Abstracts of Communications 556th Meeting of the Biochemical Society Imperial College of Science and Technology, London 30 June and 7July 7975
MASS SPECTROMETRY IN BIOCHEMISTRY: a joint Colloquium organized on behalf of the Biochemical Society and the Perkin Division of the Chemical Society by H. Morris (London) and R. A. W. Johnstone (Liverpool) Applications of Chemical Ionization Mass Spectrometry JOHN M. WILSON Department of Chemistry, University of Manchester, Manchester M13 9PL, U.K.
The first examples quoted of c.i.m.s. (chemical ionization mass spectrometry) were observed in the spectra of n-alkanes present as 1% impurities in methane at a pressure of 133Pa (1 Torr). At this pressure, under electron impact, the following processes are observed (Field & Munson, 1965).
CH4 CH4+' CH4 CH3++CH4 CH4 CHz' CzH3' CH4
+
+ +
-
CH,", CH2, GIz+', CH+ CH,' + CH3' CzH,++Hz CzH3' Hz H ' C3H5' Hz
+ + +
CHs+ and CzHs+together account for over 90% of the ions produced and C3Hs+is the only other ion of importance. When another molecular species is added to the methane, the principal methods of ionization will be by reaction with CH5+and with CzHS+.For n-alkanes the reactions are
These reactions are only mildly exothermic and the [M - HI+ ion, or quasimolecularion [QM+], is very abundant compared with fragment ions produced from it. Indeed for n-octadecane the C18H37+ ion is responsible for more than 30% of total ionization. For compounds containing nitrogen and oxygen, the principal ionization process is protonation, and [M+H]+ ions of high abundance are observed for many molecules of biological importance. The c.i. spectra of amines are extremely useful. The molecular ion of amphetamine (I) cannot be detected by the electron-impact method, and the spectrum is dominated by theion at m/e 44 (CH3+CHNHz).In the methaneci spectrum the two most abundant ions are produced by the processes shown in Scheme 1.
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BIOCHEMICAL SOCIETY TRANSACTIONS
I Scheme 1. Processes producing the two most abundant ions in the c.i. spectrum
R-CO-NH-R
+ CH,’
+ R-CO-NH2-R
+ CHd
Scheme 2. Production of ‘sequence’ ions in the c.i.m.s. of acylpeptide esters
It is thus possible to analyse mixtures of amines of different molecular weights. If the intensity of the [M+H]+ peak is to be used for quantitative measurements, a system in which only this ion is produced would be an improvement. In the case of amines this can be achieved by using isobutane instead of methane as the reactant gas. The ions produced from isobutane are mostly C4H9+, probably the tertiary butyl cation, and, by using this, spectra containing only the [M+H]+ ions can be obtained from amines. Another group of nitrogen compounds where molecular ions are of low abundance are the barbiturates. In c.i.m.s. these compounds produce [M+H]+ ions with relative abundances of 4 6 4 7 % of total ionization and these can be used for the quantitative analysis of mixtures (Fales et al., 1970). There have been a number of applications to peptide structure determination. C.i.m.s. can be used for the analysis of phenylthiohydantions from the Edman degradation, and the sensitivity is 10-100 times greater than by electron impact. This is a common feature of c.i.m.s. where the spectrum is dominated by the [M+H]+ ion. The advantage over gas chromatography is that glutamate, serine, threonine, lysine, aspartate, glutamine and asparagine can all be analysed as their free phenylthiohydantoin derivatives without further treatment (Fales et al., 1971). One must, however, set against this advantage the cost of a mass spectrometer. Various mass-spectrometric techniques have been used over the past 10 years to obtain information on the sequence of peptides from the fragments present in the spectra of simple derivatives. The difficulty of this approach using electron-impact is the multiplicity of possible fragmentation processes and in some cases the absence of the ‘significant’ fragments, i.e. those produced by simple cleavage of the peptide bond. In the c.i.m.s. of acyl peptide esters there are two series of ‘sequence’ ions produced as shown in Scheme 2 and the spectra show a much lower abundance of ions produced by alternative cleavages (Gray et al., 1970; Kiryushkin et al., 1971). An advantage of c.i.m.s. for use with gas chromatography is that the ion source can take all or most of the column effluent, and the reactant gas may be used as the carrier gas (Arsenault et al., 1970; Schoengold & Munson, 1970). It may in the future be possible to use c.i.m.s. in conjunction with liquid chromatography. Experiments using a solution input to the mass spectrometer have shown that it is possible to maintain 1975
556th MEETING, LONDON
455
chemical ionization conditions with the solvent as the ionizing gas, Ammonia, water, methanol and hexane are all suitable solvents (Baldwin & McLafferty, 1973). A further development of c.i.m.s. has been the construction of an ion source that operates at atmospheric pressure. Instead of an electron beam the primary ionization source is 63Ni radiation. The ions produced in the atmospheric-pressure region are accelerated into a vacuum systemwith a mass analyser. Samplesevaporated from benzene solutions in the ionizing region are ionized by proton transfer from C6H7+to give [M+H]+ ions. By using this system it was possible to detect nicotine in the urine extracts of smokers and also of non-smokers who shared the same room (Horning et al., 1973). The sensitivity of the system has been tested by using single-ion monitoring and the lower limit that has been reached so far is l5Ofg of 2,6-dimethyl-y-pyrone(Carroll et af., 1974). This method promises to be extremely useful in the analysisof very small quantities of biological samples. Arsenault, G. P., Dolhun, J. J. & Biernann, K. (1970) J. Chem.SOC.Chem. Commm. 1542-1543 Baldwin, M. A. & McLa!Terty,F. W. (1973) Org. Muss Spectrom. 7,1111-1 112 Carroll, D. I., Dzidic, I., Stillwell, R. N., Horning, M. G. & Horning, E. C. (1974) Anal. Chem. 46,707-710.
Fales, H. M., Milne, G. W. A. & Axenrod, T. (1970) Anal. Chem. 42,1432-1435 Fales, H. M., Nagai, Y.,Milne, G. W. A., Brewer, H. B., Bronzert, T. J. & Pisano, J. J. (1971) Anal. Biochem.43,288-299
Field, F. H. & Munson, M. S. B. (1965) J. Am. Chem. SOC.87,3289-3299 Gray, W. R.,Wojcik, L. H. & Futrell, J. H. (1970) Biochem. Biophys. Res. Commun. 41,111 11119
Homing, E. C., Horning, M. G., Carroll, D. I., Stillwell, R. N. & Dzidic, I. (1973) Life Sci. 13, 1331-1346
Kjushkin, A. A., Fales, H. M., Axenrod, T., Gilbert, E. J. & Milne, G. W. A. (1971) Org. Mass Spectrom. 5,19-3 1 Schoengold, D. M. & Munson, B. (1970) Anal. Chem. 42,1811-1813
Field Desorption and Field Ionization Mass Spectrometry DAVID E. GAMES Department of Chemistry, University College, P.O. Box 18, Cardir CF1 1XL, U.K. For many years mass-spectrometricanalysis of biologically important compounds has been carried out almost exclusively with electron-impact sources. One problem which arises when this type of ionization is used is that certain structural features in a molecule can facilitate cleavage of the molecule to such an extent that the molecular ion is either absent from the mass spectrum, or is of such low intensity that it is easily confused with ions arising from impurities in the sample, or with background ions in the mass spectrometer. Electron-impact spectra run at low-electron voltages, field ionization (Beckey, 1971) and chemical ionization (Field, 1968) have been used as methods of minimizing fragmentation and providing molecular or quasi-molecular ions of high intensity. A disadvantage of these techniques is that the substance under investigation must have some volatility and may be subject to thermal decomposition. Beckey (1969), reported a new method of sample supply for field ionization called field desorption whereby solid samples are ionized without prior vaporization. The technique gives mass spectra which have molecular or quasi-molecular ions and some fragment ions for compounds which are thermally labile and/or of low volatility. Beckey, Schulten and Winkler and their colleagues have shown the wide applicability of this method of ionization, and recently, with the availability of commercial and ‘home-made’ sources, a number of other groups have become active in this area. In the present paper the utility of field desorption in some studies of compounds of biological interest will be discussed together with some developmentsin field ionization. Recent review articles by Beckey & Schulten (1975a, 6)give a detailed discussion of the more theoretical aspects of the technique together with many examples of the application of the technique.
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MASS SPECTROMETRY IN BIOCHEMISTRY: a joint Colloquium organized on behalf of the Biochemical Society and the Perkin Division of the Chemical Society by H. Morris (London) and R. A. W. Johnstone (Liverpool) Applications of Chemical Ionization Mass Spectrometry JOHN M. WILSON Department of Chemistry, University of Manchester, Manchester M13 9PL, U.K.
The first examples quoted of c.i.m.s. (chemical ionization mass spectrometry) were observed in the spectra of n-alkanes present as 1% impurities in methane at a pressure of 133Pa (1 Torr). At this pressure, under electron impact, the following processes are observed (Field & Munson, 1965).
CH4 CH4+' CH4 CH3++CH4 CH4 CHz' CzH3' CH4
+
+ +
-
CH,", CH2, GIz+', CH+ CH,' + CH3' CzH,++Hz CzH3' Hz H ' C3H5' Hz
+ + +
CHs+ and CzHs+together account for over 90% of the ions produced and C3Hs+is the only other ion of importance. When another molecular species is added to the methane, the principal methods of ionization will be by reaction with CH5+and with CzHS+.For n-alkanes the reactions are
These reactions are only mildly exothermic and the [M - HI+ ion, or quasimolecularion [QM+], is very abundant compared with fragment ions produced from it. Indeed for n-octadecane the C18H37+ ion is responsible for more than 30% of total ionization. For compounds containing nitrogen and oxygen, the principal ionization process is protonation, and [M+H]+ ions of high abundance are observed for many molecules of biological importance. The c.i. spectra of amines are extremely useful. The molecular ion of amphetamine (I) cannot be detected by the electron-impact method, and the spectrum is dominated by theion at m/e 44 (CH3+CHNHz).In the methaneci spectrum the two most abundant ions are produced by the processes shown in Scheme 1.
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454
BIOCHEMICAL SOCIETY TRANSACTIONS
I Scheme 1. Processes producing the two most abundant ions in the c.i. spectrum
R-CO-NH-R
+ CH,’
+ R-CO-NH2-R
+ CHd
Scheme 2. Production of ‘sequence’ ions in the c.i.m.s. of acylpeptide esters
It is thus possible to analyse mixtures of amines of different molecular weights. If the intensity of the [M+H]+ peak is to be used for quantitative measurements, a system in which only this ion is produced would be an improvement. In the case of amines this can be achieved by using isobutane instead of methane as the reactant gas. The ions produced from isobutane are mostly C4H9+, probably the tertiary butyl cation, and, by using this, spectra containing only the [M+H]+ ions can be obtained from amines. Another group of nitrogen compounds where molecular ions are of low abundance are the barbiturates. In c.i.m.s. these compounds produce [M+H]+ ions with relative abundances of 4 6 4 7 % of total ionization and these can be used for the quantitative analysis of mixtures (Fales et al., 1970). There have been a number of applications to peptide structure determination. C.i.m.s. can be used for the analysis of phenylthiohydantions from the Edman degradation, and the sensitivity is 10-100 times greater than by electron impact. This is a common feature of c.i.m.s. where the spectrum is dominated by the [M+H]+ ion. The advantage over gas chromatography is that glutamate, serine, threonine, lysine, aspartate, glutamine and asparagine can all be analysed as their free phenylthiohydantoin derivatives without further treatment (Fales et al., 1971). One must, however, set against this advantage the cost of a mass spectrometer. Various mass-spectrometric techniques have been used over the past 10 years to obtain information on the sequence of peptides from the fragments present in the spectra of simple derivatives. The difficulty of this approach using electron-impact is the multiplicity of possible fragmentation processes and in some cases the absence of the ‘significant’ fragments, i.e. those produced by simple cleavage of the peptide bond. In the c.i.m.s. of acyl peptide esters there are two series of ‘sequence’ ions produced as shown in Scheme 2 and the spectra show a much lower abundance of ions produced by alternative cleavages (Gray et al., 1970; Kiryushkin et al., 1971). An advantage of c.i.m.s. for use with gas chromatography is that the ion source can take all or most of the column effluent, and the reactant gas may be used as the carrier gas (Arsenault et al., 1970; Schoengold & Munson, 1970). It may in the future be possible to use c.i.m.s. in conjunction with liquid chromatography. Experiments using a solution input to the mass spectrometer have shown that it is possible to maintain 1975
556th MEETING, LONDON
455
chemical ionization conditions with the solvent as the ionizing gas, Ammonia, water, methanol and hexane are all suitable solvents (Baldwin & McLafferty, 1973). A further development of c.i.m.s. has been the construction of an ion source that operates at atmospheric pressure. Instead of an electron beam the primary ionization source is 63Ni radiation. The ions produced in the atmospheric-pressure region are accelerated into a vacuum systemwith a mass analyser. Samplesevaporated from benzene solutions in the ionizing region are ionized by proton transfer from C6H7+to give [M+H]+ ions. By using this system it was possible to detect nicotine in the urine extracts of smokers and also of non-smokers who shared the same room (Horning et al., 1973). The sensitivity of the system has been tested by using single-ion monitoring and the lower limit that has been reached so far is l5Ofg of 2,6-dimethyl-y-pyrone(Carroll et af., 1974). This method promises to be extremely useful in the analysisof very small quantities of biological samples. Arsenault, G. P., Dolhun, J. J. & Biernann, K. (1970) J. Chem.SOC.Chem. Commm. 1542-1543 Baldwin, M. A. & McLa!Terty,F. W. (1973) Org. Muss Spectrom. 7,1111-1 112 Carroll, D. I., Dzidic, I., Stillwell, R. N., Horning, M. G. & Horning, E. C. (1974) Anal. Chem. 46,707-710.
Fales, H. M., Milne, G. W. A. & Axenrod, T. (1970) Anal. Chem. 42,1432-1435 Fales, H. M., Nagai, Y.,Milne, G. W. A., Brewer, H. B., Bronzert, T. J. & Pisano, J. J. (1971) Anal. Biochem.43,288-299
Field, F. H. & Munson, M. S. B. (1965) J. Am. Chem. SOC.87,3289-3299 Gray, W. R.,Wojcik, L. H. & Futrell, J. H. (1970) Biochem. Biophys. Res. Commun. 41,111 11119
Homing, E. C., Horning, M. G., Carroll, D. I., Stillwell, R. N. & Dzidic, I. (1973) Life Sci. 13, 1331-1346
Kjushkin, A. A., Fales, H. M., Axenrod, T., Gilbert, E. J. & Milne, G. W. A. (1971) Org. Mass Spectrom. 5,19-3 1 Schoengold, D. M. & Munson, B. (1970) Anal. Chem. 42,1811-1813
Field Desorption and Field Ionization Mass Spectrometry DAVID E. GAMES Department of Chemistry, University College, P.O. Box 18, Cardir CF1 1XL, U.K. For many years mass-spectrometricanalysis of biologically important compounds has been carried out almost exclusively with electron-impact sources. One problem which arises when this type of ionization is used is that certain structural features in a molecule can facilitate cleavage of the molecule to such an extent that the molecular ion is either absent from the mass spectrum, or is of such low intensity that it is easily confused with ions arising from impurities in the sample, or with background ions in the mass spectrometer. Electron-impact spectra run at low-electron voltages, field ionization (Beckey, 1971) and chemical ionization (Field, 1968) have been used as methods of minimizing fragmentation and providing molecular or quasi-molecular ions of high intensity. A disadvantage of these techniques is that the substance under investigation must have some volatility and may be subject to thermal decomposition. Beckey (1969), reported a new method of sample supply for field ionization called field desorption whereby solid samples are ionized without prior vaporization. The technique gives mass spectra which have molecular or quasi-molecular ions and some fragment ions for compounds which are thermally labile and/or of low volatility. Beckey, Schulten and Winkler and their colleagues have shown the wide applicability of this method of ionization, and recently, with the availability of commercial and ‘home-made’ sources, a number of other groups have become active in this area. In the present paper the utility of field desorption in some studies of compounds of biological interest will be discussed together with some developmentsin field ionization. Recent review articles by Beckey & Schulten (1975a, 6)give a detailed discussion of the more theoretical aspects of the technique together with many examples of the application of the technique.
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