- Email: [email protected]
Analysis of nucleosides, nucleotides and oligonucleotides using fast atom bombardment mass spectrometry
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trends in analytical chemistry, vol. 7, no. I, 1988
Analysis of nucleosides, nucleotides and oligonucleotides using fast atom bombardment mass spectrometry Karl H. Schram Tucson,AZ, U.S.A. Mass spectrometry, especially fast atom bombardment mass spectrometry, is playing an ever increasing role in the identification of naturally occurring and synthetic nucleic acid components. Practical and applied aspects of this technique are discussed using examples taken from the recent literature describing the mass spectral analysis of nucleosides, nucleotides and oligonucleotides.
The closing sentence of a previous review in this journal concerning the mass spectrometry of nucleic acid components’ has been shown to be prophetic indeed, stating that “nucleic acid chemistry needs mass spectrometry and the demand is growing”. A number of factors are responsible for this expanding role of mass spectrometry into areas previously intractable to this analytical technique, with one of the most important being the development of fast atom bombardment (FAB)* mass spectrometry2. This is not to imply that other methods in mass spectrometry have not been important, they have been3T4 and other new techniques and methods are being intensively investigated in many laboratories. However, because of widespread interest, the focus of this review will be the development and application of FAB mass spectrometry to the analysis of nucleosides, nucleotides and oligonucleotides5. FAB: general comments FAB ionization offers a number of advantages, and disadvantages, relative to the classical ionization techniques of electron impact (EI) and chemical ionization (CI) for the analysis of nucleic acid components4T5. Among these advantages are its simplicity of use, the relatively low cost of the necessary equipment, the ease of attaching a FAB gun onto an existing mass spectrometer, and the extended time
l
The acronym FAB is used in this review because of its widespread name recognition by persons not trained as mass spectroscopists. Strong arguments have, however, been made for replacing the term ‘FAB’ with ‘liquid SIMS’ (secondary-ion mass spectrometry). See ref. 3 for an expanded discussion of this point.
01659936/88/$03.00.
over which ions may be detected. The latter aspect is unique to FAB and important when other mass spectrometric techniques, such as high resolution or metastable ion analysis, are to be performed. The above mentioned advantages have combined to make FAB mass spectrometry a both widely available and commonly used technique. Among the limitations of the FAB method are the presence of intense ions derived from the matrix (solvent), a loss in structural information and sensitivity relative to EI or CI, the difficulty sometimes encountered in identifying an appropriate matrix for dissolution of the sample, and the necessity of performing the analysis on purified, individual components. Although this list of disadvantages may appear rather long, techniques are now, or are becoming, available which offset or eliminate these limitations and the use of FAB for the characterization of nucleic acid components is a highly successful and important technique. FAB of nucleosides Compared to results obtained using EI or CI, the FAB spectrum of an underivatized nucleoside is simple and is dominated by the MH+ and BH; ions6. .Even though a general loss of structural information results from this simplification of the mass spectrum, FAB provides unambiguous molecular weight determination, identification of the aglycone and, by difference, the weight of the sugar moiety. Most importantly, nucleosides which cannot be analyzed in their free form using EI or CI provide FAB mass spectra with little difficulty. No instance has yet been encountered in the author’s laboratory where a nucleoside, even highly polarized mesoionic nucleosides’, has failed to provide information on these basic structural features. The requirement that the sample be dissolved in a liquid matrix in order to obtain a spectrum leads to one of the greatest difficulties of the FAB method. Intense ions are generated by the matrix which can hinder identification of sample related ions, especially in the case of a nucleoside of unknown structure. A number of approaches can, however, be used to overcome this difficulty. Probably the easiest 0 Elsevier Science Publishers B .V.
trends inanalytical chemistry, vol. 7, no. 1,1988
Fig. 1. The “matrixless” FAB mass spectrum of adenosine obtained by melting the sample on the tip of the probe. No matrix ions are present to mask sample related ions. B = ion formed by cleavage of the glycosidic bond, representing the aglycone (base) portion of the molecule. For B + 30 and B f 44 structures see ref. 5.
approach is to re-run the sample in a second matrix. By comparing the two spectra, ions related to the nucleoside should be easily identified. Another approach, being investigated in the author’s laboratory, is to melt the nucleoside which will then act as its own matrix. A “pure” FAB mass spectrum of the sample can thus be obtained. However, this method requires the use of a heatable FAB probe and some of the advantage of not having to heat the sample, as is the case in normal FAB operation, is lost. On the other hand, the amount of energy required to melt a sample is considerably less than that required to vaporize the sample, so decomposition is not as extensive as in the case of the vapor phase techniques. This approach has been used to obtain a “neat” FAB spectrum of adenosine, shown in Fig. 1, which displays a strong MH’ ion and other structurally important fragment ions, some of which are not observed in the normal FAB spectrum. How useful this method will be requires further investigation into the quantity of sample required and the amount of decomposition experienced by more polar samples, e.g., deoxyguanosine or cytosine nucleosides. By far the best approach to use in overcoming the major disadvantages of FAB is tandem mass spectrometry (MS-MS)s. For example, Crow et aL9 have shown that FAB MS-MS with collisionally activated dissociation (CAD) not only eliminates matrix derived artifact ions from the mass spectrum, it also restores a considerable amount of structural information lost in the normal FAB spectrum. A limitation of the use of tandem mass spectrometry is the cost of such instruments which may be expected to reduce
29
somewhat availability to this type of instrumentation. An alternative approach, at least as far as restoration of structural information to the FAB spectrum of nucleosides using normal FAB instrumentation, is to perform the analysis on the trimethylsilyl (TMS) derivative of the nucleoside. This approach is being investigated in the author’s laboratory”. Following preparation of the derivative the normal FAB spectrum is acquired using an appropriate matrix. A significant enhancement in the number and intensity of structurally significant fragment ions is observed, relative to the FAB spectrum of the free nucleoside. In fact, as shown in Fig. 2, a high degree of similarity is observed when the FAB mass spectrum is compared to the EI spectrum. For example, the FAB spectrum of the TMS derivative of adenosine displays 8 of 10 sugar ions and 13 of 16 aglycone related fragments observed in the EI spectrum of the same sample. The presence of a number of odd-electron ions in this FAB spectrum suggests that fragmentation is proceeding via ion-radical mechanisms. Structure elucidation of nucleosides using FAB Based on the structure-fragmentation studies mentioned above, the use of FAB mass spectrometry for the determination of the structure of an unknown nucleoside is relatively straight forward. However, interactions of the sample with the atom beam or solvent have been reported4 and, thus, the FAB spectrum of an unknown should be analyzed carefully to insure that no FAB-induced or solventgenerated reactions have taken place. A technique of great practical importance which can be used in connection with the structure elucidation of unknown nucleosides is the direct determination of the number of exchangeable hydrogen atoms in molecule. When the FAB spectrum of any unknown compound is obtained in 0-perdeuterated glycerol and 2H20, exchange of all active hydrogens is observed which permits a precise determination of the total number of exchangeable hydrogens4. The structure elucidation of adducts formed in the reaction of chemicals and drugs with various components of DNA, or DNA itself, is of fundamental importance in understanding both carcinogenesis and the mechanism of action of antitumor agents. In the last few years mass spectrometry has been applied to a number of these structural problems, with the most recent work utilizing FAB mass spectrometry495. An illustrative example of the approach used in solving such problems involved the use FAB CAD MS-MS” for the characterization of adducts of pyrrolizidine alkaloids with nucleosides and nucleotides. The conclusion of this work was that FAB
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,/
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m/z Fig. 2. Comparison of the FAB and EI spectra of the TMS derivative of adenosine. The FAB spectrum of the TMS derivative shows a large number of structurally significant ions, most of which are absent in the FAB spectrum of the free nucleoside. DG represents diglyme, the matrix used in these experiments and the ions at mlz 704 and 776 are the DG adducts of the MH’(TMS), and MH+(TMS), ions, respectively. S represents the sugar portion of the molecule and B the base. Data obtained as described in ref. IO.
MS-MS provides information relating to the molecular weight of the covalently bonded alkaloid and to the nature and sites of modification of the base, sugar and alkaloid. Cleavages leading to the major ions in the positive ion FAB spectrum of the dihydropyrrolizinol-deoxyguanosine adduct are shown in Fig. 3l’. lH+
f(-H)
RB+44
Fig. 3. Major fragment ions of structural significance observed in the FAB CAD MS-MS spectrum of the dihydropyrrolizinol adduct of guanosine. Nomenclature: R = alkaloid, B = nucleic acid base, S = sugar. Adapted from ref. II.
FAB of mononucleotides and dinucleotide phosphates In general, the positive ion FAB spectra of nucleotides is more complex than that of the corresponding nucleoside, a result of metal ion, e.g., Na+, exchange for hydrogen on the phosphate hydroxyl groups. For this reason, most of the literature describing the FAB mass spectrometry of nucleotides has been performed using the negative ion detection mode. The negative ion FAB mass spectra of nucleotides display a strong [M-H]- ion which permits assignment of molecular weight. Other ions observed using normal FAB conditions include the B- ion and phosphate ions corresponding to (H,POJ and (PO,)). A difference in intensity of the B- ion has been used as a means of distinguishing 3’- from 5’-nucleotide monophosphates, with the B- ion being more intense in the case of the 3’-isomer. A mechanistic rational for this behavioral difference has been recently proposed based on the FAB CAD MS-MS analysis of a large number of mononucleotides and dinucleotide phosphates12. Likewise, FAB MS-MS has revealed differences in fragmentation of isomeric 3’S’- and 2’,3’-cyclic mononucleotides’3.
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The normal negative ion FAB spectra of the dinucleotide phosphates resemble those of the mononucleotides and a comparison of the spectra of isomerit d(ApT) and d(TpA), where A is adenosine and T is thymidine, are quite similar, with abundant [M-H]- ions being observed along with ions corresponding to [M-H-AH]-, [M-H-TH -, A-, T- and “sequence ions” at m/z 321 and 33011. In contrast, the metastable ion spectrum of the [M-H]- ion of each of the isomers is unique, thus permitting assignment to the sequence of the dinucleotide monophosphates. The conclusion drawn from this work is that, of the currently available methods, CAD coupled with MS-MS is the preferred method of analysis, since this approach provides all information necessary to completely characterize dinucleotide monophosphates as to the nucleic acid bases present and assign the position of each base as being in either the 3’- or 5’-position. FAB of oligonucleotides Although the capability of using negative ion FAB for the analysis and sequencing of oligodeoxynucleotides has been established for some time, the role of mass spectrometry is not expected to be great in this area because of the speed and sensitivity of conventional methods. However, the use of FAB for characterizing synthetic oligonucleotide “building blocks”, either free or partially blocked, may be expected to increase in the future. Free oligodeoxynucleotides up to 10 residues in length have been sequenced using FAB mass spectrometry14. Relatively intense [M-H]- ions are observed and molecular weight assignments may be checked by the presence of a glycerol adduction and a doubly charged deprotonated molecular ion at [M-2H]*-, i.e., m/z = (MW-2)/2. Sequence ions arise from cleavages originating at both ends of the oligodeoxynucleotide chain permitting bidirectional sequence analysis. Distinction of the 3’- from the 5’sequence ions is, however, possible. While the analysis of fully protected oligodeoxynucleotides has not been successful, the presence of at least one free phosphate group restores the structural and sequence information observed in the free compounds and the FAB spectra of the partially protected and free samples are very similar, but more complex in the former case because of additional fragmentations arising from the protecting groups. Conclusions and outlook FAB mass spectrometry has been shown to have important applications in the analysis of nucleosides, nucleotides and oligodeoxynucleotides. Major applications of this technique are expected to continue
in the structure elucidation of modified nucleosides (nucleotides) isolated from natural sources and in the confirmation of structure of synthetic oligonucleotides. Little work has been done in the area of quantitation of nucleosides or nucleotides using FAB. If methods can be developed to decrease the detection limits of FAB, the technique could become important in clinical studies of nucleoside antitumor and antiviral agents, where, if coupled with CAD MS-MS capability, the direct analysis of crude biological samples may be possible - or at least the time spent in sample preparation greatly reduced. Availability of stable isotope labeled internal standards will also be important in any FAB quantitation studies and general methods for the synthesis of such compounds need to be explored. Liquid chromatography-mass spectrometry (LC-MS) is also expected to play a more important role in both basic biochemical problems and in clinical studies involving metabolite identification and pharmacokinetic studies. Instruments and methods used in LC-MS must, however, be capable of routine operation. As new methods and instrumental capabilities are developed, and combined with the classical EUCI ionization and gas chromatographic-mass spectrometric (GC-MS) techniques, the range of application of mass spectrometry to problems involving nucleic acids and their components continues to grow. The 25 years since the first paper describing the analysis of nucleosides15 by mass spectrometry has seen an almost inconceivable, in retrospect, expansion of instrumental capabilities and applications in the area of nucleic acid analysis. Similar growth, especially in the biomedical field, can be expected in the future. Acknowledgements This work is supported and 43068.
by NIH Grants CA 42309
References M. Linscheid, Trends Anal. Chem., 2 (No. 2) (1983) 32. M. Barber, R. S. Bordoli, R. D. Sedgwick and A. N. Tyler, Nature (London), 293 (1981) 270. A. L. Burlingame, T. A. Ballie and P. J. Derrick, Anal. Chem. Fund. Rev., 58 (1986) 165R. J. A. McCloskey, in A. L. Burlingame and N. Castagnoli, Jr. (Editors), Mass Spectrometry In the Health and Life Sciences, Elsevier, Amsterdam, 1985, p. 521. D. L. Slowikowski and K. H. Schram, Nucleosides Nucleotides, 4 (1985) 309. D. L. Slowikowski and K. H. Schram, Nucleosides Nucleotides, 4 (1985) 347. E. M. Schubert, K. H. Schram and R. A. Glennon, J. Heterocycl. Chem., 22 (1985) 889. L. G. Wright, J. C. Schwartz and R. G. Cooks, Trends Anal. Chem., 5 (No. 9) (1986) 236.
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9 F. W. Crow, K. B. Tomer, M. L. Gross, J. A. McCloskey and D. E. Bergstrom, Anal. Biochem., 139 (1984) 243. 10 K. H. Schram and D. L. Slowikowski, Biomed. Environ. Mass Spectrom., 13 (1986) 263. 11 K. B. Tomer, M. L. Gross and M. L. Deinzer, Anal. Chem.,
58 (1986) 2527. 12 R. L. Cerny, M. L. Gross and L. Grotjahn,
Anal. Biochem.,
156 (1986) 424. 13 E. E. Kingston, J. H. Beynon, R. P. Newton and J. G. Liehr, Biomed. Mass Spectrom., 12 (1985) 525. 14 L. Grotjahn, H. Blocker and R. Frank, Biomed. Mass Spectrom., 12 (1985) 514.
15 K. Biemann and J. A. McCloskey, J. Am. Chem. Sot., 84 (1962) 2005. Dr. Karl Schram received his Ph.D. in Medicinal Chemistry from the University of Utah. Originally trained as a synthetic nucleoside chemist, Dr. Schram became interested in mass spectrometry while talking with Dr. J. A. McCloskey. He later joined his group as an NIH postdoctoral fellow. He is currently an Associate Professor of Pharmaceutical Sciences in the College of Pharmacy at the University of Arizona. His research interests center on the application of mass spectrometry to biomedical problems, with a focus on the analysis of nucleic acid components.
Use of simplex optimization to improve the performance of an inductively coupled plasma in atomic emission spectrometry G. L. Moore Randburg, South Africa
Simplex methods are used in the optimization of many analytical techniques. In inductively coupled plasma-atomic emission spectrometry, various performance criteria can be used, including signal-to-background ratios, signal-to-noise ratios, detection limits, and the minimization of matrix effects. The simplex method can indicate deficiencies in equipment design that restrain perJormance. The threshold of auto-optimization of analytical instruments has been reached.
Simplex optimization has been used extensively in industry as a mathematical method for the optimization of interdependent variables, and has been applied to the optimization of such diverse operations as the tanning of hides, the establishment of the wear resistance of carbide coatings, and the removal of copper from anodic slags, to the determination of the compositions of clinker, rocket propellants, and lowfoaming household detergent. Over the past decade, its application to analytical techniques has been appreciated, and it has been widely used to improve the performance of many analytical instruments. It has also been applied to pattern recognition, the interpretation of spectral data’ and the fitting of non-linear curves to laboratory data2. In 1962 Spendley et al3 published what has become known as the basic simplex method, involving a fixed stepsize simplex. A simplex is a geometrical 0165-9936/88/$03.00.
figure that can move over a response surface to locate the optimum position. Spendley et al. foresaw that the advent of digital computers would facilitate the adaptation of the simplex method to other areas of research. A simplex always has one more vertex (leg) than the number of variable parameters, ~1, to be optimized. A simplex can be regarded as a multi-legged device that moves over a response surface by lifting one detachable leg (the worst response) and replacing it on the surface in a direction expected to be towards the region of optimum response. If the initial simplex is large it moves rapidly but cannot define the optimum position accurately. If it is small its progress is slow but it can accurately determine the optimum position. The modified simplex method (MSM), which allows for expansion and contraction of the simplex, was introduced by Nelder and Mead4 in 1965 and well described by Morgan and Deming’ in 1974. When the fixed-size simplex is used, it is sometimes difficult to recognize when an optimum has been reached, whereas the variable-size simplex moves rapidly towards the optimum region and then contracts or ‘homes in’ on the optimum. Subsequently, modifications to improve the speed and reduce the number of experiments required (super MSMs) were published along with suggestions for improvements?‘. A recent computer search of Chemical Abstracts from 1966 to 1985 for papers featuring simplex optimization showed that, apart from a burst of activity in 1974 and 1975, there has been a steady increase in OElsevier Science Publishers B.V.
trends in analytical chemistry, vol. 7, no. I, 1988
Analysis of nucleosides, nucleotides and oligonucleotides using fast atom bombardment mass spectrometry Karl H. Schram Tucson,AZ, U.S.A. Mass spectrometry, especially fast atom bombardment mass spectrometry, is playing an ever increasing role in the identification of naturally occurring and synthetic nucleic acid components. Practical and applied aspects of this technique are discussed using examples taken from the recent literature describing the mass spectral analysis of nucleosides, nucleotides and oligonucleotides.
The closing sentence of a previous review in this journal concerning the mass spectrometry of nucleic acid components’ has been shown to be prophetic indeed, stating that “nucleic acid chemistry needs mass spectrometry and the demand is growing”. A number of factors are responsible for this expanding role of mass spectrometry into areas previously intractable to this analytical technique, with one of the most important being the development of fast atom bombardment (FAB)* mass spectrometry2. This is not to imply that other methods in mass spectrometry have not been important, they have been3T4 and other new techniques and methods are being intensively investigated in many laboratories. However, because of widespread interest, the focus of this review will be the development and application of FAB mass spectrometry to the analysis of nucleosides, nucleotides and oligonucleotides5. FAB: general comments FAB ionization offers a number of advantages, and disadvantages, relative to the classical ionization techniques of electron impact (EI) and chemical ionization (CI) for the analysis of nucleic acid components4T5. Among these advantages are its simplicity of use, the relatively low cost of the necessary equipment, the ease of attaching a FAB gun onto an existing mass spectrometer, and the extended time
l
The acronym FAB is used in this review because of its widespread name recognition by persons not trained as mass spectroscopists. Strong arguments have, however, been made for replacing the term ‘FAB’ with ‘liquid SIMS’ (secondary-ion mass spectrometry). See ref. 3 for an expanded discussion of this point.
01659936/88/$03.00.
over which ions may be detected. The latter aspect is unique to FAB and important when other mass spectrometric techniques, such as high resolution or metastable ion analysis, are to be performed. The above mentioned advantages have combined to make FAB mass spectrometry a both widely available and commonly used technique. Among the limitations of the FAB method are the presence of intense ions derived from the matrix (solvent), a loss in structural information and sensitivity relative to EI or CI, the difficulty sometimes encountered in identifying an appropriate matrix for dissolution of the sample, and the necessity of performing the analysis on purified, individual components. Although this list of disadvantages may appear rather long, techniques are now, or are becoming, available which offset or eliminate these limitations and the use of FAB for the characterization of nucleic acid components is a highly successful and important technique. FAB of nucleosides Compared to results obtained using EI or CI, the FAB spectrum of an underivatized nucleoside is simple and is dominated by the MH+ and BH; ions6. .Even though a general loss of structural information results from this simplification of the mass spectrum, FAB provides unambiguous molecular weight determination, identification of the aglycone and, by difference, the weight of the sugar moiety. Most importantly, nucleosides which cannot be analyzed in their free form using EI or CI provide FAB mass spectra with little difficulty. No instance has yet been encountered in the author’s laboratory where a nucleoside, even highly polarized mesoionic nucleosides’, has failed to provide information on these basic structural features. The requirement that the sample be dissolved in a liquid matrix in order to obtain a spectrum leads to one of the greatest difficulties of the FAB method. Intense ions are generated by the matrix which can hinder identification of sample related ions, especially in the case of a nucleoside of unknown structure. A number of approaches can, however, be used to overcome this difficulty. Probably the easiest 0 Elsevier Science Publishers B .V.
trends inanalytical chemistry, vol. 7, no. 1,1988
Fig. 1. The “matrixless” FAB mass spectrum of adenosine obtained by melting the sample on the tip of the probe. No matrix ions are present to mask sample related ions. B = ion formed by cleavage of the glycosidic bond, representing the aglycone (base) portion of the molecule. For B + 30 and B f 44 structures see ref. 5.
approach is to re-run the sample in a second matrix. By comparing the two spectra, ions related to the nucleoside should be easily identified. Another approach, being investigated in the author’s laboratory, is to melt the nucleoside which will then act as its own matrix. A “pure” FAB mass spectrum of the sample can thus be obtained. However, this method requires the use of a heatable FAB probe and some of the advantage of not having to heat the sample, as is the case in normal FAB operation, is lost. On the other hand, the amount of energy required to melt a sample is considerably less than that required to vaporize the sample, so decomposition is not as extensive as in the case of the vapor phase techniques. This approach has been used to obtain a “neat” FAB spectrum of adenosine, shown in Fig. 1, which displays a strong MH’ ion and other structurally important fragment ions, some of which are not observed in the normal FAB spectrum. How useful this method will be requires further investigation into the quantity of sample required and the amount of decomposition experienced by more polar samples, e.g., deoxyguanosine or cytosine nucleosides. By far the best approach to use in overcoming the major disadvantages of FAB is tandem mass spectrometry (MS-MS)s. For example, Crow et aL9 have shown that FAB MS-MS with collisionally activated dissociation (CAD) not only eliminates matrix derived artifact ions from the mass spectrum, it also restores a considerable amount of structural information lost in the normal FAB spectrum. A limitation of the use of tandem mass spectrometry is the cost of such instruments which may be expected to reduce
29
somewhat availability to this type of instrumentation. An alternative approach, at least as far as restoration of structural information to the FAB spectrum of nucleosides using normal FAB instrumentation, is to perform the analysis on the trimethylsilyl (TMS) derivative of the nucleoside. This approach is being investigated in the author’s laboratory”. Following preparation of the derivative the normal FAB spectrum is acquired using an appropriate matrix. A significant enhancement in the number and intensity of structurally significant fragment ions is observed, relative to the FAB spectrum of the free nucleoside. In fact, as shown in Fig. 2, a high degree of similarity is observed when the FAB mass spectrum is compared to the EI spectrum. For example, the FAB spectrum of the TMS derivative of adenosine displays 8 of 10 sugar ions and 13 of 16 aglycone related fragments observed in the EI spectrum of the same sample. The presence of a number of odd-electron ions in this FAB spectrum suggests that fragmentation is proceeding via ion-radical mechanisms. Structure elucidation of nucleosides using FAB Based on the structure-fragmentation studies mentioned above, the use of FAB mass spectrometry for the determination of the structure of an unknown nucleoside is relatively straight forward. However, interactions of the sample with the atom beam or solvent have been reported4 and, thus, the FAB spectrum of an unknown should be analyzed carefully to insure that no FAB-induced or solventgenerated reactions have taken place. A technique of great practical importance which can be used in connection with the structure elucidation of unknown nucleosides is the direct determination of the number of exchangeable hydrogen atoms in molecule. When the FAB spectrum of any unknown compound is obtained in 0-perdeuterated glycerol and 2H20, exchange of all active hydrogens is observed which permits a precise determination of the total number of exchangeable hydrogens4. The structure elucidation of adducts formed in the reaction of chemicals and drugs with various components of DNA, or DNA itself, is of fundamental importance in understanding both carcinogenesis and the mechanism of action of antitumor agents. In the last few years mass spectrometry has been applied to a number of these structural problems, with the most recent work utilizing FAB mass spectrometry495. An illustrative example of the approach used in solving such problems involved the use FAB CAD MS-MS” for the characterization of adducts of pyrrolizidine alkaloids with nucleosides and nucleotides. The conclusion of this work was that FAB
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M-15
540 I’““““I”“‘““I’““““I’““““1 400 500
-L
,/
M 555 600
m/z Fig. 2. Comparison of the FAB and EI spectra of the TMS derivative of adenosine. The FAB spectrum of the TMS derivative shows a large number of structurally significant ions, most of which are absent in the FAB spectrum of the free nucleoside. DG represents diglyme, the matrix used in these experiments and the ions at mlz 704 and 776 are the DG adducts of the MH’(TMS), and MH+(TMS), ions, respectively. S represents the sugar portion of the molecule and B the base. Data obtained as described in ref. IO.
MS-MS provides information relating to the molecular weight of the covalently bonded alkaloid and to the nature and sites of modification of the base, sugar and alkaloid. Cleavages leading to the major ions in the positive ion FAB spectrum of the dihydropyrrolizinol-deoxyguanosine adduct are shown in Fig. 3l’. lH+
f(-H)
RB+44
Fig. 3. Major fragment ions of structural significance observed in the FAB CAD MS-MS spectrum of the dihydropyrrolizinol adduct of guanosine. Nomenclature: R = alkaloid, B = nucleic acid base, S = sugar. Adapted from ref. II.
FAB of mononucleotides and dinucleotide phosphates In general, the positive ion FAB spectra of nucleotides is more complex than that of the corresponding nucleoside, a result of metal ion, e.g., Na+, exchange for hydrogen on the phosphate hydroxyl groups. For this reason, most of the literature describing the FAB mass spectrometry of nucleotides has been performed using the negative ion detection mode. The negative ion FAB mass spectra of nucleotides display a strong [M-H]- ion which permits assignment of molecular weight. Other ions observed using normal FAB conditions include the B- ion and phosphate ions corresponding to (H,POJ and (PO,)). A difference in intensity of the B- ion has been used as a means of distinguishing 3’- from 5’-nucleotide monophosphates, with the B- ion being more intense in the case of the 3’-isomer. A mechanistic rational for this behavioral difference has been recently proposed based on the FAB CAD MS-MS analysis of a large number of mononucleotides and dinucleotide phosphates12. Likewise, FAB MS-MS has revealed differences in fragmentation of isomeric 3’S’- and 2’,3’-cyclic mononucleotides’3.
trends in analyticalchemistry,
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The normal negative ion FAB spectra of the dinucleotide phosphates resemble those of the mononucleotides and a comparison of the spectra of isomerit d(ApT) and d(TpA), where A is adenosine and T is thymidine, are quite similar, with abundant [M-H]- ions being observed along with ions corresponding to [M-H-AH]-, [M-H-TH -, A-, T- and “sequence ions” at m/z 321 and 33011. In contrast, the metastable ion spectrum of the [M-H]- ion of each of the isomers is unique, thus permitting assignment to the sequence of the dinucleotide monophosphates. The conclusion drawn from this work is that, of the currently available methods, CAD coupled with MS-MS is the preferred method of analysis, since this approach provides all information necessary to completely characterize dinucleotide monophosphates as to the nucleic acid bases present and assign the position of each base as being in either the 3’- or 5’-position. FAB of oligonucleotides Although the capability of using negative ion FAB for the analysis and sequencing of oligodeoxynucleotides has been established for some time, the role of mass spectrometry is not expected to be great in this area because of the speed and sensitivity of conventional methods. However, the use of FAB for characterizing synthetic oligonucleotide “building blocks”, either free or partially blocked, may be expected to increase in the future. Free oligodeoxynucleotides up to 10 residues in length have been sequenced using FAB mass spectrometry14. Relatively intense [M-H]- ions are observed and molecular weight assignments may be checked by the presence of a glycerol adduction and a doubly charged deprotonated molecular ion at [M-2H]*-, i.e., m/z = (MW-2)/2. Sequence ions arise from cleavages originating at both ends of the oligodeoxynucleotide chain permitting bidirectional sequence analysis. Distinction of the 3’- from the 5’sequence ions is, however, possible. While the analysis of fully protected oligodeoxynucleotides has not been successful, the presence of at least one free phosphate group restores the structural and sequence information observed in the free compounds and the FAB spectra of the partially protected and free samples are very similar, but more complex in the former case because of additional fragmentations arising from the protecting groups. Conclusions and outlook FAB mass spectrometry has been shown to have important applications in the analysis of nucleosides, nucleotides and oligodeoxynucleotides. Major applications of this technique are expected to continue
in the structure elucidation of modified nucleosides (nucleotides) isolated from natural sources and in the confirmation of structure of synthetic oligonucleotides. Little work has been done in the area of quantitation of nucleosides or nucleotides using FAB. If methods can be developed to decrease the detection limits of FAB, the technique could become important in clinical studies of nucleoside antitumor and antiviral agents, where, if coupled with CAD MS-MS capability, the direct analysis of crude biological samples may be possible - or at least the time spent in sample preparation greatly reduced. Availability of stable isotope labeled internal standards will also be important in any FAB quantitation studies and general methods for the synthesis of such compounds need to be explored. Liquid chromatography-mass spectrometry (LC-MS) is also expected to play a more important role in both basic biochemical problems and in clinical studies involving metabolite identification and pharmacokinetic studies. Instruments and methods used in LC-MS must, however, be capable of routine operation. As new methods and instrumental capabilities are developed, and combined with the classical EUCI ionization and gas chromatographic-mass spectrometric (GC-MS) techniques, the range of application of mass spectrometry to problems involving nucleic acids and their components continues to grow. The 25 years since the first paper describing the analysis of nucleosides15 by mass spectrometry has seen an almost inconceivable, in retrospect, expansion of instrumental capabilities and applications in the area of nucleic acid analysis. Similar growth, especially in the biomedical field, can be expected in the future. Acknowledgements This work is supported and 43068.
by NIH Grants CA 42309
References M. Linscheid, Trends Anal. Chem., 2 (No. 2) (1983) 32. M. Barber, R. S. Bordoli, R. D. Sedgwick and A. N. Tyler, Nature (London), 293 (1981) 270. A. L. Burlingame, T. A. Ballie and P. J. Derrick, Anal. Chem. Fund. Rev., 58 (1986) 165R. J. A. McCloskey, in A. L. Burlingame and N. Castagnoli, Jr. (Editors), Mass Spectrometry In the Health and Life Sciences, Elsevier, Amsterdam, 1985, p. 521. D. L. Slowikowski and K. H. Schram, Nucleosides Nucleotides, 4 (1985) 309. D. L. Slowikowski and K. H. Schram, Nucleosides Nucleotides, 4 (1985) 347. E. M. Schubert, K. H. Schram and R. A. Glennon, J. Heterocycl. Chem., 22 (1985) 889. L. G. Wright, J. C. Schwartz and R. G. Cooks, Trends Anal. Chem., 5 (No. 9) (1986) 236.
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9 F. W. Crow, K. B. Tomer, M. L. Gross, J. A. McCloskey and D. E. Bergstrom, Anal. Biochem., 139 (1984) 243. 10 K. H. Schram and D. L. Slowikowski, Biomed. Environ. Mass Spectrom., 13 (1986) 263. 11 K. B. Tomer, M. L. Gross and M. L. Deinzer, Anal. Chem.,
58 (1986) 2527. 12 R. L. Cerny, M. L. Gross and L. Grotjahn,
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15 K. Biemann and J. A. McCloskey, J. Am. Chem. Sot., 84 (1962) 2005. Dr. Karl Schram received his Ph.D. in Medicinal Chemistry from the University of Utah. Originally trained as a synthetic nucleoside chemist, Dr. Schram became interested in mass spectrometry while talking with Dr. J. A. McCloskey. He later joined his group as an NIH postdoctoral fellow. He is currently an Associate Professor of Pharmaceutical Sciences in the College of Pharmacy at the University of Arizona. His research interests center on the application of mass spectrometry to biomedical problems, with a focus on the analysis of nucleic acid components.
Use of simplex optimization to improve the performance of an inductively coupled plasma in atomic emission spectrometry G. L. Moore Randburg, South Africa
Simplex methods are used in the optimization of many analytical techniques. In inductively coupled plasma-atomic emission spectrometry, various performance criteria can be used, including signal-to-background ratios, signal-to-noise ratios, detection limits, and the minimization of matrix effects. The simplex method can indicate deficiencies in equipment design that restrain perJormance. The threshold of auto-optimization of analytical instruments has been reached.
Simplex optimization has been used extensively in industry as a mathematical method for the optimization of interdependent variables, and has been applied to the optimization of such diverse operations as the tanning of hides, the establishment of the wear resistance of carbide coatings, and the removal of copper from anodic slags, to the determination of the compositions of clinker, rocket propellants, and lowfoaming household detergent. Over the past decade, its application to analytical techniques has been appreciated, and it has been widely used to improve the performance of many analytical instruments. It has also been applied to pattern recognition, the interpretation of spectral data’ and the fitting of non-linear curves to laboratory data2. In 1962 Spendley et al3 published what has become known as the basic simplex method, involving a fixed stepsize simplex. A simplex is a geometrical 0165-9936/88/$03.00.
figure that can move over a response surface to locate the optimum position. Spendley et al. foresaw that the advent of digital computers would facilitate the adaptation of the simplex method to other areas of research. A simplex always has one more vertex (leg) than the number of variable parameters, ~1, to be optimized. A simplex can be regarded as a multi-legged device that moves over a response surface by lifting one detachable leg (the worst response) and replacing it on the surface in a direction expected to be towards the region of optimum response. If the initial simplex is large it moves rapidly but cannot define the optimum position accurately. If it is small its progress is slow but it can accurately determine the optimum position. The modified simplex method (MSM), which allows for expansion and contraction of the simplex, was introduced by Nelder and Mead4 in 1965 and well described by Morgan and Deming’ in 1974. When the fixed-size simplex is used, it is sometimes difficult to recognize when an optimum has been reached, whereas the variable-size simplex moves rapidly towards the optimum region and then contracts or ‘homes in’ on the optimum. Subsequently, modifications to improve the speed and reduce the number of experiments required (super MSMs) were published along with suggestions for improvements?‘. A recent computer search of Chemical Abstracts from 1966 to 1985 for papers featuring simplex optimization showed that, apart from a burst of activity in 1974 and 1975, there has been a steady increase in OElsevier Science Publishers B.V.