ANALYTICAL
67, 3.59-37 1 ( 1975)
BIOCHEMISTRY
Sequence Determination of Peptides by Mass Spectrometry: Methods for Combining “Wet” Separation Techniques with Mass Spectrometry H. FALTER' Department
of Chemistry,
Laurentian
University,
K. JAYASIMHULU Department
of Chemistry,
University
Sudbury.
Ontario,
Canada
AND R. A. DAY of Cincinnati,
Cincinnati,
Ohio
4.5221
Received December 26, 1973; accepted January 17, 1975 Methods are described that allow the combination of established techniques for peptide separation, paper chromatography and electrophoresis, with mass spectrometry. The development of these methods is part of an ongoing effort in the search for a methodology for the systematic utilization of mass spectrometry for the elucidation of primary structure of proteins and peptides. Peptides and ammo acids are detected on chromatograms by conversion to covalent derivatives that are also suitable for mass spectrometry. The most useful reagents for detection and derivatization of peptides reported here are dansyl chloride, N,Ndimethylaminobenzaldehyde. N,N-dimethylaminocinnamaldehyde, and N-hy droxysuccinimido /3-naphthoate. Detection limits and mass spectra for some of these derivatives are reported.
Recently there have been some significant successes in obtaining the sequences of peptides from proteins and, in some cases, complete sequences of small proteins by mass spectrometry using various techniques. These either require sophisticated equipment or use of elaborate sample preparations (l-3). Proteins and large peptides themselves do not yield mass spectra suitable for sequence analysis. In order to employ mass spectrometry for amino acid sequence determination, it is necessary to degrade larger peptides into smaller ones, which can be converted to volatile derivatives. Generally speaking therefore, a large number of peptides would have to be generated in order to provide sufficient information for the assignment of the amino acid sequence of a protein. The analysis of complex mixtures of peptides, without prior separation, is a difficult matter that requires exact mass, partial vaporization, and metastable-ion data (4). While a degree of success in the analysis of 1 To whom correspondence concerning this paper should be addressed. 359 CopYright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
360
FALTER,
JAYASIMHULU
AND
DAY
peptide mixtures by a variety of approaches (5-10) gives cause for optimism, it is clear that these methods are not yet generally applicable. The separation of mixtures of peptides by paper chromatography and electrophoresis is well known. Peptides are usually located on chromatograms by means of the ninhydrin reaction. Unfortunately, the formation of Ruhemann’s purple is accompanied by the degradation of peptides (11). We have endeavored to utilize reagents for the detection of amino acids and peptides which do not cause their degradation (12). These reagents must form covalently bonded derivatives, also suitable for mass spectrometry (13). The derivatives can then be eluted, estermed, and subjected to mass spectrometry. MATERIALS
AND
METHODS
Materials
The peptide tetra-r.-phenylalanine was obtained from Cyclochemical. NJ-Dimethylaminobenzaldehyde was obtained from Fisher Scientific Co. Dansyl chloride (5,dimethylamino1-naphthalenesulfonyl chloride), lysozyme, trypsin, and a-chymotrypsin were obtained from Sigma Chemical Co. Solvents used for elution of the peptide derivatives from the paper were glass distilled, reagent grade. Paper Chromatography
and Electrophoresis
Commercially available amino acids and peptides were used without further purification. Varying amounts (characteristically 0.02, 0.05 and 0.10 pmole) were spotted on Whatman 3MM chromatography paper and then chromatographed in the descending direction using a butanol : acetic acid: H,O (4 : 1: 5, v/v/v) solvent system. The chromatograms were dried extensively before reaction with the detection reagents. In the case of chymotryptic peptides of lysozyme, electrophoresis was carried out at a voltage of 15 V/cm using a pyridine:acetic acid:water (100:4:900, v/v/v) system at pH 6.5 before chromatographing in the second dimension as described above. Peptide maps were prepared from tryptic digests of carboxymethylated egg-white lysozyme essentially by the method of Canfield (14). Approximately 3 mg of carboxymethylated lysozyme digest were used for the preparation of each map. Reagents
for the Detection
of Amino
Acids and Peptides
a) Dunsyl chloride. Dansyl chloride (100 mg) was dissolved in acetone (100 ml) and the solution was filtered to remove traces of insoluble particles. Immediately prior to use, 3 ml of triethylamine was added to
AMINO
ACID
SEQUENCING
BY
MASS
SPECTROMETRY
361
the solution. Chromatograms were dipped in an open tray for about 1 min and then dried in an oven at 65°C for 10 min. Spots were detected by their yellow color and, in the dark, with a long-wavelength uv lamp (Black Ray, UVL-21, Ultraviolet Products Inc., San Gabriel, CA). b) N,N-Dimethyluminobenzaldehyde (DMB). DMB (100 mg) was dissolved in 100 ml of anhydrous ethyl ether. Acetic acid ( 1 ml) was added immediately before the solution was used. Chromatograms were dipped, or sprayed uniformly, and dried in an oven for 10 min at 105-l 10°C. The more intense spots were yellow against a white background. Greater sensitivity could be achieved with a short-wavelength uv light (Mineralight UVL-12, Ultraviolet Products, Inc.). The best contrasts were obtained by placing the chromatograms in an open tray containing liquid nitrogen and removing the uv light after a short exposure. Brilliantly phosphorescent spots persisted long enough to allow marking with a pencil. c) N,N-Dimethylaminocinnama~dehyde (DMC). DMC (5 mg) was dissolved in 100 ml of dioxane. Triethylamine (1 ml) was added immediately before the solution was used. Chromatograms were dipped, then dried in an oven for a short time. Spots were a deep red to reddish brown. 6) N,-Hydroxysuccinimido p-naphthoate (HSN). HSN (60 mg) was dissolved in 100 ml of chloroform, and 1 ml of triethylamine was added immediately before use. Chromatograms were dipped in an open tray for about 1 min. The chromatograms were then rinsed with benzene by chromatographing in this solvent overnight. After drying in an oven at 110°C for a few minutes, spots could be seen with a long-wavelength uv lamp. Elution
and EsteriJication
of Peptides
The spots were cut out, placed in 17 X 1.4-cm distilling columns with 14/20 standard taper joints and indented supports and eluted with 2-3 ml of refluxing methanol or ethanol contained in 5- or IO-ml roundbottom flasks. The distilling columns were removed after an hour, two drops of thionyl chloride were added to the alcoholic extracts, and the refluxing was continued for 30 min. The solutions were then concentrated in a rotary evaporator, and appropriate portions were transferred to the sample probes used in the solid inlet probe of the mass spectrometer by successive transfers of small volumes and intermittent drying with a stream of hot air. Typically - 0.1 pmole of each sample was transferred to the sample probe. Mass Spectrometq
Mass spectra were obtained with a Hitachi-Perkin-Elmer RMU-7 mass spectrometer operating in a low resolution mode with a resolution
362
FALTER,
JAYASIMHULU
AND
DAY
setting of 1200 (calculated at two adjacent peaks with about 90% separation). The source temperature (chamber temperature) is usually maintained at 250°C while working with peptides, and the sample heater into which the sample probe is inserted is heated slowly until the molecular ion is seen, at which time the spectra were recorded repeatedly. The temperature at which an intense molecular ion was observed is recorded. The temperature, the electron beam energy and the accelerating voltage used are noted in the captions to the various figures. RESULTS
Table 1 shows a number of reagents, that can be used to detect amino acids on paper chromatograms. All of these reagents form covalent derivatives with the free amino group(s) of amino acids and peptides. Therefore, detection and blocking of the amino terminus and any sidechain amino group can be accomplished in one step. Not all amino-blocking groups are equally suitable from the point of view of yielding mass spectra that contain sequence information (13,15,16). The mass spectrometric suitability of these blocking groups has been investigated previously (13,16) by comparing the spectra obtained from derivatives of the model peptide Val-Ile-Ala. Partially as a result of this earlier study (13) and partially because of less sensitive detection levels, a number of other reagents which can be used to detect amino acids on paper chromatograms were rejected. These reagents included: phthalic anhydride, N-hydroxysuccinimido (Ynaphthoate, naphthalene-1,8-dicarboxylic acid anhydride, cr- and pDETECTION
AGENTS
TABLE FOR AMINO
Reagent
Materials detected
Dansyl chloride a
All common amino acids Maps of tryptic digest of carboxymethylated lysozyme Selected amino acids Maps of chymotryptic digest of carboxymethylated lysozyme All common amino acids Maps of tryptic digest of carboxymethylated lysozyme All common amino acids
DMB DMC HSN
1 ACIDS
AND PEPTIDES
Comment Detection level less than 0.05 ~moleb~c Most ninhydrin spots are detectable: see text Detection level less than 0.02 pmoleb All ninhydrin spots are detectable; see text Detection level less than 0.05 pmole b,c Most ninhydrin spots are detectable Detection level less than 0. I pmole
a Abbreviations: 5-Dimethylamino-I-naphthalenesulfonyl chloride (dansyl N,N-dimethylaminobenzaldehyde (DMB); N,N-dimethylaminocinnamaldehyde N-hydroxysuccinimido p-naphthoate (HSN). b After chromatography in one dimension. c Most amino acids are still detected in amounts of 0.02 umole.
chloride); (DMC);
AMINO
ACID
SEQUENCING
BY
MASS
SPECTROMETRY
363
naphthalenesulfonyl chloride, a+ and @chloromethylnaphthalene, and 2,3-his-(bromomethyl)-naphthalene. All of the reagents listed in Table 1 can be used to detect amino acids on chromatograms in amounts of less than 0.10 pmole after these have been chromatographed in one dimension. Many of the amino acids can be detected at substantially lower levels. After separation in two dimensions, especially if one of these involves electrophoresis, the contrast between the spots and the background decreases somewhat. Peptide maps from tryptic and chymotryptic digestion of carboxymethylated lysozyme were used to test the feasibility of detecting peptides from the proteolytic digest of a protein. The maps obtained were compared with maps developed with ninhydrin and, in the case of the tryptic digests, to the published map of Canfield (14). When the tryptic peptide maps were developed with dansyl chloride, the contrast in each case was less than when duplicate maps were developed with ninhydrin. Clearly separated spots could readily be recognized on the dansyl maps. However, mainly in the central region of the map, where a number of relatively large peptides are crowded together, the contrast did not permit the unambiguous assignment of all the spots detected with ninhydrin. When somewhat larger amounts of material can be used, and especially if separation in only one dimension is required, the technique is highly satisfactory. Figure 1 shows the mass spectrum of the peptide tetra-Phe, which was spotted in amounts of 0.5 pmole on Whatman 3MM paper, chromatographed in one dimension and then detected on the chromatogram with dansyl chloride. This mass spectrum illustrates some of the aspects, which make the dansyl group a desirable amino-terminus blocking group. The M* ion is prominent and the ions produced by the fragmentation of the side chains are lower than might be anticipated. In a peptide containing four phenylalanine side-chains it might have been quite reasonable to expect these peaks to be very intense. It can be easily shown, by chromatography followed by color development with ninhydrin, that in the detection procedure with dansyl chloride only a fraction of a given peptide is derivatized. When relatively large amounts of a peptide are used, i.e., 0.5 pmole of tetraphenylalanine, a satisfactory mass spectrum can be obtained directly. When it is essential that smaller amounts be used, a further step might be added in which that fraction of the peptide which was not derivatized during detection is derivatized subsequent to elution from paper. DMB is an excellent detection reagent for peptides and amino acids. All amino acids tested could be detected clearly at the 0.02-pmole level. Contrast between the sample spots and the background is excellent when chromatograms are placed in an open tray containing liquid ni-
l"bI
h',
550
1
544
200
203
lL,L
563
277
600
650
647
300
675
700
,,(,,,,,,,,,,,,,,,,,
350
I
x3
methyl ester. The mass temperature of the solid
,,,,,,,,,,,,,,,,,)
250
of dansyl-tetraphenylalanine beam energy of 12 eV. The
500
I
8,
I'I'I'I
527
500
150
FIG. I. Mass spectrum 0.9 kV and at an electron
450
100
171
/
r,~
800
,,,,,,,,
450
,,,,,,,
M+
850
lji,,l
038
spectrometer was operated at an accelerating sample heater block was 190°C.
750
400
voltage
of
900 m/e
m
AMINO
ACID
SEQUENCING
XI/SN3f
BY
MASS
/vi 3Auf73c9
SPECTROMETRY
365
366
FALTER,
JAYASIMHULU
AND DAY
trogen and the phosphorescence generated by a brief exposure to shortwavelength uv light is observed. Figure 2 shows the mass spectrum of DMB-Ala-Ala-Ala-Met methyl ester. The peptide fragment was obtained from a chymotryptic digest of carboxymethylated lysozyme. The resultant peptide mixture was subjected to analysis on a Spherix XX2 peptide column using a pyridine-acetic acid-water buffer system of pH 3.1-5.0 on a suitable gradient system. The peptide thus isolated was further chromatographed on Whatman 3MM paper and derivatized with DMB. The eluted peptide derivative was estertied and the mass spectrum was obtained. The amino acid analysis of the peptide hydrolyzate indicated the presence of three alanine residues and one methionine residue. The mass spectrum analysis indicated the sequence to be Ala-Ala-Ala-Met, corresponding to the amino acid sequence of residues 9-12 of lysozyme. The N-terminal sequence peaks m/e 175,218,246,273,290,448, and 476 as well as the molecular ion (M+) m/e 507 are prominent. The peaks at m/e 433 and 446 are ascribed to methionine side-chain and thioether loss, respectively, from M+. Peptide maps from chymotryptic digestion of carboxymethylated lysozyme were developed with DMB. The same number of spots could be detected in each case, when duplicate maps were developed with ninhydrin, 2,4,6-trinitrobenzenesulfonic acid or with DMB. Quantitative amino acid analyses were carried out on the hydrolyzates of the eluted %
120
100
147
161 169
204
274
HC =\/
302
CH
3
415
446 M+
150 200 250 300 350 400 450 m/e 3. Mass spectrum of N,N-dimethylaminobenzylidene glycylisoleucylleucine methyl ester. The mass spectrometer was operated at an accelerating voltage of I.2 kV and at an electron beam energy of 70 eV. The temperature of the solid sample heater block was 300°C. FIG.
AMINO
ACID
SEQUENCING
BY
MASS
SPECTROMETRY
367
368
FALTER,
JAYASIMHULU
AND
DAY
spots from maps developed with 2.4,6-trinitrobenzenesulfonic acid ( 17). The percentage recovery of the peptide from the chromatogram as determined by the amino acid analysis of the hydrolyzate of the eluted peptide or peptide derivative was found to be about 50%. The spots obtained by spraying the map with DMB reagent solution could also be eluted and hydrolyzed to obtain the quantitative amino acid composition of the peptide. The presence of DMB did not interfere with quantitative amino acid analysis. The tryptophan-containing peptides were however detected by the short wavelength uv light before the maps were subjected to any of the derivatization procedures (17). Figures 3 and 4 show the mass spectra of DMB-Cly-Ile-Leu methyl ester and di-DMB-Lys-Val-Phe methyl ester, respectively. Both peptides were obtained from the chymotryptic digestion of carboxymethylated lysozyme ( 18). The peptide Lys-Val-Phe is the N-terminal peptide containing amino acid residues 1-3 and the peptide Gly-Ile-Leu corresponds to amino acid residues 54-56 of lysozyme. These peptides had been separated on a map as described above. The spectrum of DMB-Gly-Ile-Leu methyl ester displays prominent N-terminal sequence peaks m/e 161, 189, 204, 274, 30 1 and 415 as well as M+ at m/e 446. The spectrum of (DMB),-Lys-Val-Phe methyl ester is more complex because of fragmentation of the hydrocarbon portion of the lysine side-chain. The N-terminal sequence peaks at m/e 363, 407, 490,505, and 609 and M+ at m/e 668 are prominent. Peak m/e 537 corresponds to M+ of DMB-Lys-Val-Phe-OCH,. Peak m/e 520 can be M+ - 148 which is attributed to the loss of p-N,N-dimethylbenzaldimine from the M+ ion and m/e 5 18 appears to correspond to m/e 609 - 9 1 (GH,). The mass spectra of Schiff-base derivatives of peptides have been described recently ( 13,16). In agreement with the results of those studies both derivatives showed intense M+ peaks and the necessary peaks to allow the assignment of the amino acid sequence, with the exception of the positions of leucine and isoleucine, which cannot be distinguished in the mass spectrum. The sequence shown is in agreement with the published amino acid sequence for lysozyme (20). The remaining derivatives shown in Table 1 may be of interest in certain circumstances. The DMC derivatives are intensely colored and are also suitable for mass spectrometry (13). The derivatives formed with HSN can be detected only after the background on the chromatogram has been reduced by rechromatography with organic solvents, such as benzene, and subsequently heating the chromatogram at 1 10°C for a short period. The fragmentation pattern for the HSN derivatives are different in the sense that elimination of the side chains is not suppressed
AMINO
ACID
SEQUENCING
(13). This fact may aid sometimes trum.
BY
MASS
SPECTROMETRY
in the interpretation
369
of a mass spec-
DISCUSSION
We have shown that amino acids and peptides can be detected on chromatograms by reagents that convert these to stable derivatives. Some of these derivatives are highly suitable from the point of view of yielding mass spectra that contain sequence information. This offers an attractive alternative for the determination of the amino acid sequence of mixtures of peptides, since the separation of peptides by paper chromatography and electrophoresis is well known. All of the reagents listed in Table 1 can be used to detect amino acids in amounts of less than 0.10 pmole after chromatography in one dimension. Generally speaking, somewhat larger amounts are desirable for mass spectrometry, especially since some losses are encountered. Only a fraction of the amino acids are derivatized in the detection procedure. If the available quantity of the amino acid is very small, a second step might be added after elution from paper, in which the remaining amino acid is derivatized. Some background in the mass spectra apparently results from the elution of samples from the chromatography paper. It is, however, not necessary to purify the samples after elution, since the background usually occurs at relatively low m/e values. Peaks below the m/e value of the amino-terminal blocking group are generally speaking less important for an interpretation of the spectrum than those occurring at higher m/e values. Phthalaldehyde, recently reported as a reagent for monitoring column chromatography of amino acids (21), has been shown to give peptide derivatives suitable for mass spectrometry (Hamilton and Day, unpublished). The reagent did not give consistently good results as a detection reagent on paper (Jayasimhulu, unpublished). Dansyl, DMB, and DMC derivatives yield mass spectra with intense M+ peaks. This aids in the interpretation of mass spectra. Fragments due to the elimination of side chains are virtually absent in many of these derivatives ( 13,16). Sometimes, especially when dealing with unknown compounds, it may be desirable to observe such fragments. In such cases, derivatives formed with HSN may be more useful since the elimination of side chains is commonly observed in these derivatives. In principle it should be possible to detect any compound which contains functional groups capable of reacting with the various reagents. It is therefore evident that the method presented here has the potential for wider application in the identification of biological materials. These methods of separation of the peptides obtained from enzyme digestion of
370
FALTER,
JAYASIMHULU
AND
DAY
lysozyme by (a) electrophoresis coupled with chromatography and (b) column chromatography coupled with paper chromatography are used to isolate pure peptide fractions. These were then eluted, esterified and derivatized suitably for mass spectrometric analysis to obtain sequence information. These methods were developed to generate peptide derivatives suitable for mass spectrometric analysis. The gas chromatographic and mass spectrometric properties of these peptide derivatives are under investigation. The overall procedures outlined above are basically the techniques that are being adopted for complete sequencing of proteins using mass spectrometry in our laboratories. ACKNOWLEDGMENTS This work was supported by grants from the American Cancer Society (No. P-343). National Science Foundation (No. GP-8490 and GP-34264), National Institutes of Health (No. GM-18756), and a grant from the National Science Council of Canada. RAD is a Career Development Grant awardee of the USPHS (Grant No. 5 K04 GM0 7654). We thank Miss Gisela Diercus and Miss Lizette Lavoie for technical assistance.
REFERENCES 1. Das, B. C.. and Lederer, E. (1970) in Topics in Organic Mass Spectrometry (Burlingame, A. L., ed.), p. 255, Wiley-lnterscience, New York. 2. Shemyakin, M. M., Ovchinnikov, Yu. A., and Kiryushkin, A. A., (1921) in Mass Spectrometry, Techniques and Applications (Milne, G. W. A., ed.), p. 289, WileyInterscience, New York. 3. Biemann, K., (1972) in Biochemical Applications of Mass Spectrometry (Wailer. G. R., ed.), p. 405, Wiley-Interscience, New York. 4. McLafferty, F. W., Venkataraghavan, R., and Irving, P. (1970) Biochem. Biophys. Res. Commun.
39, 274,.
5. Wipf, H. K., Irving, P., McCamish, M., Venkataraghavan, R., McLaIferty, F. W. (1973) J. Amer. Chem. Sot. 95, 3369. 6. Biemann, K. (1969) Abstract 9, Division of Biological Chemistry, Abstracts Volume, 158th Amer. Chem. Sot. Meeting, New York (1969); Nau, A., Kelley, J. A., and Biemann, K. ( 1973) J. Amer. Chem. Sot. 95, 7162. 7. Morris, H. R., Dickinson, R. J., and Williams, D. H. (1973) Biochem. Biophys. Res. Commrrn.
51, 247.
8. Ovchinnikov, Yu. A., and Kiryushkin, A. A. (1972) FEBS Left. 21, 300. 9. Caprioli, R. M., Seifert, I”.. E., Jr., and Sutherland, D. E. (1973) Biochem. Biophys. Res. Commun. 55, 67. 10. Ling, N., Rivier. J., Burgus, R., and Guillemin. R. (1973) Biochemisfry 12, 5305. Il. Ruhemann, S. (1911) J. Chem. Sot. 992, 1486: for a recent discussion of this reaction see Lamothe, P. J., and McCormick, P. G., Anal. Chem. 45, 1908. 12. For preliminary accounts of this work see Falter, H., and Day, R. A. (1970) Abstract 74, Division of Biological Chemistry. Abstracts Volume, 160th Amer. Chem. Sot. Nat. Meeting, Chicago; Falter, H., Lehman, J. P., and Day, R. A. (1971) Abstracts Volume, XXIII Int. Congr. Pure Appl. Chem., Boston, MA, July 25-30, p. 90. 13. Day, R. A., Falter, H., Lehman, J. P., and Hamilton, R. E. (1973) J. Org. Chem. 38, 782. 14. Canfield, R. E. (1963) J. Biol. Chem. 238, 2691. 15. Prox, A., and Sun, K. K. (1966) 2. Naturforsch. 21b, 1028.
AMINO
16. 17. 18. 19.
ACID
SEQUENCING
BY
MASS
SPECTROMETRY
371
Patil, G. V., Hamilton, R. E., and Day, R. A. ( 1973) J. Ovg. Mass Specrrom. Neumann, H., Gurari, D., and Sokolovsky, M. (1971) fnt. J. Biochem. 2, 481. Canfield, R. E. (1963) J. Biol. Chem. 238, 2684. Budzikiewicz, H., Djerassi, C., and Williams, D. H. (1967) Mass Spectrometry of Organic Compounds, pp. 174 ff., Holden-Day, San Franciso. 20. Canfield, R. E. (1963) J. Biol. Chem. 238, 2698. 21. Roth, M. (1971) Anal. Chtm. 43, 880.
67, 3.59-37 1 ( 1975)
BIOCHEMISTRY
Sequence Determination of Peptides by Mass Spectrometry: Methods for Combining “Wet” Separation Techniques with Mass Spectrometry H. FALTER' Department
of Chemistry,
Laurentian
University,
K. JAYASIMHULU Department
of Chemistry,
University
Sudbury.
Ontario,
Canada
AND R. A. DAY of Cincinnati,
Cincinnati,
Ohio
4.5221
Received December 26, 1973; accepted January 17, 1975 Methods are described that allow the combination of established techniques for peptide separation, paper chromatography and electrophoresis, with mass spectrometry. The development of these methods is part of an ongoing effort in the search for a methodology for the systematic utilization of mass spectrometry for the elucidation of primary structure of proteins and peptides. Peptides and ammo acids are detected on chromatograms by conversion to covalent derivatives that are also suitable for mass spectrometry. The most useful reagents for detection and derivatization of peptides reported here are dansyl chloride, N,Ndimethylaminobenzaldehyde. N,N-dimethylaminocinnamaldehyde, and N-hy droxysuccinimido /3-naphthoate. Detection limits and mass spectra for some of these derivatives are reported.
Recently there have been some significant successes in obtaining the sequences of peptides from proteins and, in some cases, complete sequences of small proteins by mass spectrometry using various techniques. These either require sophisticated equipment or use of elaborate sample preparations (l-3). Proteins and large peptides themselves do not yield mass spectra suitable for sequence analysis. In order to employ mass spectrometry for amino acid sequence determination, it is necessary to degrade larger peptides into smaller ones, which can be converted to volatile derivatives. Generally speaking therefore, a large number of peptides would have to be generated in order to provide sufficient information for the assignment of the amino acid sequence of a protein. The analysis of complex mixtures of peptides, without prior separation, is a difficult matter that requires exact mass, partial vaporization, and metastable-ion data (4). While a degree of success in the analysis of 1 To whom correspondence concerning this paper should be addressed. 359 CopYright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
360
FALTER,
JAYASIMHULU
AND
DAY
peptide mixtures by a variety of approaches (5-10) gives cause for optimism, it is clear that these methods are not yet generally applicable. The separation of mixtures of peptides by paper chromatography and electrophoresis is well known. Peptides are usually located on chromatograms by means of the ninhydrin reaction. Unfortunately, the formation of Ruhemann’s purple is accompanied by the degradation of peptides (11). We have endeavored to utilize reagents for the detection of amino acids and peptides which do not cause their degradation (12). These reagents must form covalently bonded derivatives, also suitable for mass spectrometry (13). The derivatives can then be eluted, estermed, and subjected to mass spectrometry. MATERIALS
AND
METHODS
Materials
The peptide tetra-r.-phenylalanine was obtained from Cyclochemical. NJ-Dimethylaminobenzaldehyde was obtained from Fisher Scientific Co. Dansyl chloride (5,dimethylamino1-naphthalenesulfonyl chloride), lysozyme, trypsin, and a-chymotrypsin were obtained from Sigma Chemical Co. Solvents used for elution of the peptide derivatives from the paper were glass distilled, reagent grade. Paper Chromatography
and Electrophoresis
Commercially available amino acids and peptides were used without further purification. Varying amounts (characteristically 0.02, 0.05 and 0.10 pmole) were spotted on Whatman 3MM chromatography paper and then chromatographed in the descending direction using a butanol : acetic acid: H,O (4 : 1: 5, v/v/v) solvent system. The chromatograms were dried extensively before reaction with the detection reagents. In the case of chymotryptic peptides of lysozyme, electrophoresis was carried out at a voltage of 15 V/cm using a pyridine:acetic acid:water (100:4:900, v/v/v) system at pH 6.5 before chromatographing in the second dimension as described above. Peptide maps were prepared from tryptic digests of carboxymethylated egg-white lysozyme essentially by the method of Canfield (14). Approximately 3 mg of carboxymethylated lysozyme digest were used for the preparation of each map. Reagents
for the Detection
of Amino
Acids and Peptides
a) Dunsyl chloride. Dansyl chloride (100 mg) was dissolved in acetone (100 ml) and the solution was filtered to remove traces of insoluble particles. Immediately prior to use, 3 ml of triethylamine was added to
AMINO
ACID
SEQUENCING
BY
MASS
SPECTROMETRY
361
the solution. Chromatograms were dipped in an open tray for about 1 min and then dried in an oven at 65°C for 10 min. Spots were detected by their yellow color and, in the dark, with a long-wavelength uv lamp (Black Ray, UVL-21, Ultraviolet Products Inc., San Gabriel, CA). b) N,N-Dimethyluminobenzaldehyde (DMB). DMB (100 mg) was dissolved in 100 ml of anhydrous ethyl ether. Acetic acid ( 1 ml) was added immediately before the solution was used. Chromatograms were dipped, or sprayed uniformly, and dried in an oven for 10 min at 105-l 10°C. The more intense spots were yellow against a white background. Greater sensitivity could be achieved with a short-wavelength uv light (Mineralight UVL-12, Ultraviolet Products, Inc.). The best contrasts were obtained by placing the chromatograms in an open tray containing liquid nitrogen and removing the uv light after a short exposure. Brilliantly phosphorescent spots persisted long enough to allow marking with a pencil. c) N,N-Dimethylaminocinnama~dehyde (DMC). DMC (5 mg) was dissolved in 100 ml of dioxane. Triethylamine (1 ml) was added immediately before the solution was used. Chromatograms were dipped, then dried in an oven for a short time. Spots were a deep red to reddish brown. 6) N,-Hydroxysuccinimido p-naphthoate (HSN). HSN (60 mg) was dissolved in 100 ml of chloroform, and 1 ml of triethylamine was added immediately before use. Chromatograms were dipped in an open tray for about 1 min. The chromatograms were then rinsed with benzene by chromatographing in this solvent overnight. After drying in an oven at 110°C for a few minutes, spots could be seen with a long-wavelength uv lamp. Elution
and EsteriJication
of Peptides
The spots were cut out, placed in 17 X 1.4-cm distilling columns with 14/20 standard taper joints and indented supports and eluted with 2-3 ml of refluxing methanol or ethanol contained in 5- or IO-ml roundbottom flasks. The distilling columns were removed after an hour, two drops of thionyl chloride were added to the alcoholic extracts, and the refluxing was continued for 30 min. The solutions were then concentrated in a rotary evaporator, and appropriate portions were transferred to the sample probes used in the solid inlet probe of the mass spectrometer by successive transfers of small volumes and intermittent drying with a stream of hot air. Typically - 0.1 pmole of each sample was transferred to the sample probe. Mass Spectrometq
Mass spectra were obtained with a Hitachi-Perkin-Elmer RMU-7 mass spectrometer operating in a low resolution mode with a resolution
362
FALTER,
JAYASIMHULU
AND
DAY
setting of 1200 (calculated at two adjacent peaks with about 90% separation). The source temperature (chamber temperature) is usually maintained at 250°C while working with peptides, and the sample heater into which the sample probe is inserted is heated slowly until the molecular ion is seen, at which time the spectra were recorded repeatedly. The temperature at which an intense molecular ion was observed is recorded. The temperature, the electron beam energy and the accelerating voltage used are noted in the captions to the various figures. RESULTS
Table 1 shows a number of reagents, that can be used to detect amino acids on paper chromatograms. All of these reagents form covalent derivatives with the free amino group(s) of amino acids and peptides. Therefore, detection and blocking of the amino terminus and any sidechain amino group can be accomplished in one step. Not all amino-blocking groups are equally suitable from the point of view of yielding mass spectra that contain sequence information (13,15,16). The mass spectrometric suitability of these blocking groups has been investigated previously (13,16) by comparing the spectra obtained from derivatives of the model peptide Val-Ile-Ala. Partially as a result of this earlier study (13) and partially because of less sensitive detection levels, a number of other reagents which can be used to detect amino acids on paper chromatograms were rejected. These reagents included: phthalic anhydride, N-hydroxysuccinimido (Ynaphthoate, naphthalene-1,8-dicarboxylic acid anhydride, cr- and pDETECTION
AGENTS
TABLE FOR AMINO
Reagent
Materials detected
Dansyl chloride a
All common amino acids Maps of tryptic digest of carboxymethylated lysozyme Selected amino acids Maps of chymotryptic digest of carboxymethylated lysozyme All common amino acids Maps of tryptic digest of carboxymethylated lysozyme All common amino acids
DMB DMC HSN
1 ACIDS
AND PEPTIDES
Comment Detection level less than 0.05 ~moleb~c Most ninhydrin spots are detectable: see text Detection level less than 0.02 pmoleb All ninhydrin spots are detectable; see text Detection level less than 0.05 pmole b,c Most ninhydrin spots are detectable Detection level less than 0. I pmole
a Abbreviations: 5-Dimethylamino-I-naphthalenesulfonyl chloride (dansyl N,N-dimethylaminobenzaldehyde (DMB); N,N-dimethylaminocinnamaldehyde N-hydroxysuccinimido p-naphthoate (HSN). b After chromatography in one dimension. c Most amino acids are still detected in amounts of 0.02 umole.
chloride); (DMC);
AMINO
ACID
SEQUENCING
BY
MASS
SPECTROMETRY
363
naphthalenesulfonyl chloride, a+ and @chloromethylnaphthalene, and 2,3-his-(bromomethyl)-naphthalene. All of the reagents listed in Table 1 can be used to detect amino acids on chromatograms in amounts of less than 0.10 pmole after these have been chromatographed in one dimension. Many of the amino acids can be detected at substantially lower levels. After separation in two dimensions, especially if one of these involves electrophoresis, the contrast between the spots and the background decreases somewhat. Peptide maps from tryptic and chymotryptic digestion of carboxymethylated lysozyme were used to test the feasibility of detecting peptides from the proteolytic digest of a protein. The maps obtained were compared with maps developed with ninhydrin and, in the case of the tryptic digests, to the published map of Canfield (14). When the tryptic peptide maps were developed with dansyl chloride, the contrast in each case was less than when duplicate maps were developed with ninhydrin. Clearly separated spots could readily be recognized on the dansyl maps. However, mainly in the central region of the map, where a number of relatively large peptides are crowded together, the contrast did not permit the unambiguous assignment of all the spots detected with ninhydrin. When somewhat larger amounts of material can be used, and especially if separation in only one dimension is required, the technique is highly satisfactory. Figure 1 shows the mass spectrum of the peptide tetra-Phe, which was spotted in amounts of 0.5 pmole on Whatman 3MM paper, chromatographed in one dimension and then detected on the chromatogram with dansyl chloride. This mass spectrum illustrates some of the aspects, which make the dansyl group a desirable amino-terminus blocking group. The M* ion is prominent and the ions produced by the fragmentation of the side chains are lower than might be anticipated. In a peptide containing four phenylalanine side-chains it might have been quite reasonable to expect these peaks to be very intense. It can be easily shown, by chromatography followed by color development with ninhydrin, that in the detection procedure with dansyl chloride only a fraction of a given peptide is derivatized. When relatively large amounts of a peptide are used, i.e., 0.5 pmole of tetraphenylalanine, a satisfactory mass spectrum can be obtained directly. When it is essential that smaller amounts be used, a further step might be added in which that fraction of the peptide which was not derivatized during detection is derivatized subsequent to elution from paper. DMB is an excellent detection reagent for peptides and amino acids. All amino acids tested could be detected clearly at the 0.02-pmole level. Contrast between the sample spots and the background is excellent when chromatograms are placed in an open tray containing liquid ni-
l"bI
h',
550
1
544
200
203
lL,L
563
277
600
650
647
300
675
700
,,(,,,,,,,,,,,,,,,,,
350
I
x3
methyl ester. The mass temperature of the solid
,,,,,,,,,,,,,,,,,)
250
of dansyl-tetraphenylalanine beam energy of 12 eV. The
500
I
8,
I'I'I'I
527
500
150
FIG. I. Mass spectrum 0.9 kV and at an electron
450
100
171
/
r,~
800
,,,,,,,,
450
,,,,,,,
M+
850
lji,,l
038
spectrometer was operated at an accelerating sample heater block was 190°C.
750
400
voltage
of
900 m/e
m
AMINO
ACID
SEQUENCING
XI/SN3f
BY
MASS
/vi 3Auf73c9
SPECTROMETRY
365
366
FALTER,
JAYASIMHULU
AND DAY
trogen and the phosphorescence generated by a brief exposure to shortwavelength uv light is observed. Figure 2 shows the mass spectrum of DMB-Ala-Ala-Ala-Met methyl ester. The peptide fragment was obtained from a chymotryptic digest of carboxymethylated lysozyme. The resultant peptide mixture was subjected to analysis on a Spherix XX2 peptide column using a pyridine-acetic acid-water buffer system of pH 3.1-5.0 on a suitable gradient system. The peptide thus isolated was further chromatographed on Whatman 3MM paper and derivatized with DMB. The eluted peptide derivative was estertied and the mass spectrum was obtained. The amino acid analysis of the peptide hydrolyzate indicated the presence of three alanine residues and one methionine residue. The mass spectrum analysis indicated the sequence to be Ala-Ala-Ala-Met, corresponding to the amino acid sequence of residues 9-12 of lysozyme. The N-terminal sequence peaks m/e 175,218,246,273,290,448, and 476 as well as the molecular ion (M+) m/e 507 are prominent. The peaks at m/e 433 and 446 are ascribed to methionine side-chain and thioether loss, respectively, from M+. Peptide maps from chymotryptic digestion of carboxymethylated lysozyme were developed with DMB. The same number of spots could be detected in each case, when duplicate maps were developed with ninhydrin, 2,4,6-trinitrobenzenesulfonic acid or with DMB. Quantitative amino acid analyses were carried out on the hydrolyzates of the eluted %
120
100
147
161 169
204
274
HC =\/
302
CH
3
415
446 M+
150 200 250 300 350 400 450 m/e 3. Mass spectrum of N,N-dimethylaminobenzylidene glycylisoleucylleucine methyl ester. The mass spectrometer was operated at an accelerating voltage of I.2 kV and at an electron beam energy of 70 eV. The temperature of the solid sample heater block was 300°C. FIG.
AMINO
ACID
SEQUENCING
BY
MASS
SPECTROMETRY
367
368
FALTER,
JAYASIMHULU
AND
DAY
spots from maps developed with 2.4,6-trinitrobenzenesulfonic acid ( 17). The percentage recovery of the peptide from the chromatogram as determined by the amino acid analysis of the hydrolyzate of the eluted peptide or peptide derivative was found to be about 50%. The spots obtained by spraying the map with DMB reagent solution could also be eluted and hydrolyzed to obtain the quantitative amino acid composition of the peptide. The presence of DMB did not interfere with quantitative amino acid analysis. The tryptophan-containing peptides were however detected by the short wavelength uv light before the maps were subjected to any of the derivatization procedures (17). Figures 3 and 4 show the mass spectra of DMB-Cly-Ile-Leu methyl ester and di-DMB-Lys-Val-Phe methyl ester, respectively. Both peptides were obtained from the chymotryptic digestion of carboxymethylated lysozyme ( 18). The peptide Lys-Val-Phe is the N-terminal peptide containing amino acid residues 1-3 and the peptide Gly-Ile-Leu corresponds to amino acid residues 54-56 of lysozyme. These peptides had been separated on a map as described above. The spectrum of DMB-Gly-Ile-Leu methyl ester displays prominent N-terminal sequence peaks m/e 161, 189, 204, 274, 30 1 and 415 as well as M+ at m/e 446. The spectrum of (DMB),-Lys-Val-Phe methyl ester is more complex because of fragmentation of the hydrocarbon portion of the lysine side-chain. The N-terminal sequence peaks at m/e 363, 407, 490,505, and 609 and M+ at m/e 668 are prominent. Peak m/e 537 corresponds to M+ of DMB-Lys-Val-Phe-OCH,. Peak m/e 520 can be M+ - 148 which is attributed to the loss of p-N,N-dimethylbenzaldimine from the M+ ion and m/e 5 18 appears to correspond to m/e 609 - 9 1 (GH,). The mass spectra of Schiff-base derivatives of peptides have been described recently ( 13,16). In agreement with the results of those studies both derivatives showed intense M+ peaks and the necessary peaks to allow the assignment of the amino acid sequence, with the exception of the positions of leucine and isoleucine, which cannot be distinguished in the mass spectrum. The sequence shown is in agreement with the published amino acid sequence for lysozyme (20). The remaining derivatives shown in Table 1 may be of interest in certain circumstances. The DMC derivatives are intensely colored and are also suitable for mass spectrometry (13). The derivatives formed with HSN can be detected only after the background on the chromatogram has been reduced by rechromatography with organic solvents, such as benzene, and subsequently heating the chromatogram at 1 10°C for a short period. The fragmentation pattern for the HSN derivatives are different in the sense that elimination of the side chains is not suppressed
AMINO
ACID
SEQUENCING
(13). This fact may aid sometimes trum.
BY
MASS
SPECTROMETRY
in the interpretation
369
of a mass spec-
DISCUSSION
We have shown that amino acids and peptides can be detected on chromatograms by reagents that convert these to stable derivatives. Some of these derivatives are highly suitable from the point of view of yielding mass spectra that contain sequence information. This offers an attractive alternative for the determination of the amino acid sequence of mixtures of peptides, since the separation of peptides by paper chromatography and electrophoresis is well known. All of the reagents listed in Table 1 can be used to detect amino acids in amounts of less than 0.10 pmole after chromatography in one dimension. Generally speaking, somewhat larger amounts are desirable for mass spectrometry, especially since some losses are encountered. Only a fraction of the amino acids are derivatized in the detection procedure. If the available quantity of the amino acid is very small, a second step might be added after elution from paper, in which the remaining amino acid is derivatized. Some background in the mass spectra apparently results from the elution of samples from the chromatography paper. It is, however, not necessary to purify the samples after elution, since the background usually occurs at relatively low m/e values. Peaks below the m/e value of the amino-terminal blocking group are generally speaking less important for an interpretation of the spectrum than those occurring at higher m/e values. Phthalaldehyde, recently reported as a reagent for monitoring column chromatography of amino acids (21), has been shown to give peptide derivatives suitable for mass spectrometry (Hamilton and Day, unpublished). The reagent did not give consistently good results as a detection reagent on paper (Jayasimhulu, unpublished). Dansyl, DMB, and DMC derivatives yield mass spectra with intense M+ peaks. This aids in the interpretation of mass spectra. Fragments due to the elimination of side chains are virtually absent in many of these derivatives ( 13,16). Sometimes, especially when dealing with unknown compounds, it may be desirable to observe such fragments. In such cases, derivatives formed with HSN may be more useful since the elimination of side chains is commonly observed in these derivatives. In principle it should be possible to detect any compound which contains functional groups capable of reacting with the various reagents. It is therefore evident that the method presented here has the potential for wider application in the identification of biological materials. These methods of separation of the peptides obtained from enzyme digestion of
370
FALTER,
JAYASIMHULU
AND
DAY
lysozyme by (a) electrophoresis coupled with chromatography and (b) column chromatography coupled with paper chromatography are used to isolate pure peptide fractions. These were then eluted, esterified and derivatized suitably for mass spectrometric analysis to obtain sequence information. These methods were developed to generate peptide derivatives suitable for mass spectrometric analysis. The gas chromatographic and mass spectrometric properties of these peptide derivatives are under investigation. The overall procedures outlined above are basically the techniques that are being adopted for complete sequencing of proteins using mass spectrometry in our laboratories. ACKNOWLEDGMENTS This work was supported by grants from the American Cancer Society (No. P-343). National Science Foundation (No. GP-8490 and GP-34264), National Institutes of Health (No. GM-18756), and a grant from the National Science Council of Canada. RAD is a Career Development Grant awardee of the USPHS (Grant No. 5 K04 GM0 7654). We thank Miss Gisela Diercus and Miss Lizette Lavoie for technical assistance.
REFERENCES 1. Das, B. C.. and Lederer, E. (1970) in Topics in Organic Mass Spectrometry (Burlingame, A. L., ed.), p. 255, Wiley-lnterscience, New York. 2. Shemyakin, M. M., Ovchinnikov, Yu. A., and Kiryushkin, A. A., (1921) in Mass Spectrometry, Techniques and Applications (Milne, G. W. A., ed.), p. 289, WileyInterscience, New York. 3. Biemann, K., (1972) in Biochemical Applications of Mass Spectrometry (Wailer. G. R., ed.), p. 405, Wiley-Interscience, New York. 4. McLafferty, F. W., Venkataraghavan, R., and Irving, P. (1970) Biochem. Biophys. Res. Commun.
39, 274,.
5. Wipf, H. K., Irving, P., McCamish, M., Venkataraghavan, R., McLaIferty, F. W. (1973) J. Amer. Chem. Sot. 95, 3369. 6. Biemann, K. (1969) Abstract 9, Division of Biological Chemistry, Abstracts Volume, 158th Amer. Chem. Sot. Meeting, New York (1969); Nau, A., Kelley, J. A., and Biemann, K. ( 1973) J. Amer. Chem. Sot. 95, 7162. 7. Morris, H. R., Dickinson, R. J., and Williams, D. H. (1973) Biochem. Biophys. Res. Commrrn.
51, 247.
8. Ovchinnikov, Yu. A., and Kiryushkin, A. A. (1972) FEBS Left. 21, 300. 9. Caprioli, R. M., Seifert, I”.. E., Jr., and Sutherland, D. E. (1973) Biochem. Biophys. Res. Commun. 55, 67. 10. Ling, N., Rivier. J., Burgus, R., and Guillemin. R. (1973) Biochemisfry 12, 5305. Il. Ruhemann, S. (1911) J. Chem. Sot. 992, 1486: for a recent discussion of this reaction see Lamothe, P. J., and McCormick, P. G., Anal. Chem. 45, 1908. 12. For preliminary accounts of this work see Falter, H., and Day, R. A. (1970) Abstract 74, Division of Biological Chemistry. Abstracts Volume, 160th Amer. Chem. Sot. Nat. Meeting, Chicago; Falter, H., Lehman, J. P., and Day, R. A. (1971) Abstracts Volume, XXIII Int. Congr. Pure Appl. Chem., Boston, MA, July 25-30, p. 90. 13. Day, R. A., Falter, H., Lehman, J. P., and Hamilton, R. E. (1973) J. Org. Chem. 38, 782. 14. Canfield, R. E. (1963) J. Biol. Chem. 238, 2691. 15. Prox, A., and Sun, K. K. (1966) 2. Naturforsch. 21b, 1028.
AMINO
16. 17. 18. 19.
ACID
SEQUENCING
BY
MASS
SPECTROMETRY
371
Patil, G. V., Hamilton, R. E., and Day, R. A. ( 1973) J. Ovg. Mass Specrrom. Neumann, H., Gurari, D., and Sokolovsky, M. (1971) fnt. J. Biochem. 2, 481. Canfield, R. E. (1963) J. Biol. Chem. 238, 2684. Budzikiewicz, H., Djerassi, C., and Williams, D. H. (1967) Mass Spectrometry of Organic Compounds, pp. 174 ff., Holden-Day, San Franciso. 20. Canfield, R. E. (1963) J. Biol. Chem. 238, 2698. 21. Roth, M. (1971) Anal. Chtm. 43, 880.
