectrum of all its daughter ions m2+. Historically, this was first achieved by use of reversed-sector instruments in which the magnetic sector precedes the electric sector; the magnetic field strength B is set to preselect m,+, and the various ions m2+ produced by fragmentation (spontaneous or collision induced) of ml+, in the field-free region immediately following the magnet, are successively detected by scanning the electric sector field E downward (2,3)while the accelerating voltage V is unchanged. Until recently, such daughter-ion spectra could not be obtained on instruments designed in the more orthodox configuration in which the electric sector precedes the magnet. However, methods are now available whereby scanning of 0 1981 American Chemical Society