An investigation of the tyrothricin complex by tandem mass spectrometry

International Journal of Mass Spectrometry and Ion Processes, Elsevier Science Publishers B.V., Amsterdam

143

122 (1992) 143-151

An investigation of the tyrothricin complex by tandem mass spectrometry* M. Barbe@, D.J. Bell”,‘, M.R. Morrisa**, L.W. Tetlera, J.J. Monaghanb, W.E. Mordenb, B.W. Bycroft” and B.N. Greend aDepartment of Chemistry, UMIST, Manchester M60 IQD (UK) bICI C&P Ltd, Runcorn, Cheshire WA7 4QD (UK) ‘Department of Pharmaceutical Science, University of Nottingham, Nottingham NG7 2RD (UK) dVG Biotech, Manchester WA17 5R.Z (UK) (First received 29 June 1992; in final form 21 August 1992)

ABSTRACT Tandem mass spectrometry has been shown to be a powerful technique for determining the structures of biological compounds. This paper details the mass spectrometric methods employed to characterise the structural variations found within a mixture of cyclic decapeptides, tyrothricin, produced by the bacterium Bacillus brevis. Keywords: tandem mass spectrometry; peptides; biological materials; FAB.

DEDICATION

The results obtained during the course of this study formed the basis of a presentation by Professor Michael Barber to the 35th meeting of the American Society for Mass Spectrometry held in Denver in 1987. (This sadly proving to be his last attendance.) Much of the following paper was written before the untimely death of Michael Barber but we feel that it is a fitting tribute to his memory that it now be published as the results clearly demonstrate the power of the techniques that he was instrumental in developing. INTRODUCTION

Tyrothricin is a mixture of cyclic decapeptides and is widely used as a topical antimicrobial Correspondence to: L.W. Tetler, Department of Chemistry, UK. * Dedicated to the memory of Professor Michael Barber.

produced by Bacillus brevis agent. These compounds, UMIST,

Manchester

‘Deceased. ‘Current address: Smith Kline Beecham, Epsom, Surrey KT18 SXQ, UK *Current address: Department of Chemistry, Purdue University, W. Lafayette, 0168-1176/92/$05.00

0

1992 Elsevier Science Publishers

M60 lQD,

IN 47907, USA

B.V. All rights reserved.

M. Barber et al./Int. J. Mass Spectrom. Ion Processes 122 (1992) 143-151

144 ,Asn

-Gin

R2 I R, \ Pro 'Phe

Fig. 1. Structures

Val I Orn -Leu'

of tyrocidins

A, B and C.

together with gramicidin S which is also produced by strains of the same organism, are significant in that they are peptide products representative of a non-ribosomal biosynthetic process. There is increasing evidence that this is characteristic of many other types of microbial peptide antibiotics. The structures of the three major components of the tyrothricin complex i.e. tyrocidins A, B and C (Fig. l), together with gramicidin S, have been established [l] and these account for approximately 75% of the peptidic material of the total complex. The biosynthesis of the tyrocidins and gramicidin S has been the subject of an intensive study by Lipman [2] who proposed a complex multifunctional enzyme system consisting of three synthetases, of increasing molecular weight, which have been isolated and shown to affect the production (in vitro) of the appropriately activated amino-acyl-adenylates. A representation of the proposed biosynthetic sequence is shown in Fig. 2. Most of these general

Tyrocidine Synthetase

Fig. 2. Proposed

biosynthetic

pathway.

M. Barber et aLlInt. J. Mass Spectrom. Ion Processes 122 (1992) 143-151

145

Tyrocidin A

1270

Tyrocidin B

1309

m/z -

Fig. 3. Molecular ion region of FAB-MS spectrum of the tyrothricin complex.

proposals have now been confirmed by detailed genetic and biological investigations [3]. The initiation of the biosynthetic process involves the formation of an enzyme bond with the D-Phe residue followed by sequential addition of amino acid residues starting with Pro. There is evidence that the remaining amino acid residues are variable within defined limits. The overall fidelity of incorporation of all the amino acids does not appear to be stringent and a variety of replacements has been claimed both within the peptides derived from natural systems and by in vitro experiments. Since structure analysis and sequencing of the closely related compounds is not trivial, these proposals have, in the main, been inferred rather than vigorously established. As a prelude to further studies with this important group of antibiotics we have identified fast atom bombardment-tandem mass spectrometry (FAB-MS-MS) as a facile and rapid analytical method of not only resolving the complexes but also establishing the structure and sequence of the individual components. If we examine the molecular ion region of the tyrothricin complex, obtained by FAB-MS, ions which are representative of the three major components are clearly seen (Fig. 3). Additionally, there exist six additional signals of varying intensity which are related to the three major components. Interestingly, assuming equal sensitivities, it would appear that these additional components account for a large proportion of the remaining 25% of material which has not been characterised. Since the tyrothricin complex appears to be an archetypal, non-ribosomal system, we felt that it would be interesting and important to attempt to sequence the six unknown compounds and, therefore, try to observe the overall pattern of point substitutions which must be incorporated into the biosynthetic system proposed. The strategy adopted was to use FAB-MS-MS on the total mixture without

146

M. Barber et al./Int. J. Mass Spectrom. Ion Processes 122 (1992) 143-151

prior separation. We would use the three known compounds, tyrocidins A, B and C, to establish a common fragmentation pattern, giving sequence information, which would then be used to carry out the structural elucidation of the unknowns. Having established the connectivities of the amino acids, the direction of the amide bond linkages is then inferred from the known structures. Without a reference structure and fragmentation pathway, the clockwise or anti-clockwise assignment of the amide bond would be much more difficult although the feasibility of such assignments has been reported by Eckhart et al. [4]. EXPERIMENTAL

The tyrothricin was purchased from Sigma Chemicals Ltd, and used as received. FAB was used as the ionisation method of choice, with xenon as the bombarding particles at 8 keV energy. The matrix which was found most applicable for this work was m-nitrobenzyl alcohol. The technique of MS-MS [5] is simply the application of two mass analysers in tandem, the first of which is used to select the ion of interest, in this case the protonated molecule ions (M +H)+ of the components of the tyrothricin complex. The selected ion is then collisionally activated by allowing it to interact in a confined region with a target gas at relatively high pressures, in our case either helium, argon or xenon. The ionic fragments produced by this collisional activation are then analysed by the second mass spectrometer. Therefore, because of the high precision with which we can isolate an ion of a given mass-to-charge ratio using the first analyser, and hence obtain an ‘uncontaminated mass spectrum’ from it, prior separation of the mixtures, such as the complex we are interested in, is not necessary. (Suppression effects [6] observed in FAB must be taken into consideration when dealing with mixtures.) Two instruments were used in this investigation. First, the VG 70SE4F(VG Analytical, Manchester, UK), in which both elements of the MSMS system are high resolution double focussing mass analysers. This was operated in the high energy collision mode (selected ions having kiloelectronvolt translational energy). Secondly, the VG ZAB-HSQ (VG Analytical, Manchester, UK), in which the first element is a high resolution double focussing mass analyser, and the second a quadrupole mass filter. This tandem arrangement was operated in low energy collision mode (selected ions having a translational energy of between 14 and 150 eV). In the case of the high energy collision work, the target gas used was helium at a pressure such as to achieve 50% attenuation of the selected ion beam. The low energy work used xenon or argon at pressures between 0.5 and 2mbar. For both instruments, MS1 was operated at a resolution of approximately 2000. One of

M. Barber et aLlInt. J. Mass Spectrom. Ion Processes 122 (1992) 143-1.51

Fig. 4. Fragmentation

pathway of protonated

147

tyrocidin A.

the major problems encountered in FAB-MS-MS concerned the low energy collisional activation of the selected precursor ion. It would appear, from our experience, that the conditions for this type of experiment were extremely compound dependent. If we were to obtain “across the board” sensitivity, giving all of the sequence ions, then the collision energy was a most sensitive parameter and had to be readjusted for each of the compounds under investigation even though they were highly related species. In our case, we had a considerable amount of material at our disposal. While we only used approximately 5 pg per loading, for some of the protonated molecule ions of interest several loadings were required to optimise collision conditions. If we had been in the situation of having only a few nanomoles of hard-won material, this could have been a major obstacle to using this very powerful method. However, using the VG 70-SE4F and high energy collisions proved a much easier method of obtaining high sequence sensitivity with no necessity to fine tune the conditions of induced fragmentation. RESULTS AND DISCUSSION

Considering the interpretation of the molecular ion region of the total complex (Fig. 3), the ions observed at 14Da higher than each major component could be a simple replacement of Val by Leu, which is often seen in mixtures of this type, or more unusually, a replacement of Asn or Orn by either Gln or Lys. The replacement of Tyr by Trp could give rise to the ions observed at 23 Da higher than each major component, or alternatively the replacement of Tyr by Phe, i.e. a loss of 16 Da for tyrocidins B and C. It can be seen therefore that there is considerable ambiguity in a trivial and nonspecific interpretation of these results. We therefore carried out the MS-MS experiments on the known structures of tyrocidins A, B and C to delineate a fragmentation pattern which was specific to the sequencing of the compound, and also specific to the clockwise or anti-clockwise arrangements of the amide bonds. The fragmentation pattern which we chose, out of several available to us, is shown in Fig. 4. Here we have protonation of the molecule, ring opening and losses indicative of the sequence of the cyclic peptide. The ions of importance are shown in Fig. 5a for the MS-MS spectrum of tyrocidin A using the VG ZAB-HSQ, and in Fig. 6a using the VG 70-SE4F. Tyrocidin B and C behave in a similar way. Using the same instruments, we then applied the MS-MS procedure to the tyrocidin A + 14, the spectra of which are shown in Figs. 5b and 6b. As can

M. Barber et aLlInt. J. Mass Spectrom. Ion Processes 122 (1992) 143-151

148

I270

I

1000

1200

Mass

(a) 3-

284

2-

392

-Mass (b) Fig. 5. Low energy (eV) CID spectrum of (a) protonated tyrocidin A + 14.

tyrocidin A and (b) protonated

be seen, below ion m/z 896 the spectra are identical to that of tyrocidin A, showing that the sequence up to and including Val is as indicated by the fragmentation scheme shown in Fig. 4. The difference can clearly be seen as the replacement of Orn by a residue of mass 128 Da. As mentioned above, this can be either Gln or Lys. Acetylation of the total mixture, using acetic

M. Barber et al./Int. J. Mass Spectrom. Ion Processes 122 (1992) 143-151

149

488 500 040 788 888 900 1000110012001300 (a)

50

0

Fig. 6. High energy (keV) CID spectrum of (a) protonated tyrocidin A + 14.

tyrocidin A and (b) protonated

anhydride, showed that the 128 Da residue contained a primary amino group indicating that the residue is Lys. Accurate mass measurements were carried out on the A + 14 using the masses of tyrocidins A and B as the internal standards. The results are shown in Table 1, confirming that the replacement residue is, indeed, Lys. Exactly the same results were obtained when the above TABLE 1

Protonated tyrocidin A Protonated tyrocidin B Unknown tyrocidin

1270.6624 1309.6733 1284.6781

Expected (replace Orn by Gln) Expected (replace Om by Lys)

1284.6417 1284.6781

150

M. Barber et al./Int. J. Mass Spectrom. Ion Processes 122 (1992) 143-151

li

50

I 1200 1300

400 500 600 ?00 300 900 1000 1100 1200 1300 (b) Fig. 7. High energy (keV) CID spectrum of (a) protonated tyrocidin A + 23.

tyrocidin A and (b) protonated

sequence of experiments was carried out on the tyrocidin B + 14 and C + 14 ions. Thus, three of the six extra compounds have been characterised. (A further possibility would be the substitution with an N-methylated derivative of Orn, however this was considered to be unlikely.) A similar MS-MS analysis was carried out on the satellite peaks 23Da above tyrocidins A, B and C. The results are shown in Figs. 7a and 7b for tyrocidin A and its satellite. These were obtained using the VG 70SE4F, utilising high energy collisional activation with helium as the target gas. (The spectra depicted have been curtailed in the mass range for the sake of clarity.) From m/z 634 downwards, the two spectra are identical, thus showing that the integrity of the sequence is maintained from Pro up to and including Gln (when using the fragmentation pathway shown in Fig. 4). However, the complex of peaks in the areas of m/z 797 and 896 in tyrocidin A, due to the Tyr/Val unit, has been shifted by 23 mass numbers indicating a replacement of Tyr by Trp. It will be noticed that since these molecules do not contain

M. Barber et al.lInt. J. Mass Spectrom. Ion Processes 122 (1992) 143-151

151

Asn-Gin, R2' I RI \ Pro 'Phe-Leu'

RJ \ val I R4

Fig. 8. Proposed structures for six “unknown”

components

of Tyrothricin.

tyrosine, we have seen fit to rename them tryptocidins. The same point substitution was obtained for the satellites of B and C. A listing of the structures of the six new compounds is shown in Fig. 8. From the listing of structures for the new tyrocidins and tryptocidins shown in Fig. 8, we can now compare these with the biosynthesis as outlined in Fig. 2. We can see immediately that the amino acid substitutions proposed do not follow any particular trend. It is known that the two Phe residues adjacent to the Pro can be substituted by Trp to give rise to the tyrocidins B and C. From the new structures elucidated the Tyr residue can be replaced by Trp giving rise to tryptocidins A, B and C. The following Val is again invariant, but the Orn can be replaced by Lys. However, once again, the final Leu and Phe remain invariant. This must be due to the conformation of the ultimate total ring system and the amino acids which the synthetases can accept. REFERENCES B.W. Bycroft (Ed.), Dictionary of Antibiotics and Related Substances, Chapman and Hall, New York, 1988, pp. 909-910. F. Lipman, Adv. Microbial. Physiol., 21 (1980) 227. W. Schlumbohm, T. Stein, C. Ullrich, J. Vater, M. Krause, M.A. Marahiel, V. Kruft and B. Wittmannliebold, J. Biol. Chem., 266 (1991) 23135. K. Eckhart, H. Schwarz, K.B. Tomer and M.L. Gross, J. Am. Chem. Sot., 107 (1985) 6765. F.W. McLafferty (Ed.), Tandem Mass Spectrometry, Wiley-Interscience, New York, 1983. M.R. Clench, G.V. Garner, D.B. Gordon and M. Barber, Biomed. Mass Spectrom., 12 (1985) 355.