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The mass spectral analysis of covalently labeled amino acid methylthiohydantoin derivatives derived from affinity-labeled proteins
ANALYTICAL
BIOCHEMISTRY
75, 682-685
(1976)
The Mass Spectral Analysis of Covalently Labeled Amino Acid Methylthiohydantoin Derivatives Derived from Affinity-Labeled Proteins This communication describes the use of mass spectrometry to identify the methylthiohydantoin derivatives of amino acids containing covalently bound 2,4-dinitrophenyl-p-aminobenzoyl and 2,4-dinitrophenyl labels resulting from the affinity labeling of anti-DNP antibodies with the photoactive labels 2,4-dinitrophenyl-p-aminobenzoyl diazoketone (DNPABDK) (1) and 2,Cdinitrophenyl-1-azide (DNP-NJ (2). The affinity labeling of a rabbit anti-2,Cdinitrophenyl (DNP) antibody with the photoactive label 2,Cdinitrophenyl-1-azide (DNP-NJ and the affinity labeling of a rabbit anti-2,4-dinitrophenyl-p-aminobenzoylglutamate (DNP-ABG) antibody with the label 2,4-dinitrophenyl-p-aminobenzoyl diazoketone (DNP-ABDK) has been described in detail in earlier publications (1,2). The separation of the labeled antibody light and heavy chains and the Nagarse (Enzymes Development Corp., N.Y.) digestion of the heavy chain preparations and subsequent isolation of the label-containing peptides have been described in an earlier communication (2). The radioactive fractions (labeled peptides and amino acids) purified from the Nagarse digests were treated with excess methyl isothiocyanate (MITC) in pyridine/water @O/20, v/v) as described by Fairwell et al. (3). After 30 min the excess MITC and pyrldine/water were removed in vacua and the residue was redissolved in 50-100 ~1 of pyridine/water solvent mixture. An aliquot of the reaction mixture containing the methylthiourea derivatives, representing about lo-30 pg of material, was transferred to a glass capillary tube and evaporated to dryness for mass spectral analysis. The remaining material was evaporated to dryness and treated with anhydrous trifluoroacetic acid (TFA) for 15 min after which the TFA was removed in VUCUO. The resulting peptide mixture was subjected to a second addition of MITC in pyridine/water and the entire procedure repeated. The mass spectra of the methylthiourea derivatives were obtained by thermally cyclizing the samples in the mass spectrometer in the manner described by Ellis (4). The samples were introduced via a solid sample inlet probe into a DuPont Model 21-490 mass spectrometer and spectra were obtained at 20°C intervals as the probe temperature was increased to 400°C. The fifth step of the sequential degradation of the peptide mixtures produced methylthiourea derivatives whose mass spectra were compatible 682 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
683
SHORT COMMUNICATIONS
100
200
400
300
500
600
m/e
FIG. 1. Mass spectrum of the methylthiourea derivatives isolated from step 5 of the Edman degradation of the labeled peptides isolated from Nagarse digestion of the anti-DNP-pABG antibody heavy chains labeled with DNP-p-aminobenzoyl diazoketone.
with that of a substituted amino acid methylthiohydantoin. The mass spectrum of the methylthiourea derivative of the fifth residue, in addition to ions at m/e 130 and 430, exhibited ions at m/e 168, 210, 258, 286, 301, 328, and 510. The spectrum is shown in Fig. 1. Since the DNP-p-aminobenzoyl diazoketone (I) reacts with the antibody protein chain via the __)
DNP-NW&H4-C-CH2:
DNP-NHC6HqCH=C=0
(m.w.
8
(m.w.
300)
299)
(III)
ketocarbene (II) or as a ketene (III) arising from the Wolff rearrangement of the ketocarbene, the presence of the label should add 300 amu to the molecular ion of the labeled amino acid MTH if it reacted as the ketocarbene and 299 amu if it bound via the ketene. The ions in the mass spectrum of the methylthiohydantoin of residue 5 suggest that the covalently labeled amino acids are histidine and glycine. The ion at m/e 510 corresponds to the molecular ion of histidine, m/e 210 plus the molecular weight of the label (300 amu). The series of ions at m/e 328,301, 286,258,210, 182, and 168 can arise from the stepwise fragmentation of the DNPp-aminobenzoyl histidine MTH, as indicated in IV. The ions at m/e 430 and 130, as well as the ions at 168, 182, 258, 286, and 301 also
684
SHORT 167
Noq
COMMUNICATIONS
182
258
286
iv3aZ 323
300
510
\z12\‘:is-m+ \ 258
224
210
(IV) suggest the presence of the labeled amino acid glycine. The molecular ion of glycine is m/e 130, to which the addition of 300 amu due to the bound label would give a molecular ion of m/e 430 for the labeled glycine MTH. The presence of the labeled glycine MTH is supported by the fact that the histidine MTH derivative does not produce an appreciable ion at m/e 130 which, since.it is present in considerable abundance, probably arises from the fragmentation of DNP-p-aminobenzoyl glycine MTH corresponding to the loss of the DNP-p-aminobenzoyl group (M+-300). The affinity label products isolated from the Nagarse digestion of heavy chains of two antibody fractions (A and C) labeled with 2,4-dinitrophenylI-azide (DNP-N,) (V) proved to be primarily single amino acids rather than peptides, the main species isolated from the Nagarse digestion of antibody
N02
-/ \79
N3
NO2
W)
labeled with DNP-p-aminobenzoyl diazoketone (1). The mass spectra of the labeled material from antibody fraction A treated with methylisothiocyanate contained ions at m/e 399, 391, 279, 271, and 130. The spectra of the material from fraction C contained an ion at m/e 325 in addition to ions at 399, 391, 279, 271, and 130. DNP-N, reacts via the nitrene, DNP-N, to form DNP-N-amino acid derivatives. Consequently, the mass spectra of amino acid MTH derivatives containing the DNP-N-label should exhibit ions corresponding to the molecular ion of the amino acid MTH plus 181 amu due to the DNP-N moiety. The ions at m/e 399 and 391 respectively, are consistent with the molecular ion of the dehydro form of DNP-N-phenylalanine MTH (M+-2 = 399) and the molecular ion of DNP-N-histidine MTH (M+ = 391) respectively. The ion at m/e 271 apparently corresponds to the molecular weight of DNP-N plus the fragment C,H5 arising from the fragmentation of the DNP-N-phenylalanine MTH. The ion at m/e 325 in the mass spectra of the methylthiourea derivatives from fraction C corresponds to the molecular ion of DNPN-alanine MTH (144 + 181). The ions at m/e 130 corroborate this since neither phenylalanine MTH nor histidine MTH exhibits appreciable m/e 130 ions.
SHORT COMMUNICATIONS
685
This preliminary study suggests that the identity of amino acids which have been covalently labeled by small organic molecules used as affinity labels of protein active sites can be established from the mass spectra of their methylthiohydantoin derivatives. The method lends itself to the analysis of small quantities of labeled amino acids, as is the case in most affinity-labeling experiments. ACKNOWLEDGMENT This work was supported by agrant (BMS 71-00742) from the National Science Foundation.
REFERENCES 1. Lindeman, J. G., Woodard, D. K., Woehler, M. E.. and Lovins, R. E. (1975)Zmmunothem. 12, 849. 2. Cannon, L. E., Woodard, D. K., Woehler, M. E.. and Lovins, R. E. (1974) Zmmunology 26, 1183. 3. Fairwell, T., Ellis, S., and Lovins. R. E. (1973) Anal. Biochem. 53, 115. 4. Ellis. S., Fairwell, T., and Lovins, R. E. (1972) Biochem. Biophys. Res. Commun. 49, 1407.
JOEL LINDEMAN ROBERT E. LOVINS Department of Biochemistry University of Georgia Athens, Georgia 30602 Received April 8, 1976; accepted June II, 1976
BIOCHEMISTRY
75, 682-685
(1976)
The Mass Spectral Analysis of Covalently Labeled Amino Acid Methylthiohydantoin Derivatives Derived from Affinity-Labeled Proteins This communication describes the use of mass spectrometry to identify the methylthiohydantoin derivatives of amino acids containing covalently bound 2,4-dinitrophenyl-p-aminobenzoyl and 2,4-dinitrophenyl labels resulting from the affinity labeling of anti-DNP antibodies with the photoactive labels 2,4-dinitrophenyl-p-aminobenzoyl diazoketone (DNPABDK) (1) and 2,Cdinitrophenyl-1-azide (DNP-NJ (2). The affinity labeling of a rabbit anti-2,Cdinitrophenyl (DNP) antibody with the photoactive label 2,Cdinitrophenyl-1-azide (DNP-NJ and the affinity labeling of a rabbit anti-2,4-dinitrophenyl-p-aminobenzoylglutamate (DNP-ABG) antibody with the label 2,4-dinitrophenyl-p-aminobenzoyl diazoketone (DNP-ABDK) has been described in detail in earlier publications (1,2). The separation of the labeled antibody light and heavy chains and the Nagarse (Enzymes Development Corp., N.Y.) digestion of the heavy chain preparations and subsequent isolation of the label-containing peptides have been described in an earlier communication (2). The radioactive fractions (labeled peptides and amino acids) purified from the Nagarse digests were treated with excess methyl isothiocyanate (MITC) in pyridine/water @O/20, v/v) as described by Fairwell et al. (3). After 30 min the excess MITC and pyrldine/water were removed in vacua and the residue was redissolved in 50-100 ~1 of pyridine/water solvent mixture. An aliquot of the reaction mixture containing the methylthiourea derivatives, representing about lo-30 pg of material, was transferred to a glass capillary tube and evaporated to dryness for mass spectral analysis. The remaining material was evaporated to dryness and treated with anhydrous trifluoroacetic acid (TFA) for 15 min after which the TFA was removed in VUCUO. The resulting peptide mixture was subjected to a second addition of MITC in pyridine/water and the entire procedure repeated. The mass spectra of the methylthiourea derivatives were obtained by thermally cyclizing the samples in the mass spectrometer in the manner described by Ellis (4). The samples were introduced via a solid sample inlet probe into a DuPont Model 21-490 mass spectrometer and spectra were obtained at 20°C intervals as the probe temperature was increased to 400°C. The fifth step of the sequential degradation of the peptide mixtures produced methylthiourea derivatives whose mass spectra were compatible 682 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
683
SHORT COMMUNICATIONS
100
200
400
300
500
600
m/e
FIG. 1. Mass spectrum of the methylthiourea derivatives isolated from step 5 of the Edman degradation of the labeled peptides isolated from Nagarse digestion of the anti-DNP-pABG antibody heavy chains labeled with DNP-p-aminobenzoyl diazoketone.
with that of a substituted amino acid methylthiohydantoin. The mass spectrum of the methylthiourea derivative of the fifth residue, in addition to ions at m/e 130 and 430, exhibited ions at m/e 168, 210, 258, 286, 301, 328, and 510. The spectrum is shown in Fig. 1. Since the DNP-p-aminobenzoyl diazoketone (I) reacts with the antibody protein chain via the __)
DNP-NW&H4-C-CH2:
DNP-NHC6HqCH=C=0
(m.w.
8
(m.w.
300)
299)
(III)
ketocarbene (II) or as a ketene (III) arising from the Wolff rearrangement of the ketocarbene, the presence of the label should add 300 amu to the molecular ion of the labeled amino acid MTH if it reacted as the ketocarbene and 299 amu if it bound via the ketene. The ions in the mass spectrum of the methylthiohydantoin of residue 5 suggest that the covalently labeled amino acids are histidine and glycine. The ion at m/e 510 corresponds to the molecular ion of histidine, m/e 210 plus the molecular weight of the label (300 amu). The series of ions at m/e 328,301, 286,258,210, 182, and 168 can arise from the stepwise fragmentation of the DNPp-aminobenzoyl histidine MTH, as indicated in IV. The ions at m/e 430 and 130, as well as the ions at 168, 182, 258, 286, and 301 also
684
SHORT 167
Noq
COMMUNICATIONS
182
258
286
iv3aZ 323
300
510
\z12\‘:is-m+ \ 258
224
210
(IV) suggest the presence of the labeled amino acid glycine. The molecular ion of glycine is m/e 130, to which the addition of 300 amu due to the bound label would give a molecular ion of m/e 430 for the labeled glycine MTH. The presence of the labeled glycine MTH is supported by the fact that the histidine MTH derivative does not produce an appreciable ion at m/e 130 which, since.it is present in considerable abundance, probably arises from the fragmentation of DNP-p-aminobenzoyl glycine MTH corresponding to the loss of the DNP-p-aminobenzoyl group (M+-300). The affinity label products isolated from the Nagarse digestion of heavy chains of two antibody fractions (A and C) labeled with 2,4-dinitrophenylI-azide (DNP-N,) (V) proved to be primarily single amino acids rather than peptides, the main species isolated from the Nagarse digestion of antibody
N02
-/ \79
N3
NO2
W)
labeled with DNP-p-aminobenzoyl diazoketone (1). The mass spectra of the labeled material from antibody fraction A treated with methylisothiocyanate contained ions at m/e 399, 391, 279, 271, and 130. The spectra of the material from fraction C contained an ion at m/e 325 in addition to ions at 399, 391, 279, 271, and 130. DNP-N, reacts via the nitrene, DNP-N, to form DNP-N-amino acid derivatives. Consequently, the mass spectra of amino acid MTH derivatives containing the DNP-N-label should exhibit ions corresponding to the molecular ion of the amino acid MTH plus 181 amu due to the DNP-N moiety. The ions at m/e 399 and 391 respectively, are consistent with the molecular ion of the dehydro form of DNP-N-phenylalanine MTH (M+-2 = 399) and the molecular ion of DNP-N-histidine MTH (M+ = 391) respectively. The ion at m/e 271 apparently corresponds to the molecular weight of DNP-N plus the fragment C,H5 arising from the fragmentation of the DNP-N-phenylalanine MTH. The ion at m/e 325 in the mass spectra of the methylthiourea derivatives from fraction C corresponds to the molecular ion of DNPN-alanine MTH (144 + 181). The ions at m/e 130 corroborate this since neither phenylalanine MTH nor histidine MTH exhibits appreciable m/e 130 ions.
SHORT COMMUNICATIONS
685
This preliminary study suggests that the identity of amino acids which have been covalently labeled by small organic molecules used as affinity labels of protein active sites can be established from the mass spectra of their methylthiohydantoin derivatives. The method lends itself to the analysis of small quantities of labeled amino acids, as is the case in most affinity-labeling experiments. ACKNOWLEDGMENT This work was supported by agrant (BMS 71-00742) from the National Science Foundation.
REFERENCES 1. Lindeman, J. G., Woodard, D. K., Woehler, M. E.. and Lovins, R. E. (1975)Zmmunothem. 12, 849. 2. Cannon, L. E., Woodard, D. K., Woehler, M. E.. and Lovins, R. E. (1974) Zmmunology 26, 1183. 3. Fairwell, T., Ellis, S., and Lovins. R. E. (1973) Anal. Biochem. 53, 115. 4. Ellis. S., Fairwell, T., and Lovins, R. E. (1972) Biochem. Biophys. Res. Commun. 49, 1407.
JOEL LINDEMAN ROBERT E. LOVINS Department of Biochemistry University of Georgia Athens, Georgia 30602 Received April 8, 1976; accepted June II, 1976