Ion MobilityâMass Spectrometry of Lasso Peptides: Signature of a

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Ion mobility - mass spectrometry of lasso peptides: signature of a rotaxane topology Kevin Jeanne Dit Fouque, Carlos Afonso, Séverine Zirah, Julian David Hegemann, Marcel Zimmermann, Mohamed A. Marahiel, Sylvie Rebuffat, and Hélène Lavanant Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac503772n • Publication Date (Web): 13 Dec 2014 Downloaded from http://pubs.acs.org on December 31, 2014

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Analytical Chemistry

Kevin Jeanne Dit Fouque,† Carlos Afonso,†* Séverine Zirah,‡ Julian D. Hegemann,§ Marcel Zimmermann,§ Mohamed A. Marahiel,§ Sylvie Rebuffat,‡ Hélène Lavanant † †

Normandie Univ, COBRA, UMR 6014 and FR 3038; Université de Rouen; INSA Rouen; CNRS, IRCOF, 1 Rue Tesnière, 76821 Mont-Saint-Aignan Cedex, France ‡ Muséum national d'Histoire naturelle, Sorbonne Universités, Centre national de la Recherche scientifique, Laboratoire Molécules de Communication et Adaptation des Microorganismes, UMR 7245 CNRS-MNHN, CP 54, 57 rue Cuvier, 75005 Paris, France. § Philipps-Universität Marburg, Fachbereich Chemie-Biochemie, Hans-Meerwein-Strasse 4 and LOEWE-Center for Synthetic Microbiology, 35032 Marburg, Germany.

Supporting Information Placeholder

ACS Paragon Plus Environment


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ABSTRACT: Ion mobility mass spectrometry data were collected on a set of five class II lasso peptides and their

branched-cyclic topoisomers prepared in denaturing solvent conditions with and without sulfolane as a supercharging agent. Sulfolane was shown not to affect ion mobility results and to allow the formation of highly charged multiply protonated molecules. Drift time values of low charged multiply protonated molecules were found to be similar for the two peptide topologies, indicating the branched-cyclic peptide to be folded in the gas phase into a conformation as compact as the lasso peptide. Conversely, high charge states enabled a discrimination between lasso and branched-cyclic topoisomers, as the former remained compact in the gas phase while the branched-cyclic topoisomer unfolded. Comparison of the ion mobility mass spectrometry data of the lasso and branched-cyclic peptides for all charge states including the higher charge states obtained with sulfolane yielded three trends that allowed differentiation of the lasso form from the branched-cyclic topology: low intensity of highly charged protonated molecules, even with the supercharging agent, low change in collision cross sections with increasing charge state of all multiply protonated molecules, and narrow ion mobility peak widths associated with the co-existence of fewer conformations and possible conformational changes.

Lasso peptides are bioactive peptides produced by bacteria that present a mechanically interlocked structure where the C-terminal tail of the peptide is threaded through and trapped within an N-terminal macrocycle.1-2 Strong sterical constraints which come from bulky side chains and/or disulfide bridges stabilize the lasso structure. Since the discovery of this class of peptides, more than 30 lasso peptides have been characterized and their discovery through genome mining is an active area of research.3-7 Lasso peptides are classified depending on the presence (class I and III) or absence (class II) of disulfide bridges. They exhibit different biological activities (enzyme inhibition, receptor antagonism, antibacterial and/or anti-HIV activities),1-2 and the lasso topology is a prerequisite for the activities reported. Thus, the extraordinary rotaxane topology of lasso peptides, together with their panel of biological activities, make them a very attractive scaffold for drug design.8-9 This compact and interlocked topology provides the peptides with outstanding stability towards carboxypeptidase digestion, temperature and denaturing conditions. However, unthreading of the C-terminal tail has been reported for certain natural lasso peptides, as well as for several truncated or substituted variants.4, 10-11 Therefore, discovering new lasso peptides and using these peptides in drug design require to unambiguously characterize the lasso topology and differentiate the lasso from the unthreaded topoisomers. Liquid chromatography is regularly used as a means to differentiate the two topoisomers especially when following heat denaturation or the effect of digestion treatments.8, 12 This method involves potentially numerous chromatographic runs and assignment of the chromatographic shifts can be ambiguous. The structural characterization of lasso structures and the differentiation of the mechanically interlocked structure from the unthreaded topoisomer (named branched-cyclic) is not a simple task. NMR13 and X-ray diffraction14-15 are methods of choice for the comprehensive characterization of lasso peptides, as illustrated by the three-dimensional structures reported. However, these techniques require relatively large amounts of samples and cannot be applied to mixtures. Tandem mass spectrometry using different

modes of activation such as collision induced dissociation (CID) and electron capture dissociation (ECD) have also been used and showed several different characteristics of fragmentation for the mechanically interlocked structures and the corresponding unthreaded branched-cyclic peptide.4, 16-19 Certain lasso peptides, such as microcin J25 yield specific product ions where the N-terminal macrocycle and part of the C-terminal tail remain interlocked. In addition, we demonstrated that time-resolved ECD experiments can be used to determine the dissociation rate of selected precursor ions and thus characterize a pattern of dissociation rates specific to lasso peptides. The dissociation rates were observed to be slow when the cleavages occur close to the ring/tail connection. This approach is time consuming and involves the use of a FT-ICR instrument, which is not widely available.19 Since the pioneer work of Clemmer and Jarrold, numerous reports have shown the potential of ion mobility coupled to mass spectrometry (IM-MS) for the characterization of biomolecules.20-23 This coupling affords an additional dimension of separation, which is related to the gasphase conformation of the ions. In the ion mobility cell, ions drift in a buffer gas under the influence of an electric field, leading to a separation in the millisecond time scale depending on the ion-neutral collision cross section.24 Since the introduction of a commercial IM-MS instrument in 2006, interest in this technique has increased even further.25 This methodology therefore comes out as an obvious choice for the differentiation of lasso peptides from the branched-cyclic topoisomers, as they are expected to produce different conformations. As the measurement occur in the gas phase, lasso peptides can be used as a test to probe the gas phase conformations compared to the conformations adopted in solution. In this work, five different class II lasso peptides (astexin1(19),11 microcin J25,26-29 capistruin,3 caulosegnin I30 and syanodin I4) have been investigated (Figure 1). A detailed investigation of the evolution of the collision cross section as a function of the charge state of multiply protonated ions was carried out confirming the potential of ion mobility-mass spectrometry for the characterization of topologically constrained peptides.


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Figure 1. Sequences and three-dimensional structures of the lasso peptides used in this study: astexin-1(19),11 capistruin,3 caulosegnin I,30 microcin J2526 and syanodin I.4 The macrolactam ring and C-terminal tail are shown in green and blue, respectively. The bulky amino acids acting as plugs of the rotaxane topology are shown in magenta.

The 3D structures of the five class II lasso peptides astexin-1(19),11 microcin J25,26-29 capistruin,3 caulosegnin I30 and syanodin I4 are represented on Figure 1. The first four are represented as determined by NMR, and the structure of syanodin I is putative based on tandem mass spectrometry results and carboxypeptidase Y assays. Microcin J25 was produced from a culture of Escherichia coli MC4100 harboring the plasmid pTUC202,31 cultivated for 16 h in M63 medium supplemented with 1 mg/mL vitamin B1, 0.02% MgSO4, 0.02% glucose and 1 g/L casamino acids. Capistruin was produced from a culture of the naturally producing strain Burkholderia thailandensis E264 incubated for 24 h at 42 °C in M20 medium containing gentamycin (8 μg/mL), as described previously.3 Astexin-1(19), caulosegnin I and syanodin I were heterologously produced in E. coli BL21 (DE3) transformed with the plasmid pET41a expression vector carrying the T7 promoter, cultivated for 3 days in M9 minimal, as already reported.4, 11, 30 Astexin-1(19) is the 19-amino acid truncated form of astexin-1, which 3D structure has been characterized by NMR. Microcin J25 and capistruin were extracted from the culture supernatants by solid phase extraction on a C8 column,3 while astexin-1(19), caulosegnin I and syanodin I were extracted from the culture pellet with methanol.2b, 9, 11 The peptides were then purified by semi-preparative reversedphase HPLC on a C18 column. The synthetic branchedcyclic peptides corresponding to the sequences of microcin J25 and capistruin, containing the macrolactam ring but without threading of the C-terminal tail, were obtained from Genepep (St Jean de Védas, France). For astexin-1(19), caulosegnin I and syanodin I, the topoisomeric variants were obtained by heating the lasso peptides

at 95°C for 3 hours and were subsequently purified by reversed-phase HPLC. Sulfolane was purchased from Sigma-Aldrich (Lyon, France) and LC-MS grade acetonitrile from VWR (Fontenay-sous-bois, France). Deionized water (18 MΩ) was obtained from a Milli-Q apparatus (Millipore, Bedford, MA, USA). 10 µM solutions were prepared in water/acetonitrile 50/50 (v/v) with 4 % formic acid to ensure complete protonation. Sulfolane (99%, Sigma-Aldrich, Saint Quentin-Fallavier, France) was added up to 0.5% volume in some cases. The experiments were carried out in a hybrid quadrupole ion-mobility time of flight mass spectrometer (Waters, Synapt G2) equipped with an ESI source and operated in the positive ion mode. The details of the instrument were described elsewhere.32 The experimental parameters are listed in detail in the supplementary information (Table S1). Collision cross sections (CCS) were estimated after using the calibration procedure described by Smith et al.33 The reference compounds used were doubly and triply protonated polyalanine peptides for which collision cross sections have been determined by Bush et al. 34 using uniform field drift tube ion mobility. The correlation used is available in the supplementary information (Figure S1).

In this study, we investigated a set of class II lasso peptides together with their unthreaded topoisomers. When the compounds were dissolved in water/acetonitrile 50/50 with 4% formic acid, [M+nH]n+ multiply protonated molecules with 2+ and 3+ charge states could be observed. When the supercharging agent35 sulfolane36 was added, an additional [M+4H]4+ ion was observed, while the intensity of the doubly protonated molecule decreased (Table S2). Ion mobility experiments were carried out for the



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lasso peptide and the branched-cyclic peptide separately and in 1/1 mixtures, both with and without sulfolane.

Figure 2. Correlation of drift times measured from solutions with and without sulfolane. Blue and red symbols correspond to the lasso and the branched-cyclic topologies, respectively; , , , ,  stand for astexin-1(19), capistruin, caulosegnin I, microcin J25 and syanodin I respectively. [M+3H]3+ ions of syanodin I were not observed without sulfolane. Error bars of double standard deviations (n=5) were too small for representation except for triply charged microcin J25.

Comparison of drift times with and without sulfolane were found extremely well correlated (Figure 2) with a slope of 0.992 to 1.002 (for [M+3H]3+ and [M+2H]2+ respectively), showing that the supercharging agent did not have any significant effect on the gas phase conformations of the lower charged multiply protonated ions of the peptides investigated in this study. Figure 3 shows ion mobility spectra of multiply protonated astexin-1(19) and its branched-cyclic topoisomer analyzed in a 1/1 mixture (Figures 3a, b, c) and separately (Figures 3d, e, f). Astexin-1(19) was chosen here as a typical representative of the results obtained for the five lasso peptides studied, as they were very similar (Table S2). The drift times of doubly and triply protonated molecules of astexin-1(19) were very similar for the lasso and branched-cyclic toposiomers (Figures 3d and 3e) and were consequently not separated when analyzed as a mixture (Figures 3a and 3b). While a compact structure was anticipated for the lasso peptide, a more unfolded conformation was expected for the branched-cyclic toposoimer because a denaturing solvent was used. However, our observations showed the lower charge state of multiply protonated ions exhibited compact conformations regardless of the topology. The non-lasso peptide displayed a gas phase compact conformation rather than an unstructured solution phase conformation, suggesting a folding of the tail onto the ring in the gas phase.

Figure 3. Extracted ion mobility spectra of multiply protonated ions of astexin-1(19) and its synthetic branched-cyclic topoisomer: (a) and (d) [M +2H]2+ m/z 1047.83, (b) and (e) [M +3H]3+ m/z 698.91 and (c) and (f) [M +4H]4+ m/z 524.45. The top figures (a), (b) and (c) were obtained from a 1/1 mixture. The bottom figures (d), (e) and (f) are superimposed extracted ion mobility spectra of the lasso (in blue) and branched-cyclic (in red) topoisomers analyzed separately.

Dugourd and co-workers have shown previously that charge solvation plays an important role in the secondary structure of protonated peptides in the gas phase.37 The role of charge state in protein conformation was the topic of several recent works.38-40 It was suggested that the initial solution phase conditions play a minor role in the final measured collision cross sections (CCS), whereas the charge state of the protein has a major role. As a result of gas phase folding, the ion mobility separation and gas phase differentiation of the lasso and branched-cyclic topoisomers was not possible for lower charge state multiply protonated ions (Figures 3a and 3b). Conversely, for the higher charge state [M+4H]4+ ions, the drift time were significantly different for the lasso and the non-lasso peptides. This made the ion mobility separation of the topoisomers possible with the expected shorter drift time value (and more compact topology) for the lasso peptide (Figure 3f). This was true for all higher charge states as shown in Figure 4, which depicts a twodimensional map of drift time and m/z values of all the peptides. In two cases (microcin J25 and capistruin), multiple values are plotted for the branched-cyclic peptides because multiple peaks were observed in the ion mobility spectra (Figures S4 and S5 and Table S2). In Figure 4, low drift time values correspond to higher charged ions and high drift times to lower charge states. The drift time values of the low charged multiply protonated molecules were found to be similar for the two peptide topologies. Conversely, high charge states enabled to discriminate between lasso and branched-cyclic topoisomers (Figure 4). The charge states of the multiply protonated peptides permitting discrimination of the two topologies were three for caulosegnin I, microcin J25 and syanodin I, and four for astexin-1(19) and capistruin. This difference of behavior most likely arises from the size of the peptides and/or the number and localization of acid


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and basic residues. With the same number of charges, a smaller peptide will present a higher change density and will undergo higher intramolecular Coulomb repulsion.

Figure 5. Relative intensities of the different charge states in the presence of sulfolane (the sum of all charge states intensities is set to 100%, blue bars correspond to the lasso peptides and red bars to the branched-cyclic peptides).

Figure 4. Two-dimensional map of drift times and m/z values of the five pairs of topoisomeric peptides. The values for n is 1 for syanodin I (which appeared only doubly and triply protonated), and 2 for all other peptides (three different charge states observed). Blue and red symbols correspond to the lasso and the branched-cyclic topologies, respectively; , , , ,  stand for astexin-1(19), capistruin, caulosegnin I, microcin J25 and syanodin I respectively.

Figure 5 shows the relative intensities of the different charge states when supercharging sulfolane was added to the solution. We observed that the relative intensities of the higher charge states were larger for the branched-cyclic peptides than for the corresponding lasso peptides, which showed an influence of the conformational flexibility on the charge state of multiply protonated peptides. This was particularly apparent for caulosegnin I and microcin J25 for which the [M+4H]4+ ions only reached 4 and 8% relative intensity for the lasso peptide, while the branched-cyclic topoisomers yielded 70 and 57% relative intensity, respectively. The role of solution conformation on the ion charge state obtained by ESI is well documented. It is generally accepted that the charge state distribution reflects the solution conformation and that the more folded molecules yield lower charge states.41-42 This behavior does not guarantee however that the actual gas phase conformation reflects the solution phase conformation.38 We also observed that in most cases the FWHM of the branched-cyclic topoisomers were larger than those of the corresponding lasso peptides (Table S2). A larger peak width could indicate either the presence of unresolved components arising from multiple conformations or the existence of a conformational change during the ion mobility separation. To examine the first hypothesis, an estimation of the maximum FWHM for a single component depending on its charge and drift time was needed.

Apart from the presence of unresolved components, the main factors that affect the width of arrival time distributions in ion mobility mass spectrometry are known to be43 (a) the time width for ion introduction in the ion mobility cell, (b) the expansion of the ion pack due to normal molecular diffusion and (c) the existence or conversion of several species of different mobilities within the drift region. If only factor (b) is taken into account, a maximum resolution obtainable by drift tube ion mobility experiments44 is: 𝑅=

𝑡𝑑 ∆𝑡𝑤

=√

𝑧𝑒𝑉 16𝑙𝑛2 𝑘𝑇

(1)

In equation (1), td is the drift time, tw is the FWHM, k the Boltzmann constant, T the temperature (K), z the charge number, e the elementary charge and V the drift tube potential difference. In traveling wave ion mobility experiments (TWIM), the electric field varies with time and space in the stacked ring ion guide where the mobility separation takes place. If we hypothesize that V the drift tube potential difference might be replaced by an average value as all ions are on average subjected to similar conditions of electric field, Equation (1) could be extended for TWIM45 and help relate a minimum expected width to drift time and charge number: 𝑀𝑖𝑛(𝐹𝑊𝐻𝑀) = 𝐴

𝑡𝑑 √𝑧

(2)

where A is a constant. Using the observed values of the lasso peptides, for which only one unique compact conformation was expected, the FWHM were plotted as a function of the drift time divided by the square root of the charge number (Figure 6). A correlation with a determination coefficient of 0.90 was found after eliminating three values (doubly protonated microcin J25, astexin-1(19) and triply protonated capistruin) where the presence of multiple components was suspected.



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Figure 6. Correlation between the FWHM and the drift time divided by the square root of the charge number. Only the circled values were used for the correlation. Values far above the correlation line are thought to derive from the presence of unresolved components in the arrival time distributions. Error bars represent double standard deviations of repeated measurements (n=5).

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Using this correlation as a rough estimate of the FWHM dependence on drift time, a peak fitting algorithm was applied to search for hidden peaks in the larger signals of the branched-cyclic peptides (Figures S3, S4 and S5). Peak fitting analyses yielded a putative number of conformations with different centroid drift times and areas, which were converted to CCS and relative intensities for each conformation (Table 2). The number and CCS value of these different conformations is, again, largely putative as the existence of conformational change during the ion mobility experiment may occur which would cause distortion of the Gaussian peak shapes. Furthermore, different conformations with similar CCS may also exist. Nevertheless, a general trend of behavior emerged. The estimated CCS values in Table 2 show that the increase in the number of charges caused an increase in CCS, as previously described for proteins.20, 38, 46-47 The increase in CCS, and consequently the range of CCS was much larger for the branched-cyclic topoisomers than for the mechanically interlocked lasso peptides, for which the CCS remained nearly constant (Figure 7).

Table 2. Estimated number of conformations, collision cross section of five class II lasso peptides and their synthetic branchedcyclic topoisomers obtained from 10-5 mol/L peptide solutions with sulfolane.(a) LASSO Peptides

Astexin-1(19)

Capistruin

Caulosegnin I

Microcin J25

Ion

BRANCHED-CYCLIC

Number of conformations

CCS (Å²) (Ratio (%))

Number of conformations

CCS (Å²) (Ratio (%))

[M+2H]2+

3

365 (29) / 372 (51) / 377 (20)

5

366 (6) / 375 (43) / 380 (25) / 385 (16) / 392 (10)

[M+3H]3+

2

374 (93) / 391 (7)

1

388 (100)

[M+4H]4+

1

418 (100)

1

488 (100)

[M+2H]2+

1

376 (100)

2

377 (29) / 385 (71)

[M+3H]3+

2

373 (74) / 385 (26)

3

378 (26) / 388 (49) / 397 (25)

[M+4H]4+

1

403 (100)

3

455 (32) / 471 (46) / 490 (22)

[M+2H]2+

2

355 (10) / 368 (90)

4

354 (40) / 364 (27) / 373 (21) / 381 (12)

[M+3H]3+

1

359 (100)

6

379 (5) / 394 (10) / 404 (20) / 414 (28) / 423 (21) / 432 (16)

[M+4H]4+

1

374 (100)

3

464 (32) / 472 (41) / 482 (27)

[M+2H]2+

3

373 (63) / 383 (27) / 393 (10)

3

388 (63) / 394 (24) / 404 (13)

[M+3H]3+

1

383 (100)

5

395 (18) / 406 (16) / 417 (17) / 428 (21) / 437 (28)

[M+4H]4+

2

404 (88) / 442 (12)

1

514 (100)

[M+2H]2+

1

289 (100)

1

297 (100)

[M+3H]3+

1

293 (100)

5

318 (14) / 327 (22) / 336 (24) / 345 (21) / 353 (19)

Syanodin I

(a) When several conformations were evidenced, the major species is in bold characters


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For the higher charge state [M+4H]4+, the branched-cyclic topoisomers had the largest CCS, indicating molecular unfolding probably due to the interplay of intramolecular coulombic repulsion and the conformational flexibility of the peptide chain. For most lasso peptides (with the already mentioned exception of doubly protonated microcin J25, doubly protonated astexin-1(19) and triply protonated capistruin), only one dominant compact conformation was presumed. In three other cases (triply protonated astexin-1(19), triply protonated microcin J25 and quadruply protonated microcin J25), the presence of a small shoulder or adjacent peak made the presence of a second minor conformation possible (12% or less in relative intensity). For the higher charge states, low FWHM and CCS values pointed toward the existence of one unique compact topology. Conversely, the observed FWHM of nearly all the branched-cyclic topoisomers were high and presented multiple components of similar relative intensities as revealed by peak fitting algorithm. The number of multiple components was higher for lower charge states. This is expected from the larger number of possible available protonation sites and coexistence of several protomers. Comparing lasso and branched-cyclic topoisomers showed that the mechanically interlocked topology reduced the number of coexisting conformations. For example, the FWHM and ion mobility spectrum profile of doubly protonated astexin-1(19) lasso peptide suggested the possible presence of three conformations, while its branched-cyclic topoisomer hinted at five different conformations for the same charge state. For triply protonated astexin-1(19), the data pointed towards one dominant conformation with very little change in CCS for the lasso peptide, with the increase in charge state. For [M+4H]4+ astexin-1(19) ions, both topoisomers yielded narrow signals pointing toward the existence of one conformation, with an increase in CCS of 12% for the lasso peptide and 28% for the branched-cyclic peptide. A similar behavior was observed for the four other lasso peptides and their corresponding branched-cyclic topoisomers, with variations depending on the length of the sequence, the size of the loop and the C-terminal tail. Capistruin and caulosegnin, which have shorter C-terminal loops with five and seven amino-acid residues above the ring respectively, showed very small increase in CCS (8 and 4% relative increase from [M+3H]3+ to [M+4H]4+), while their branched-cyclic topoisomers yielded wide peaks indicating multiple conformers with higher increase in CCS (21 to 10% relative increase from [M+3H]3+ to [M+4H]4+). Microcin J25, which has a large loop of eleven amino acids, displayed wide peaks in the ion mobility spectra of both lasso and branched-cyclic topoisomers. FWHM values and peak fitting led to a larger number of conformers with larger CCS in the branched-cyclic peptide.

Figure 7. Range of CCS observed for all the multiply protonated ions [M+nH]n+ of the lasso (blue trace) and branchedcyclic (red trace) topoisomers (n = 2-3 for syanodin I and n = 2-4 for the four other peptide pairs).

Finally, syanodin I, which is a much shorter lasso peptide with only 17 amino acids, yielded only doubly and triply protonated molecules with fewer conformations, very small increase in CCS for the lasso peptide, while 10 to 18% relative increase (from [M+2H]2+ to [M+3H]3+) and multiple conformers were detected for the branched-cyclic topoisomer.

The ion mobility mass spectrometry data collected on a set of five class II lasso peptides and their branched-cyclic topoisomers yielded three trends that allowed fast differentiation of the two topologies. The first trend distinguishing lasso peptides was their low change in collision cross section with increasing charge state of multiply protonated molecules, leading to a narrow range of values for their CCS. The second trend was that lasso peptides showed low intensity of highly charged protonated molecules even in the presence of a supercharging agent. Finally, the third trend was that lasso peptides were observed with small peak widths and consequently a low number of conformations with different charge states. More specifically, lasso peptides showed very narrow ion mobility signals for highly protonated molecules. All these observations necessitated the use of denaturing solvent conditions with a supercharging agent to allow the formation of highly protonated molecules. The addition of this supercharging agent was shown not to affect the ion mobility results. Although these three trends were observed with variable proportions in the set of class II lasso peptides tested, they could constitute a fast indication of the presence of a mechanically interlocked topology for uncharacterized lasso peptides.

Supporting Information. Experimental parameters of the TWIM MS measurements, graph and equation of the correlation used for collision cross section calibration with multiply protonated polyalanines,


7


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graph and correlation of the drift times with and without sulfolane, figures and method for the peak fitting algorithm used for hidden peaks search. This material is available free of charge via the Internet at http://pubs.acs.org.

[email protected] The manuscript was written through contributions of all authors, and all authors have given approval to the final version of the manuscript. No competing financial interests have been declared. This work was supported by the European Regional Development Fund (ERDF) N°31708, the Région Haute Normandie (Crunch Network, N°20-13) and the Labex SynOrg (ANR-11-LABX-0029). Financial support for the Marahiel Lab was provided by the DFG (MA 811/25-1)

Special thanks to Salomé Poyer for her assistance with the LC-IM MS analyses and Gaël Coadou for his assistance in 3D representations of the lasso peptides.

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(17) Wilson, K.-A.; Kalkum, M.; Ottesen, J.; Yuzenkova, J.; Chait, B. T.; Landick, R.; Muir, T.; Severinov, K.; Darst, S. A. J. Am. Chem. Soc. 2003, 125, 12475-12483 (18) Zirah, S.; Afonso, C.; Linne, U.; Knappe, T. A.; Marahiel, M. A.; Rebuffat, S.; Tabet, J.-C. J. Am. Soc. Mass Spectrom. 2011, 22, 467-479 (19) Perot-Taillandier, M.; Zirah, S.; Rebuffat, S.; Linne, U.; Marahiel, M. A.; Cole, R. B.; Tabet, J. C.; Afonso, C. Anal. Chem. 2012, 84, 4957-4964 (20) Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 1995, 117, 10141-10142 (21) Bernstein, S. L.; Wyttenbach, T.; Baumketner, A.; Shea, J.-E.; Bitan, G.; Teplow, D. B.; Bowers, M. T. J. Am. Chem. Soc. 2005, 127, 2075-2084 (22) Ruotolo, B. T.; Benesch, J. L. P.; Sandercock, A. M.; Hyung, S.J.; Robinson, C. V. Nat. Protoc. 2008, 3, 1139-1152 (23) Woods, A. S.; Ugarov, M.; Egan, T.; Koomen, J.; Gillig, K. J.; Fuhrer, K.; Gonin, M.; Schultz, J. A. Anal. Chem. 2004, 76, 218795 (24) Kanu, A. B.; Dwivedi, P.; Tam, M.; Matz, L.; Hill, H. H., Jr. J. Mass Spectrom. 2008, 43, 1-22 (25) Giles, K.; Pringle, S. D.; Worthington, K. R.; Little, D.; Wildgoose, J. L.; Bateman, R. H. Rapid Commun. Mass Spectrom. 2004, 18, 2401-2414 (26) Rosengren, K. J.; Clark, R. J.; Daly, N. L.; Goeransson, U.; Jones, A.; Craik, D. J. J. Am. Chem. Soc. 2003, 125, 12464-12474 (27) Salomon, R. A.; Farias, R. N. J. Bacteriol. 1992, 174, 7428-35 (28) Wilson, K. A.; Kalkum, M.; Ottesen, J.; Yuzenkova, J.; Chait, B. T.; Landick, R.; Muir, T.; Severinov, K.; Darst, S. A. J. Am. Chem. Soc. 2003, 125, 12475-83 (29) Bayro, M. J.; Mukhopadhyay, J.; Swapna, G. V.; Huang, J. Y.; Ma, L. C.; Sineva, E.; Dawson, P. E.; Montelione, G. T.; Ebright, R. H. J. Am. Chem. Soc. 2003, 125, 12382-3 (30) Hegemann, J. D.; Zimmermann, M.; Xie, X.; Marahiel, M. A. J. Am. Chem. Soc. 2013, 135, 210-222 (31) Solbiati, J. O.; Ciaccio, M.; Farias, R. N.; Gonzalez-Pastor, J. E.; Moreno, F.; Salomon, R. A. J. Bacteriol. 1999, 181, 2659-62 (32) Giles, K.; Williams, J. P.; Campuzano, I. Rapid Commun. Mass Spectrom. 2011, 25, 1559-1566 (33) Smith, D. P.; Knapman, T. W.; Campuzano, I.; Malham, R. W.; Berryman, J. T.; Radford, S. E.; Ashcroft, A. E. Eur. J. Mass Spectrom. 2009, 15, 113-130 (34) Bush, M. F.; Hall, Z.; Giles, K.; Hoyes, J.; Robinson, C. V.; Ruotolo, B. T. Anal Chem 2010, 82, 9557-65 (35) Sterling, H. J.; Daly, M. P.; Feld, G. K.; Thoren, K. L.; Kintzer, A. F.; Krantz, B. A.; Williams, E. R. J. Am. Soc. Mass Spectrom. 2010, 21, 1762-1774 (36) Lomeli, S. H.; Peng, I. X.; Yin, S.; Loo, R. R.; Loo, J. A. J. Am. Soc. Mass Spectrom. 2010, 21, 127-31 (37) Albrieux, F.; Calvo, F.; Chirot, F.; Vorobyev, A.; Tsybin, Y. O.; Lepere, V.; Antoine, R.; Lemoine, J.; Dugourd, P. J. Phys. Chem. A 2010, 114, 6888-6896 (38) Hall, Z.; Robinson, C. V. J. Am. Soc. Mass Spectrom. 2012, (39) Jurneczko, E.; Kalapothakis, J.; Campuzano, I. D.; Morris, M.; Barran, P. E. Anal Chem 2012, 84, 8524-31 (40) Jurneczko, E.; Barran, P. E. Analyst 2011, 136, 20-8 (41) Loo, J. A.; Edmonds, C. G.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1990, 62, 693-8 (42) Ogorzalek Loo, R. R.; Lakshmanan, R.; Loo, J. A. J. Am. Soc. Mass Spectrom. 2014, 25, 1675-1693 (43) Watts, P.; Wilders, A. Int. J. Mass Spectrom. Ion Proc. 1992, 112, 179-190 (44) Revercomb, H. E.; Mason, E. A. Anal. Chem. 1975, 47, 970-983 (45) Shvartsburg, A. A.; Smith, R. D. Anal Chem 2008, 80, 9689-99 (46) Shelimov, K. B.; Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 1997, 119, 2240-2248 (47) Thalassinos, K.; Slade, S. E.; Jennings, K. R.; Scrivens, J. H.; Giles, K.; Wildgoose, J.; Hoyes, J.; Bateman, R. H.; Bowers, M. T. Int. J. Mass Spectrom. 2004, 236, 55-63


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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Figure 1. Sequences and three-dimensional structures of the lasso peptides used in this study: astexin1(19),11 capistruin,3 caulosegnin I,30 microcin J2526 and syanodin I.4 The macrolactam ring and C-terminal tail are shown in green and blue, respectively. The bulky amino acids acting as plugs of the rotaxane topology are shown in magenta. 64x18mm (300 x 300 DPI)


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Figure 2. Correlation of drift times measured from solu-tions with and without sulfolane. Blue and red symbols correspond to the lasso and the branched-cyclic topologies, respectively; the square, triangle, circle, diamond, star stand for astexin-1(19), capistruin, caulosegnin I, microcin J25 and syanodin I respectively. [M+3H]3+ ions of syanodin I were not observed without sulfolane. Error bars of double standard deviations (n=5) were too small for representation except for triply charged microcin J25. 170x132mm (300 x 300 DPI)



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Figure 3. Extracted ion mobility spectra of multiply protonated ions of astexin-1(19) and its synthetic branched-cyclic topoisomer: (a) and (d) [M +2H]2+ m/z 1047.83, (b) and (e) [M +3H]3+ m/z 698.91 and (c) and (f) [M +4H]4+ m/z 524.45. The top figures (a), (b) and (c) were obtained from a 1/1 mixture. The bottom figures (d), (e) and (f) are superimposed extracted ion mobility spectra of the lasso (in blue) and branched-cyclic (in red) topoisomers analyzed separately. 390x251mm (300 x 300 DPI)


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Figure 4. Two-dimensional map of drift times and m/z values of the five pairs of topoisomeric peptides. The values for n is 1 for syanodin I (which appeared only doubly and triply protonated), and 2 for all other peptides (three different charge states observed). Blue and red symbols correspond to the lasso and the branched-cyclic topologies, respectively; the square, triangle, circle, diamond, star stand for astexin-1(19), capistruin, caulosegnin I, microcin J25 and syanodin I respectively. 247x167mm (300 x 300 DPI)



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Figure 5. Relative intensities of the different charge states in the presence of sulfolane (the sum of all charge states intensities is set to 100%, blue bars correspond to the lasso peptides and red bars to the branchedcyclic peptides). 189x93mm (300 x 300 DPI)


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Figure 6. Correlation between the FWHM and the drift time divided by the square root of the charge number. Only the circled values were used for the correlation. Values far above the correlation line are thought to derive from the presence of unresolved components in the arrival time distributions. Error bars represent double standard devia-tions of repeated measurements (n=5). 153x121mm (300 x 300 DPI)



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Figure 7. Range of CCS observed for all the multiply pro-tonated ions [M+nH]n+ of the lasso (blue trace) and branched-cyclic (red trace) topoisomers (n = 2-3 for syanodin I and n = 2-4 for the four other peptide pairs). 144x94mm (300 x 300 DPI)


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