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Peptide sequencing by low resolution mass spectrometry I. The use of acetylacetonyl derivatives to identify N-terminal residues
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 37, No. 6, 1969
PEPTIDESEQUENCING BY LOWRESOLUTION MASSSPECTROMETRY I.
THEUSE OF ACETYLACETONYL DERIVATIVESTO IDENTIFY N-TERMINALRESIDUES V. Bacon, E. Jellum, W. Patton, W. Pereira and B. Halpern Department of Genetics, Stanford University Medical Center Stanford, California, 94305
Received September 12, 1969 The mass spectrometric sequence determination of oligopeptides via their acetylacetonyl derivatives gives reliable results with small peptides (2-10 residues) regardless of the amino acids present. The main advantages are the ease of derivative formation and the mass spectral fragmentation patterns which are optimal for N-terminal amino acid identification. Many published methods for sequence determination use,manually interpreted, recognition (1-5).
low resolution
mass spectra,
of peptides which depend on a
of the molecular ion and a search for ionized amide bond cleavages
Since an unambiguous identification
a mass spectrum is often difficult,
of a small molecular ion peak in
sequencing procedures based on a
recognition
of the more prominent N-terminal amino acyl ion are to be
preferred.
Whilst mixtures of long chain fatty
have been used for distinctive spectrometry of peptides, all recognition
N-terminal
acids or CH3C0and CD3C0(6)
tags for low resolution
automated procedures have relied
mass
on the
of elemental composition of the N-acyl fragment by high resolution
mass spectrometry.
(7,8)
In view of the high cost of this instrumentation,
we have re-examined the sequencing of peptides by low resolution spectrometry with derivatives identification.
which are optfmal for N-terminal amino acid
Wenow report on the sequencing of acetylacetonyl
(ACA-peptides),
which gives reliable
acid residues),
regardless of the amino acids present.
on the identification
mass
peptides
results with small peptides (2-10 amino The method depends
of the N-terminal amino acid residue 'A' in the spectra.
878
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 37, No. 6, 1969
WA A1 CH3 I 1; ; CH3COCH = C - NH - CHL COINH: ; Since, in this system, these ions unambiguousstarting *I,
Cl, Dl).
R4 ,D D R2 P *I R3C Cl I; I 1:; I; ?CH,- CO!NH - CH;- CO;NH- CH;- Co!OCH2CH3 I 8 I I 8 ar,' quite prominent,' it affoids a'n
point for the search for the sequence ions (B, C, D and
Preparation of the volatile
derivatives
involves esterification
of the peptide followed by treatment with acefylacetone.
Under the experimental
conditions used, arginine peptides are converted to 6-N-(2-pyrimidinyl) ornithine
peptides (9) and the E amino group of lysine is also derivatised.
All other functional and are left
groups present in the protein amino acids remain intact
unprotected.
A typical
the peptide (0.5-1.0 mg) with e
sample preparation
ethanolic hydrogen chloride
w/w) for 10 min and removing the solvent -in vacua. dry ethanol (100 ul), X8, Bio-Rad Cal., at pH 7-8.
involves refluxing
acetylacetone
After
(1 ml, 25-30X
redissolving
in
(30 ul) and dry ion exchange beads (AGl-
in the bicarbonate form) are added till
Molecular sieve (3A, Matheson, N.J.)
of water and the reaction mixture is left
the solution
is
is added to remove traces The supernatant liquid
overnight.
is then withdrawn with a syringe and the solvent evaporated -in vacua. residue is then inserted directly mass spectrometer.
The
into the ion source of a low resolution
On the basis of the mass spectra of almost 80 model
peptides, we find that the "A" fragment is the most prominent peak in the spectra of ACA-peptide esters, which have an N-terminal aliphaticamino acid.
(Table 1)
In somecases partial
loss of the side chain is
observed with N-terminal methionine, serine, threonine, acid.
Somelarger seryl-
and threonyl-peptides
probe temperatures and this makes recognition N-terminal position
difficult.
Histidyl-,
tryptophyl-peptides
show someelimination
or acidic-
aspartic and glutamic
tend to dehydrate at higher of these residues in the
phenylalanyl-,
tyrosyl-
and
of the side chain as ArCHz or
ArCH2O, but these ions help to confirm the presence of these residues. and arginyl
peptides yield
the very characteristic
[A-99] fragments, and show
only very small "A" fragments in the massspectra (Figure 2), whilst 879
Lysyl
cystine
BIOCHEMICAL
Vol. 37, No. 6, 1969
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 Characteristic
Fragments Used for Identification
N-Terminal Amino Acid
"A" Fragment (m/e) [CH3-CO-CH=C(CH~)-NH-CHR]+
GLY ALA SER PRO VAL THR LRIJ ILE HYP ASN MET HIS ASP PHE GLU TYR TRY LYS ARG CYS
112 126 142 152 154 156 168 168 168 169 186 192 198 202 212 218 241" 265" 275"
of N-Terminal Amino Acids Other Ions (m/e) Used for Identification
112 (A-CH20)t 138 (A-H20)?,112 (A-CH3CHO)* 150 (A-H20)f 138 (A-CH3SH)t
130 166 176 156
(Ar-CH2)+ (A-NH2-C(CH3)=CH-COCH3)+ (&NH2-C(CH3)=CH-COCH )? (A/2-H)?, 158 (A/2+H) 3
*Minor fragment.
derivatives
undergo S-S bond rupture accompanied by hydrogen transfer
(10).
Although peptides containing unmodified asparagine have been sequenced, diaaomethane has to be used instead of alcoholic esterification peptide. yield
(11) to prevent hydrolysis
interpretable
and penta-peptides.
and limits
enamino ketone function
the sequencing
Experiments to increase the
of the ACA-peptide esters by permethylation The potential
yielded a complex mixture of products.
con-
The presence of basic
amino acids decreases the volatility
procedure to tetra-
alkylation
high vapor pressure and
mass spectra from hepta- and octa-peptides
only the neutral amino acids (Figure 1).
polyfunctional
volatility
to the corresponding aspartyl
The ACA-peptide esters have a relatively
readily
taining
hydrogen chloride for
(12-15) invariably difficulty
is the
which reacts with methyl iodide to give 0 and C
(16) and a non volatile
quaternary salt. 880
4m
Fipure
128
140
Figure
1.
2.
160
17,
mm
(Finnigan
24a
Ethyl
2tIm
Temperature
1015 Mass Spectrometer)
of ACA-Arg-Ala
220
CH 4 Mass Spectrometer,
of ACA-Ala-Leu-Ala-Val-Gly-Ala-Phe
Mass Spectrum
A-99
(Atlas
Mass Spectrum
Ester
301
321
260°
C)
Ethyl
340
3fim
Ester
389
4w
421
Vol. 37, No. 6, 1969
BIOCHEMICAL
AND BIOF’HYSICAL RESEARCH COMMUNICATIONS
Acknowledgments. This work was supported by a fellowship
to E. Jellum from
the Norwegian Cancer Society and by National Aeronautics and Space Administration
grant NGR-05-020-004. REFERENCES
1. 2. 3. 4. 5. 6. 7. ;: 10. 11. 12. 13. 14. 15. 16.
Weygand, F., Prox, A,, Ktinig, W. and Fessel, H. H. Angew. Chem.2, 724 (1963). Heyns, K. and Grutsmacher, H. F. Tetrahedron Letters 1761 (1963). Wulfson, N. S., Bochkarev, V. N., Rozinov, B. V., Shemyakin, M. M., Ovchinnikov, Yu. A., Kiryushkin, A. A. and Miroshnikov, A. I. Tetrahedron Letters, 39 (1966). Barber, M., Jolles, P., Vilkas, E. and Lederer, E. Biochem. Biophys. Res. Cosm~.l.g-, 4h9 (1965). Kiryushkin, A. A., Ovchinnikov, Yu. A., Shemyakin, M. M., Bochkarev, V.N., Rosinov, B. V. and Wulfson, N. S. Tetrahedron Letters 33 (1966). Bricas, E., Van Heijenoort, J., Barber, M., Wolstenholme, W. A., Das, B. C. and Lederer, E. Biochemistry 5, 2254 (1965). Biemann, K., Cone, C., Webster, B. R. and Arsenault, G. P. 2. &. M. Sot. EtJ, 5598 (1966). Senn, M., Venkataraghavan, R. and McLafferty, F. M. Ibid 18, 5593 (1966). King, T. P. Biochemistry 2, 3454 (1966). Kiryushkin, A. A., Gorlenko, V. A., Agadshanyan, Ts E., Rosinov, B. V., Ovchinnikov, Yu. A. and Shemyakin, M. M. Experientia 2, 884 (1968). Bayer, E. and Koenig, W. A. Advances in Chromatography, Ed1tor.A. Zlatkis. Preston Technical Abstracts Co., Evanston, Ill. 187 (1969). Das, B. C., Gero, S. D. and Lederer, E. Biochem. Biophys. Res. Comm. 2, 211 (1967). Thomas, D. W. Ibid 2, 483 (1968). Thomas, D. W., Das, B. C., Gero, S. D. and Lederer, E. Ibid 32, 199, 519 (1968). Agarwal, K. L., Kenner, G. W. and Sheppard, R. C. J. Am. Chem. Sot. !X, 3096 (1969). Meyers, A. I., Reine, A. H. and Gault, R. J. Org. Chem.34, 698 (1969).
882
Vol. 37, No. 6, 1969
PEPTIDESEQUENCING BY LOWRESOLUTION MASSSPECTROMETRY I.
THEUSE OF ACETYLACETONYL DERIVATIVESTO IDENTIFY N-TERMINALRESIDUES V. Bacon, E. Jellum, W. Patton, W. Pereira and B. Halpern Department of Genetics, Stanford University Medical Center Stanford, California, 94305
Received September 12, 1969 The mass spectrometric sequence determination of oligopeptides via their acetylacetonyl derivatives gives reliable results with small peptides (2-10 residues) regardless of the amino acids present. The main advantages are the ease of derivative formation and the mass spectral fragmentation patterns which are optimal for N-terminal amino acid identification. Many published methods for sequence determination use,manually interpreted, recognition (1-5).
low resolution
mass spectra,
of peptides which depend on a
of the molecular ion and a search for ionized amide bond cleavages
Since an unambiguous identification
a mass spectrum is often difficult,
of a small molecular ion peak in
sequencing procedures based on a
recognition
of the more prominent N-terminal amino acyl ion are to be
preferred.
Whilst mixtures of long chain fatty
have been used for distinctive spectrometry of peptides, all recognition
N-terminal
acids or CH3C0and CD3C0(6)
tags for low resolution
automated procedures have relied
mass
on the
of elemental composition of the N-acyl fragment by high resolution
mass spectrometry.
(7,8)
In view of the high cost of this instrumentation,
we have re-examined the sequencing of peptides by low resolution spectrometry with derivatives identification.
which are optfmal for N-terminal amino acid
Wenow report on the sequencing of acetylacetonyl
(ACA-peptides),
which gives reliable
acid residues),
regardless of the amino acids present.
on the identification
mass
peptides
results with small peptides (2-10 amino The method depends
of the N-terminal amino acid residue 'A' in the spectra.
878
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 37, No. 6, 1969
WA A1 CH3 I 1; ; CH3COCH = C - NH - CHL COINH: ; Since, in this system, these ions unambiguousstarting *I,
Cl, Dl).
R4 ,D D R2 P *I R3C Cl I; I 1:; I; ?CH,- CO!NH - CH;- CO;NH- CH;- Co!OCH2CH3 I 8 I I 8 ar,' quite prominent,' it affoids a'n
point for the search for the sequence ions (B, C, D and
Preparation of the volatile
derivatives
involves esterification
of the peptide followed by treatment with acefylacetone.
Under the experimental
conditions used, arginine peptides are converted to 6-N-(2-pyrimidinyl) ornithine
peptides (9) and the E amino group of lysine is also derivatised.
All other functional and are left
groups present in the protein amino acids remain intact
unprotected.
A typical
the peptide (0.5-1.0 mg) with e
sample preparation
ethanolic hydrogen chloride
w/w) for 10 min and removing the solvent -in vacua. dry ethanol (100 ul), X8, Bio-Rad Cal., at pH 7-8.
involves refluxing
acetylacetone
After
(1 ml, 25-30X
redissolving
in
(30 ul) and dry ion exchange beads (AGl-
in the bicarbonate form) are added till
Molecular sieve (3A, Matheson, N.J.)
of water and the reaction mixture is left
the solution
is
is added to remove traces The supernatant liquid
overnight.
is then withdrawn with a syringe and the solvent evaporated -in vacua. residue is then inserted directly mass spectrometer.
The
into the ion source of a low resolution
On the basis of the mass spectra of almost 80 model
peptides, we find that the "A" fragment is the most prominent peak in the spectra of ACA-peptide esters, which have an N-terminal aliphaticamino acid.
(Table 1)
In somecases partial
loss of the side chain is
observed with N-terminal methionine, serine, threonine, acid.
Somelarger seryl-
and threonyl-peptides
probe temperatures and this makes recognition N-terminal position
difficult.
Histidyl-,
tryptophyl-peptides
show someelimination
or acidic-
aspartic and glutamic
tend to dehydrate at higher of these residues in the
phenylalanyl-,
tyrosyl-
and
of the side chain as ArCHz or
ArCH2O, but these ions help to confirm the presence of these residues. and arginyl
peptides yield
the very characteristic
[A-99] fragments, and show
only very small "A" fragments in the massspectra (Figure 2), whilst 879
Lysyl
cystine
BIOCHEMICAL
Vol. 37, No. 6, 1969
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 Characteristic
Fragments Used for Identification
N-Terminal Amino Acid
"A" Fragment (m/e) [CH3-CO-CH=C(CH~)-NH-CHR]+
GLY ALA SER PRO VAL THR LRIJ ILE HYP ASN MET HIS ASP PHE GLU TYR TRY LYS ARG CYS
112 126 142 152 154 156 168 168 168 169 186 192 198 202 212 218 241" 265" 275"
of N-Terminal Amino Acids Other Ions (m/e) Used for Identification
112 (A-CH20)t 138 (A-H20)?,112 (A-CH3CHO)* 150 (A-H20)f 138 (A-CH3SH)t
130 166 176 156
(Ar-CH2)+ (A-NH2-C(CH3)=CH-COCH3)+ (&NH2-C(CH3)=CH-COCH )? (A/2-H)?, 158 (A/2+H) 3
*Minor fragment.
derivatives
undergo S-S bond rupture accompanied by hydrogen transfer
(10).
Although peptides containing unmodified asparagine have been sequenced, diaaomethane has to be used instead of alcoholic esterification peptide. yield
(11) to prevent hydrolysis
interpretable
and penta-peptides.
and limits
enamino ketone function
the sequencing
Experiments to increase the
of the ACA-peptide esters by permethylation The potential
yielded a complex mixture of products.
con-
The presence of basic
amino acids decreases the volatility
procedure to tetra-
alkylation
high vapor pressure and
mass spectra from hepta- and octa-peptides
only the neutral amino acids (Figure 1).
polyfunctional
volatility
to the corresponding aspartyl
The ACA-peptide esters have a relatively
readily
taining
hydrogen chloride for
(12-15) invariably difficulty
is the
which reacts with methyl iodide to give 0 and C
(16) and a non volatile
quaternary salt. 880
4m
Fipure
128
140
Figure
1.
2.
160
17,
mm
(Finnigan
24a
Ethyl
2tIm
Temperature
1015 Mass Spectrometer)
of ACA-Arg-Ala
220
CH 4 Mass Spectrometer,
of ACA-Ala-Leu-Ala-Val-Gly-Ala-Phe
Mass Spectrum
A-99
(Atlas
Mass Spectrum
Ester
301
321
260°
C)
Ethyl
340
3fim
Ester
389
4w
421
Vol. 37, No. 6, 1969
BIOCHEMICAL
AND BIOF’HYSICAL RESEARCH COMMUNICATIONS
Acknowledgments. This work was supported by a fellowship
to E. Jellum from
the Norwegian Cancer Society and by National Aeronautics and Space Administration
grant NGR-05-020-004. REFERENCES
1. 2. 3. 4. 5. 6. 7. ;: 10. 11. 12. 13. 14. 15. 16.
Weygand, F., Prox, A,, Ktinig, W. and Fessel, H. H. Angew. Chem.2, 724 (1963). Heyns, K. and Grutsmacher, H. F. Tetrahedron Letters 1761 (1963). Wulfson, N. S., Bochkarev, V. N., Rozinov, B. V., Shemyakin, M. M., Ovchinnikov, Yu. A., Kiryushkin, A. A. and Miroshnikov, A. I. Tetrahedron Letters, 39 (1966). Barber, M., Jolles, P., Vilkas, E. and Lederer, E. Biochem. Biophys. Res. Cosm~.l.g-, 4h9 (1965). Kiryushkin, A. A., Ovchinnikov, Yu. A., Shemyakin, M. M., Bochkarev, V.N., Rosinov, B. V. and Wulfson, N. S. Tetrahedron Letters 33 (1966). Bricas, E., Van Heijenoort, J., Barber, M., Wolstenholme, W. A., Das, B. C. and Lederer, E. Biochemistry 5, 2254 (1965). Biemann, K., Cone, C., Webster, B. R. and Arsenault, G. P. 2. &. M. Sot. EtJ, 5598 (1966). Senn, M., Venkataraghavan, R. and McLafferty, F. M. Ibid 18, 5593 (1966). King, T. P. Biochemistry 2, 3454 (1966). Kiryushkin, A. A., Gorlenko, V. A., Agadshanyan, Ts E., Rosinov, B. V., Ovchinnikov, Yu. A. and Shemyakin, M. M. Experientia 2, 884 (1968). Bayer, E. and Koenig, W. A. Advances in Chromatography, Ed1tor.A. Zlatkis. Preston Technical Abstracts Co., Evanston, Ill. 187 (1969). Das, B. C., Gero, S. D. and Lederer, E. Biochem. Biophys. Res. Comm. 2, 211 (1967). Thomas, D. W. Ibid 2, 483 (1968). Thomas, D. W., Das, B. C., Gero, S. D. and Lederer, E. Ibid 32, 199, 519 (1968). Agarwal, K. L., Kenner, G. W. and Sheppard, R. C. J. Am. Chem. Sot. !X, 3096 (1969). Meyers, A. I., Reine, A. H. and Gault, R. J. Org. Chem.34, 698 (1969).
882