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MS based scanning methodologies applied to Conus venom
MS Based Scanning Methodologies Applied to Conus Venom t A. Grey Craig, Wolfgang H. Fischer, Jean E. Rivier, J. Michael Mcintosh; and William R. Grayt The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, San Diego, CA 92138-9216 and tDepartments of Psychiatry and Biology, University of Utah, Salk Lake City, UT 84112
I. Introduction Known biologically active agents in the venom produced by marine cone snails (Conus)y are small, highly constrained and specialized peptides. These venoms are a rich source of unique neuroactive molecules (1). Although the venoms from different species of cone snails may contain homologous peptides (e.g. both C. geographus, and C. striatus make peptides targeted to acetylcholine receptors and voltage sensitive calcium channels) they may also contain a number of distinct specialized peptides (e.g. the activity of conantokin-G of C. geographus which targets NMDA receptors has not been observed in C. striatus venom) (1). We describe a number of strategies (including derivatizations) that allow the identification of as yet uncharacterized toxins in these venoms. The extent of the challenge lies in the fact that most Conus venoms are complex mixtures containing over 100 peptides. With the advent of very sensitive ionization techniques such as matrix assisted laser desorption (MALDI) coupled with time-of-flight (TOF) mass analysis, measurement of the intact mass of peptides at sub-pmol levels has become a reality (2) of which we have taken advantage for the systematic screening of HPLC fractions. Partial sequence information can be obtained by carrying out enzymatic hydrolysis with exoproteinases (e.g. carboxypeptidases and aminopeptidases) (3, 4). More recently, MALDI has been used to measure metastable decomposition occurring in the first field free region of a reflectron TOF instrument (referred to as post source decay (PSD)) with only marginally more sample (5-7). While significantly less material is required for MALDI than for either UV detection, chemical sequencing or amino acid analysis, the nature of the information derived from MALDI spectra is also different. Clearly, obtaining unambiguous composition or sequence information is not a simple task. This is due to the fact that (i) the mass accuracy of MALDI-TOF measurements is generally lower than that of liquid secondary ionization (LSI) with a magnetic sector mass spectrometer, (ii) enzymatic sequencing is affected by the varying rates of cleavages at different amino acid residues and the reduced activity of most enzymes towards particular residues (e.g., tyrosine and proline) and (iii) TECHNIQUES IN PROTEIN CHEMISTRY VI Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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fragmentation information from PSD suffers from the ambiguities of assigning fragment ions as being derived from the N-terminus, C-terminus or "internal sequence". We have implemented scanning methodologies using MALDI-TOF mass spectrometry to partially purified venom from C striatus and C. ermineus. We have carried out specific derivatizations in order to deduce composition and sequence information. Together with an intact mass these measurements are used to determine whether an ionized species observed in the MALDI mass spectrum corresponds with the intact protonated molecule of a previously characterized conotoxin. The information obtained from derivatizations is also important when the ionized species does not correspond with the intact mass of peptides of known sequence. In that case, post source decay of the native and derivatized species may help assign the fragment ions.
II. Materials and Methods MALDI mass spectra were measured with a Bruker Reflex time-of-flight mass spectrometer fitted with a gridless reflectron energy analyzer and a nitrogen laser. Accelerating and reflectron voltage of +31 kV and +30 kV were employed unless otherwise specified. Typically, the amount of sample necessary for MALDI analysis was 100 fold less than that used for LSI analysis. All MALDI samples were prepared in six or more different sample preparation formats including three different UV absorbing matrices (a-cyano-4hydroxycinnamic acid, sinapinic acid and 2,5-dihydroxybenzoic acid) and two methods of preparation. In the first method peptide solution was pre-mixed with a solution of each matrix prior to application onto the probe tip (see refs. (8-10) for preparation of matrix solutions). In the second method a solution of the matrix in acetone was dried on the probe tip and then a separate sample of peptide was applied and left to dry onto the matrix (11) As noted previously, the second preparation was found to give more reliable analyses of samples which required a rinse with H2O (12, 13). No ions were observed with any other sample preparation which were not observed using a combination of the second procedure, a-cyano-4-hydroxycinnamic acid as the matrix, and rinsing of the samples. All tabulated data are for samples prepared in this manner. LSIMS spectra were measured with a JEOL IMS HXllO mass spectrometer fitted with a Cs"*" ion gun. An accelerating voltage of +10 kV and Cs"^ ion gun voltage of +30 kV were employed. An electric field scan over a narrow mass range was used to measure segments of the mass spectrum corresponding with appropriate regions of the MALDI mass spectrum. The samples, prior to the dilution used for MALDI analysis, (1 |il; 100 pmol; 0.1 % TFA solution) were added directly to a 1:1 mixture of m-nitrobenzyl alcohol and glycerol. The mass accuracy of LSIMS for measurement of the unresolved isotopic cluster is typically within 100 p.p.m. of the calculated average [M+H]+ mass. The accuracy of the observed masses listed in Tables I and II for the MALDI mass spectra benefited from the use of a reflectron instrument which generally reduced the deviation observed between spectra measured under different experimental conditions (e.g. different matrix or laser power) from ±1000 p.p.m. to ±300 p.p.m. Reflecting this level of mass accuracy we present the MALDI measurements with only 4 significant figures, compared with 5 significant figures for the LSIMS measurements. For calculation of possible amino acid substitutions, the 20 most common amino acids were used together with y-carboxyglutamate (Gla) and hydroxyproline (Hyp) which are commonly found in conotoxins (14).
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Peptide Modification : lodination was carried out on a stainless steel probe target by adding 0.1 % aq. I2 (1 |il) to the dried peptide (ca. 1 pmol). The reaction was stopped after 1 minute by addition of ascorbic acid and the MALDI matrix, a-cyano cinnamic acid in excess. Esterification with ethanol was carried out using the method of Hunt et al. (15), where an acetylchloride and ethanol solution (1:6, v:v) was added (5 |il) to the peptide dried in a microcentrifuge tube (ca. 1 pmol). After incubation for 15 minutes at room temperature a 2 mM p-mercaptoethanol (in ethanol) solution, was added (1 [i\) and the mixture was dried. The matrix, a-cyano-4-hydroxycinnamic acid (2 |j.l), was added to the micro tube and after 5 minutes 1 |il of this matrix was removed and applied to a target.
IIL Results and Discussion Figure 1 shows the HPLC profile of semi-purified venom from C. striatus (fractions labeled 3, 5-18,20 and 22). A summary of the observed masses in the MALDI and LSI mass spectra for each of these fractions is given in Table I. A data base of known conotoxins was searched for correspondence (±3 Da) with the observed masses: "matches" are scored irrespective of whether the peptide in question was originally isolated from the particular venom. Figure 2 shows the HPLC profile of semi-purified venom from C. ermineus (fractions labeled 4a, 4b, 5-11 and 14). A summary of the masses of the major species observed in the LSIMS and MALDI mass spectra for each of these fractions is given in Table II. Our finding that the preparation of a-cyano-4-hydroxycinnamic acid in acetone and subsequent rinsing (see Materials & Methods) produced all ion species which were observed with a variety of other MALDI procedures is important for further scanning of the Conus venoms for novel conotoxins. Generally, we observe at least one major species in the MALDI mass spectra corresponding to each HPLC component. The increased mass accuracy available when the instrument was operated in the reflectron mode was important for the analysis carried out. For example, fractions 4b and 7 or fractions 5 and 8 from C. ermineus appeared to be the same species when measured with the instrument operated in the linear mode. Only in the reflectron mode were we able to reliably distinguish the masses of each species. The high sensitivity of MALDI-TOF is particularly important for the analysis of native peptides such as conotoxins where often the venom of many milkings must be collected to obtain sufficient material for sequence analysis. The increased sensitivity of MALDI over LSIMS is illustrated in the analysis of fraction 5 from C. striatus venom (see Table I). Despite the two orders of magnitude difference in the amount of material consumed in the LSI experiment we did not discern any intact species in fraction 5, whereas the MALDI measurement yielded useful information. However, the comparisons in Tables I and II reveal that some components may be detected by LSIMS but not observed in the MALDI mass spectrum (measured with any of the matrices or sample preparation methods). The contrary is most likely more prevalent, i.e. that a large number of the species detected by MALDI with one or more of the matrices are difficult species to ionize with LSIMS.
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Table I. Observed masses in the LSIMS and MALDI mass spectra of fractions of C striatus [RTI 1 match CaJc! 1 MALDI LSIMS mass (m/z) Obs. mass (m/z) Obs. mass^ ISVIB 2740.2 12739t 12739.5 2544 5 NO 2579 NO 6 SVIA 2494.9 2494t 2494.9 2521 7 2521.4 8 SII 1792.0 1240 1241.5 1791.2 NO 1794t 1813 10 SI 1814 2786 1354.4 1791.3 NO 1354.6 1354t NO 2782.7 11 sm 2497.5 1456.7 1457t 2498 NO 12 2500 4099 9218 1396.4 NO 4098.6 NA 1397 13 NA 2498 3886 3940 NA NA NA 1367 14 NO 4898 4952 4968 NO 4947.0 4965.9 4882 15 4082.0 4098.5 NA 4084 4100 4792 NA 3175 16 2498 3924 4758 2176.6 2497.4 NO 17 4743 5025 NA 3938 18 3778.1 3782 NO 3713 20 3400.0 3416.0 3432.4 3418 NO NO 3348 NO 122 1 INA 1^ calculated average [M+H]+ mass. NO indicates corresponding ion in ]^ALDI or LSIMS spec trum not observecI. NAindi :ates noi analyze5d. t ind icates the obs. species which matched.
ffn
It is apparent from Table I that masses of several previously known peptides from C. striatus correspond to those found for major UV absorbance peaks. Similarly, Table II shows that the C. ermineus venom contained a peptide in fraction 7 that matched the mass of conotoxin GVIB from C. geographus — in this case, the peptide has since been analyzed and found to be completely unrelated to GVIB, whereas the putative match to SI in fraction 9 has been confirmed with chemical sequencing and mass spectrometry.
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Figure 1. UV trace of HPLC of C. striatus venom.
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Table II. Observed masses in the LSIMS and MALDI mass spectra of C. ermineus fractions 1 LSIMS Fr. 1 match Calc. 1 MALDI (m/z) mass Obs. mass (m/z)J L Obs. mass^J 3451.1 NO 2513 3105 NO 1 12496.1 NO [2497'" ^ 3100.7 3101 4b 5 3099.6 3085 3085 NA 2495.4 NA NA 2111 NO 2094 6 7 GVIB 3095.4 3098t 3096.2 3082.3 3082 8 1792 1944 3068 1354.0 1790.8 1943.6 3065.6 SI 9 1354.6 1353t NA 2094 3047 3558 NO 2094.2 NA 1803 10 2766 2781 1397.2 2765.6 2781.4 1398 11 NA 3512 3528 12369.9 NA [2370 1 14 1 ^ calculated average [M+H]+ mass. NO indicates corresponding ion in MALDI or LSIMS spectrum not observed. NA indicates not analyzed, t indicates the obs. species which matched.
From these results it is clear that useful information can be obtained from MALDI, but that it cannot be used directly to establish the identity of peptides — our ultimate aim is to obtain sequence information from these fractions. Towards that goal we are currendy developing protocols that allow reduction of cysteine residues, alkylation and MALDI measurement without the need for further purification (19). In the simplest version, measurement of the peptide before and after modification reveals the number of disulfide bridges present in the peptide. With the linear alkylated peptide, we can more easily interpret the metastable decomposition occurring in the first field free region of a time-of-flight instrument to measure the fragment ions. This protocol is shown in Figure 3 for reduced and S-carboxamidomethylated (Cam) conotoxin GIA(H-Glu-Cam-Cam-Asn-Pro-Ala-Cam-Gly-Arg-His-Tyr-Ser-Cam-Gly-LysNH2) (16, 17). The spectrum shown in Figure 3 was obtained from a derivatization of 10 pmol of peptide in which 1 pmol of peptide was applied to the target. The PSD spectrum is a composite of scans measured at reflectron voltages between 1.25 and 29.9 kV: the total ion current and therefore the baseline noise varies between individual scans. The 'b' and 'y+2' type fragment ions (18) are the most prolific series observed for this peptide and are therefore identified in Figure 3. However, significantly more sequence information is present in this spectrum (19).
time (min)
Figure 2. UV trace of HPLC of C. ermineus venom
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At this point in time it is impractical to sequence every peptide, given the complexity of the venoms. A strategy that directs the sequencing effort to selected peptides can be based on the rapidly growing number of conotoxin sequences which have previously been determined. In 1991, there were over 70 conotoxin peptides characterized from over 10 species (1); this number is now above 200, and growing rapidly with the acquisition of sequences from DNA cloning (20). As described above, we used a database of conotoxin sequences to assign fractions 3, 6, 8 and 11 of C. striatus venom as possibly containing peptides corresponding to SVIB, SVIA, SI and SIB respectively. More accurate mass measurement with LSIMS confirmed that the intact mass was consistent with this assignment in 3 of these cases (no signal was observed with LSIMS in one case). This type of data-base scanning is also being employed in reverse, to search among venom fractions for candidate peptides to match predicted translation products corresponding to cloned cDNAs. The Conus venoms often contain several minor sequence variants of toxins, arising from genetic polymorphisms, multi-gene families, and variation in post-translational processing. When the mass difference between two closely related peptides (in terms of HPLC retention times and mass) corresponds to a single amino acid substitution, a simple experiment may suffice to choose among alternatives. Consider for example fractions 5 and 7 from C. striatus venom, satellites of the major fraction tentatively identified as conotoxin SVIA (Fig 1 and Table I). The mass difference of 50±1 Da between peptides in fractions 5 and 6 could be explained by any of the following changes (i) Tyr to either Hyp, Leu or He (50 Da); (ii) His to Ser (50 Da) (iii) Phe to Pro (50 Da) or (iv) Trp to His (49 Da). Although options (ii) to (iv) are formally possible, SVIA does not contain His, Phe, or Trp, so option (i) would be favored. In order to test this directly, we iodinated small samples of approx. 2 pmol each of fractions 5 and 6. After treatment with I2, fraction 6 was shifted towards higher mass by 126 Da This shift was consistent with the presence of a tyrosine residue in this peptide (we have determined that iodination under these conditions modifies tyrosine but not histidine residues (21)). 73+2 yi3+2
' — I — ^ — I — ^ — I — ^ — r -
400
600
800
1000
1200
1400
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Figure 3. The PSD spectrum (200-1600 Da) of alkylated Conotoxin GIA.
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In contrast, fraction 5 was not modified by this protocol, indicating that the mass difference of 50 Da could be attributed to the tyrosine residue in the peptide in fraction 6, being replaced by either hydroxyproline, leucine or isoleucine in the peptide in fraction 5. Similarly, assuming that SVIA is the major component in fraction 6, the additional 27±1 Da of the peptide in fraction 7 could be attributed to change of Ser to (Leu, He or Hyp), or of Lys to Arg. Esterification of fractions 6 and 7 verified that neither component contain acidic groups, which was consistent with C-terminal amidation of SVIA. These derivatizations are highly selective, and may thus allow PSD measurements to be carried out on peptides after modification. Such a protocol would significantly enhance our ability to derive sequence information from PSD spectra, because the mass shifts observed in fragments help locate the particular residue within the peptide, and also confirm assignments of fragments as arising from N- or C-terminal regions. In addition to derivatizations that may modify the C- and N-termini and the derivatization of tyrosine residues, we have carried out oxidation of methionine residues with sufficient specificity to enable measurement of PSD spectra.
IV. Conclusion We have gained an appreciation for the ionization bias observed between MALDI and LSIMS. The utilization of PSD to identify known peptides and provide sequence information has been investigated for conotoxins. This approach to obtaining sequence information on novel peptides is attractive because of the low amount of material required. A number of mass spectrometric based derivatizations have been used to scan fractions of venoms in order to characterize peptides of interest. For closely related components (based on HPLC retention time and mass), the small scale derivatization schemes can be used to test hypotheses about peptides with otherwise novel masses (i.e. which may be homologs). The mass accuracy of the TOP technique, with a gridless reflector, was important for identifying and calling these substitutions.
Acknowledgments We would like to thank Drs. B. Olivera and UCruz, University of Utah for stimulating discussions. This work was supported by the National Institute of Health (K20MH00929, lSlORR-8425, HD-13527, DK-26741, CA-54418, HL41910, GM-48677) and supported in part by the Foundation for Medical Research, Inc. (AGC and WHF).
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References 1. B. M. Olivera, J. Rivier, J. K. Scott, D. R. Hillyard and L. J. Cruz (1991) Journal of Biological Chemistry 266,22067. 2. M. Karas, A. Ingendoh, U. Bahr and F. Hillenkamp (1989) Biomed. Mass Spectrom. 18,841. 3. M. Schar, K. O. Bomsen and E. Gassmann (1991) Rapid Commun Mass Spectrom 5,319. 4. A. S. Woods, W. Gibson and R. J. Cotter, (1994). In " Time of Flight Mass Spectrometry" (R. J. Cotter, eds.) ACS, Washington D.C., 5. B. Spengler, D. Kirsch, R. Kaufmann and E. Jaeger (1992) Rapid Commun Mass Spectrom 6, 105. 6. M. C. Huberty, J. E. Vath, W. Yu and S. A. Martin (1993) Anal Chem 65,2791. 7. W. Yu, J. E. Vath, M. C. Huberty and S. A. Martin (1993) Anal Chem 65,3015. 8. R. C. Beavis and B. T. Chait (1992) Org Mass Spectrom 27,156. 9. R. C. Beavis and B. C. Chait (1989) Rapid Commun Mass Spectrom. 3,432. 10. K. Strupat, M. Karas and F. Hillenkamp (1991) Int. J. Mass Spectrom. Ion Proc. Ill, 89. 11. O. Vorm, P. Roepstorff and M. Mann (1994). 42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, ILL, May 29- June 3 1994. 12.0. Vorm and M. Mann (1994) J Am Soc Mass Spectrom in press, 13. R. C. Beavis and F. Xiang (1994). 42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, ILL, May 29- June 3 1994. 14. B. M. Olivera, W. R. Gray, R. Zeikus, J. M. Mcintosh, J. Varga, J. Rivier, V. de Santos and L. J. Cruz (1985) Science. 230,1338. 15. D. Hunt, J. R. Yates III, J. Shabanowitz, S. Winston and C. R. Hauer (1986) Proc. Natl. Acad. Sci. USA S3,6233. 16. W. R. Gray, A. Luque, B. M. Olivera, J. Barrett and L. D. Cruz (1981) /. Biol. Chem. 256, 4734. 17. L. J. Cruz, W. R. Gray and B. M. Olivera (1978) Arch. Biochem. Biophys. 190.539. 18. P. Roepstorff and J. Fohlman (1984) Biomed. Mass Spectrom. 11,601. 19. A. G. Craig, W.H. Fischer, W. R. Gray, J. Dykert, J. E. Rivier (unpublished results). 20. D. R. Hillyard, B. M. Olivera, S. Woodward, G. P. Corpuz, W. R. Gray, C. R. Ramilo and L. J. Cruz (1989) Biochemistry 28,358. 21. A. G. Craig, J. E. Rivier, W. R. Gray and W. H. Fischer (1994). 42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, ILL, May 29- June 3 1994
I. Introduction Known biologically active agents in the venom produced by marine cone snails (Conus)y are small, highly constrained and specialized peptides. These venoms are a rich source of unique neuroactive molecules (1). Although the venoms from different species of cone snails may contain homologous peptides (e.g. both C. geographus, and C. striatus make peptides targeted to acetylcholine receptors and voltage sensitive calcium channels) they may also contain a number of distinct specialized peptides (e.g. the activity of conantokin-G of C. geographus which targets NMDA receptors has not been observed in C. striatus venom) (1). We describe a number of strategies (including derivatizations) that allow the identification of as yet uncharacterized toxins in these venoms. The extent of the challenge lies in the fact that most Conus venoms are complex mixtures containing over 100 peptides. With the advent of very sensitive ionization techniques such as matrix assisted laser desorption (MALDI) coupled with time-of-flight (TOF) mass analysis, measurement of the intact mass of peptides at sub-pmol levels has become a reality (2) of which we have taken advantage for the systematic screening of HPLC fractions. Partial sequence information can be obtained by carrying out enzymatic hydrolysis with exoproteinases (e.g. carboxypeptidases and aminopeptidases) (3, 4). More recently, MALDI has been used to measure metastable decomposition occurring in the first field free region of a reflectron TOF instrument (referred to as post source decay (PSD)) with only marginally more sample (5-7). While significantly less material is required for MALDI than for either UV detection, chemical sequencing or amino acid analysis, the nature of the information derived from MALDI spectra is also different. Clearly, obtaining unambiguous composition or sequence information is not a simple task. This is due to the fact that (i) the mass accuracy of MALDI-TOF measurements is generally lower than that of liquid secondary ionization (LSI) with a magnetic sector mass spectrometer, (ii) enzymatic sequencing is affected by the varying rates of cleavages at different amino acid residues and the reduced activity of most enzymes towards particular residues (e.g., tyrosine and proline) and (iii) TECHNIQUES IN PROTEIN CHEMISTRY VI Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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fragmentation information from PSD suffers from the ambiguities of assigning fragment ions as being derived from the N-terminus, C-terminus or "internal sequence". We have implemented scanning methodologies using MALDI-TOF mass spectrometry to partially purified venom from C striatus and C. ermineus. We have carried out specific derivatizations in order to deduce composition and sequence information. Together with an intact mass these measurements are used to determine whether an ionized species observed in the MALDI mass spectrum corresponds with the intact protonated molecule of a previously characterized conotoxin. The information obtained from derivatizations is also important when the ionized species does not correspond with the intact mass of peptides of known sequence. In that case, post source decay of the native and derivatized species may help assign the fragment ions.
II. Materials and Methods MALDI mass spectra were measured with a Bruker Reflex time-of-flight mass spectrometer fitted with a gridless reflectron energy analyzer and a nitrogen laser. Accelerating and reflectron voltage of +31 kV and +30 kV were employed unless otherwise specified. Typically, the amount of sample necessary for MALDI analysis was 100 fold less than that used for LSI analysis. All MALDI samples were prepared in six or more different sample preparation formats including three different UV absorbing matrices (a-cyano-4hydroxycinnamic acid, sinapinic acid and 2,5-dihydroxybenzoic acid) and two methods of preparation. In the first method peptide solution was pre-mixed with a solution of each matrix prior to application onto the probe tip (see refs. (8-10) for preparation of matrix solutions). In the second method a solution of the matrix in acetone was dried on the probe tip and then a separate sample of peptide was applied and left to dry onto the matrix (11) As noted previously, the second preparation was found to give more reliable analyses of samples which required a rinse with H2O (12, 13). No ions were observed with any other sample preparation which were not observed using a combination of the second procedure, a-cyano-4-hydroxycinnamic acid as the matrix, and rinsing of the samples. All tabulated data are for samples prepared in this manner. LSIMS spectra were measured with a JEOL IMS HXllO mass spectrometer fitted with a Cs"*" ion gun. An accelerating voltage of +10 kV and Cs"^ ion gun voltage of +30 kV were employed. An electric field scan over a narrow mass range was used to measure segments of the mass spectrum corresponding with appropriate regions of the MALDI mass spectrum. The samples, prior to the dilution used for MALDI analysis, (1 |il; 100 pmol; 0.1 % TFA solution) were added directly to a 1:1 mixture of m-nitrobenzyl alcohol and glycerol. The mass accuracy of LSIMS for measurement of the unresolved isotopic cluster is typically within 100 p.p.m. of the calculated average [M+H]+ mass. The accuracy of the observed masses listed in Tables I and II for the MALDI mass spectra benefited from the use of a reflectron instrument which generally reduced the deviation observed between spectra measured under different experimental conditions (e.g. different matrix or laser power) from ±1000 p.p.m. to ±300 p.p.m. Reflecting this level of mass accuracy we present the MALDI measurements with only 4 significant figures, compared with 5 significant figures for the LSIMS measurements. For calculation of possible amino acid substitutions, the 20 most common amino acids were used together with y-carboxyglutamate (Gla) and hydroxyproline (Hyp) which are commonly found in conotoxins (14).
MS Scanning Methodologies
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Peptide Modification : lodination was carried out on a stainless steel probe target by adding 0.1 % aq. I2 (1 |il) to the dried peptide (ca. 1 pmol). The reaction was stopped after 1 minute by addition of ascorbic acid and the MALDI matrix, a-cyano cinnamic acid in excess. Esterification with ethanol was carried out using the method of Hunt et al. (15), where an acetylchloride and ethanol solution (1:6, v:v) was added (5 |il) to the peptide dried in a microcentrifuge tube (ca. 1 pmol). After incubation for 15 minutes at room temperature a 2 mM p-mercaptoethanol (in ethanol) solution, was added (1 [i\) and the mixture was dried. The matrix, a-cyano-4-hydroxycinnamic acid (2 |j.l), was added to the micro tube and after 5 minutes 1 |il of this matrix was removed and applied to a target.
IIL Results and Discussion Figure 1 shows the HPLC profile of semi-purified venom from C. striatus (fractions labeled 3, 5-18,20 and 22). A summary of the observed masses in the MALDI and LSI mass spectra for each of these fractions is given in Table I. A data base of known conotoxins was searched for correspondence (±3 Da) with the observed masses: "matches" are scored irrespective of whether the peptide in question was originally isolated from the particular venom. Figure 2 shows the HPLC profile of semi-purified venom from C. ermineus (fractions labeled 4a, 4b, 5-11 and 14). A summary of the masses of the major species observed in the LSIMS and MALDI mass spectra for each of these fractions is given in Table II. Our finding that the preparation of a-cyano-4-hydroxycinnamic acid in acetone and subsequent rinsing (see Materials & Methods) produced all ion species which were observed with a variety of other MALDI procedures is important for further scanning of the Conus venoms for novel conotoxins. Generally, we observe at least one major species in the MALDI mass spectra corresponding to each HPLC component. The increased mass accuracy available when the instrument was operated in the reflectron mode was important for the analysis carried out. For example, fractions 4b and 7 or fractions 5 and 8 from C. ermineus appeared to be the same species when measured with the instrument operated in the linear mode. Only in the reflectron mode were we able to reliably distinguish the masses of each species. The high sensitivity of MALDI-TOF is particularly important for the analysis of native peptides such as conotoxins where often the venom of many milkings must be collected to obtain sufficient material for sequence analysis. The increased sensitivity of MALDI over LSIMS is illustrated in the analysis of fraction 5 from C. striatus venom (see Table I). Despite the two orders of magnitude difference in the amount of material consumed in the LSI experiment we did not discern any intact species in fraction 5, whereas the MALDI measurement yielded useful information. However, the comparisons in Tables I and II reveal that some components may be detected by LSIMS but not observed in the MALDI mass spectrum (measured with any of the matrices or sample preparation methods). The contrary is most likely more prevalent, i.e. that a large number of the species detected by MALDI with one or more of the matrices are difficult species to ionize with LSIMS.
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Table I. Observed masses in the LSIMS and MALDI mass spectra of fractions of C striatus [RTI 1 match CaJc! 1 MALDI LSIMS mass (m/z) Obs. mass (m/z) Obs. mass^ ISVIB 2740.2 12739t 12739.5 2544 5 NO 2579 NO 6 SVIA 2494.9 2494t 2494.9 2521 7 2521.4 8 SII 1792.0 1240 1241.5 1791.2 NO 1794t 1813 10 SI 1814 2786 1354.4 1791.3 NO 1354.6 1354t NO 2782.7 11 sm 2497.5 1456.7 1457t 2498 NO 12 2500 4099 9218 1396.4 NO 4098.6 NA 1397 13 NA 2498 3886 3940 NA NA NA 1367 14 NO 4898 4952 4968 NO 4947.0 4965.9 4882 15 4082.0 4098.5 NA 4084 4100 4792 NA 3175 16 2498 3924 4758 2176.6 2497.4 NO 17 4743 5025 NA 3938 18 3778.1 3782 NO 3713 20 3400.0 3416.0 3432.4 3418 NO NO 3348 NO 122 1 INA 1^ calculated average [M+H]+ mass. NO indicates corresponding ion in ]^ALDI or LSIMS spec trum not observecI. NAindi :ates noi analyze5d. t ind icates the obs. species which matched.
ffn
It is apparent from Table I that masses of several previously known peptides from C. striatus correspond to those found for major UV absorbance peaks. Similarly, Table II shows that the C. ermineus venom contained a peptide in fraction 7 that matched the mass of conotoxin GVIB from C. geographus — in this case, the peptide has since been analyzed and found to be completely unrelated to GVIB, whereas the putative match to SI in fraction 9 has been confirmed with chemical sequencing and mass spectrometry.
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Figure 1. UV trace of HPLC of C. striatus venom.
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Table II. Observed masses in the LSIMS and MALDI mass spectra of C. ermineus fractions 1 LSIMS Fr. 1 match Calc. 1 MALDI (m/z) mass Obs. mass (m/z)J L Obs. mass^J 3451.1 NO 2513 3105 NO 1 12496.1 NO [2497'" ^ 3100.7 3101 4b 5 3099.6 3085 3085 NA 2495.4 NA NA 2111 NO 2094 6 7 GVIB 3095.4 3098t 3096.2 3082.3 3082 8 1792 1944 3068 1354.0 1790.8 1943.6 3065.6 SI 9 1354.6 1353t NA 2094 3047 3558 NO 2094.2 NA 1803 10 2766 2781 1397.2 2765.6 2781.4 1398 11 NA 3512 3528 12369.9 NA [2370 1 14 1 ^ calculated average [M+H]+ mass. NO indicates corresponding ion in MALDI or LSIMS spectrum not observed. NA indicates not analyzed, t indicates the obs. species which matched.
From these results it is clear that useful information can be obtained from MALDI, but that it cannot be used directly to establish the identity of peptides — our ultimate aim is to obtain sequence information from these fractions. Towards that goal we are currendy developing protocols that allow reduction of cysteine residues, alkylation and MALDI measurement without the need for further purification (19). In the simplest version, measurement of the peptide before and after modification reveals the number of disulfide bridges present in the peptide. With the linear alkylated peptide, we can more easily interpret the metastable decomposition occurring in the first field free region of a time-of-flight instrument to measure the fragment ions. This protocol is shown in Figure 3 for reduced and S-carboxamidomethylated (Cam) conotoxin GIA(H-Glu-Cam-Cam-Asn-Pro-Ala-Cam-Gly-Arg-His-Tyr-Ser-Cam-Gly-LysNH2) (16, 17). The spectrum shown in Figure 3 was obtained from a derivatization of 10 pmol of peptide in which 1 pmol of peptide was applied to the target. The PSD spectrum is a composite of scans measured at reflectron voltages between 1.25 and 29.9 kV: the total ion current and therefore the baseline noise varies between individual scans. The 'b' and 'y+2' type fragment ions (18) are the most prolific series observed for this peptide and are therefore identified in Figure 3. However, significantly more sequence information is present in this spectrum (19).
time (min)
Figure 2. UV trace of HPLC of C. ermineus venom
100
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At this point in time it is impractical to sequence every peptide, given the complexity of the venoms. A strategy that directs the sequencing effort to selected peptides can be based on the rapidly growing number of conotoxin sequences which have previously been determined. In 1991, there were over 70 conotoxin peptides characterized from over 10 species (1); this number is now above 200, and growing rapidly with the acquisition of sequences from DNA cloning (20). As described above, we used a database of conotoxin sequences to assign fractions 3, 6, 8 and 11 of C. striatus venom as possibly containing peptides corresponding to SVIB, SVIA, SI and SIB respectively. More accurate mass measurement with LSIMS confirmed that the intact mass was consistent with this assignment in 3 of these cases (no signal was observed with LSIMS in one case). This type of data-base scanning is also being employed in reverse, to search among venom fractions for candidate peptides to match predicted translation products corresponding to cloned cDNAs. The Conus venoms often contain several minor sequence variants of toxins, arising from genetic polymorphisms, multi-gene families, and variation in post-translational processing. When the mass difference between two closely related peptides (in terms of HPLC retention times and mass) corresponds to a single amino acid substitution, a simple experiment may suffice to choose among alternatives. Consider for example fractions 5 and 7 from C. striatus venom, satellites of the major fraction tentatively identified as conotoxin SVIA (Fig 1 and Table I). The mass difference of 50±1 Da between peptides in fractions 5 and 6 could be explained by any of the following changes (i) Tyr to either Hyp, Leu or He (50 Da); (ii) His to Ser (50 Da) (iii) Phe to Pro (50 Da) or (iv) Trp to His (49 Da). Although options (ii) to (iv) are formally possible, SVIA does not contain His, Phe, or Trp, so option (i) would be favored. In order to test this directly, we iodinated small samples of approx. 2 pmol each of fractions 5 and 6. After treatment with I2, fraction 6 was shifted towards higher mass by 126 Da This shift was consistent with the presence of a tyrosine residue in this peptide (we have determined that iodination under these conditions modifies tyrosine but not histidine residues (21)). 73+2 yi3+2
' — I — ^ — I — ^ — I — ^ — r -
400
600
800
1000
1200
1400
m/z
Figure 3. The PSD spectrum (200-1600 Da) of alkylated Conotoxin GIA.
MS Scanning Methodologies
37
In contrast, fraction 5 was not modified by this protocol, indicating that the mass difference of 50 Da could be attributed to the tyrosine residue in the peptide in fraction 6, being replaced by either hydroxyproline, leucine or isoleucine in the peptide in fraction 5. Similarly, assuming that SVIA is the major component in fraction 6, the additional 27±1 Da of the peptide in fraction 7 could be attributed to change of Ser to (Leu, He or Hyp), or of Lys to Arg. Esterification of fractions 6 and 7 verified that neither component contain acidic groups, which was consistent with C-terminal amidation of SVIA. These derivatizations are highly selective, and may thus allow PSD measurements to be carried out on peptides after modification. Such a protocol would significantly enhance our ability to derive sequence information from PSD spectra, because the mass shifts observed in fragments help locate the particular residue within the peptide, and also confirm assignments of fragments as arising from N- or C-terminal regions. In addition to derivatizations that may modify the C- and N-termini and the derivatization of tyrosine residues, we have carried out oxidation of methionine residues with sufficient specificity to enable measurement of PSD spectra.
IV. Conclusion We have gained an appreciation for the ionization bias observed between MALDI and LSIMS. The utilization of PSD to identify known peptides and provide sequence information has been investigated for conotoxins. This approach to obtaining sequence information on novel peptides is attractive because of the low amount of material required. A number of mass spectrometric based derivatizations have been used to scan fractions of venoms in order to characterize peptides of interest. For closely related components (based on HPLC retention time and mass), the small scale derivatization schemes can be used to test hypotheses about peptides with otherwise novel masses (i.e. which may be homologs). The mass accuracy of the TOP technique, with a gridless reflector, was important for identifying and calling these substitutions.
Acknowledgments We would like to thank Drs. B. Olivera and UCruz, University of Utah for stimulating discussions. This work was supported by the National Institute of Health (K20MH00929, lSlORR-8425, HD-13527, DK-26741, CA-54418, HL41910, GM-48677) and supported in part by the Foundation for Medical Research, Inc. (AGC and WHF).
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