Topoisomer Differentiation of Molecular Knots by FTICR MS: Lessons from Class II Lasso Peptides

Lasso peptides constitute a class of bioactive peptides sharing a knotted structure where the C-terminal tail of the peptide is threaded through and trapped within an N-terminalmacrolactamring. The structural characterization of lasso structures and differentiation from their unthreaded topoisomers is not trivial and generally requires the use of complementary biochemical and spectroscopic methods. Here we investigated two antimicrobial peptides belonging to the class II lasso peptide family and their corresponding unthreaded topoisomers: microcin J25 (MccJ25), which is known to yield two-peptide product ions specific of the lasso structure under collisioninduced dissociation (CID), and capistruin, for which CID does not permit to unambiguously assign the lasso structure. The two pairs of topoisomers were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) upon CID, infrared multiple photon dissociation (IRMPD), and electron capture dissociation (ECD). CID and ECDspectra clearly permitted to differentiate MccJ25 from its non-lasso topoisomer MccJ25-Icm, while for capistruin, only ECD was informative and showed different extent of hydrogen migration (formation of c\bullet/z from c/z\bullet) for the threaded and unthreaded topoisomers. The ECD spectra of the triply-charged MccJ25 and MccJ25-lcm showed a series of radical b-type product ions {\eth}b0In{\TH}. We proposed that these ions are specific of cyclic-branched peptides and result from a dual c/z\bullet and y/b dissociation, in the ring and in the tail, respectively. This work shows the potentiality of ECD for structural characterization of peptide topoisomers, as well as the effect of conformation on hydrogen migration subsequent to electron capture

Traveling-wave ion mobility mass spectrometry of protein complexes: accurate calibrated collision cross-sections of human insulin oligomers.

RATIONALE: The collision cross-section (Ω) of a protein or protein complex ion can be measured using traveling-wave (T-wave) ion mobility (IM) mass spectrometry (MS) via calibration with compounds of known Ω. The T-wave Ω-values depend strongly on instrument parameters and calibrant selection. Optimization of instrument parameters and calibration standards are crucial for obtaining accurate T-wave Ω-values. METHODS: Human insulin and the fast-acting insulin aspart under native-like conditions (ammonium acetate, physiological pH) were analyzed on Waters SYNAPT G1 and G2 HDMS instruments. The calibrated T-wave Ω-values of insulin monomer, dimer, and hexamer ions were measured using many different combinations of denatured and native-like calibrants (masses between 2.85 and 256 kDa) and T-wave conditions. Drift-tube Ω-values were obtained on a modified SYNAPT G1. RESULTS: Insulin T-wave Ω-values were measured at 26 combinations of T-wave velocity and amplitude. Optimal sets of calibrants were identified that yield Ω-values with minimal dependence on T-wave conditions and calibration plots with high R(2)-values. The T-wave Ω-values determined under conditions satisfying these criteria had absolute errors <2%. Structural differences between human insulin and fast-acting insulin aspart were probed with IM-MS. Insulin aspart monomers have increased flexibility, while hexamers are more compact than human insulin. CONCLUSIONS: Accurate T-wave Ω-values that are indistinguishable from drift-tube values are obtained when using (1) native-like calibrants with masses that closely bracket that of the analyte, (2) T-wave velocities that maximize the R(2) of the calibration plot for those calibrants, and (3) at least three replicates at T-wave velocities that yield calibration plots with high R(2)

Structural heterogeneity of doubly-charged peptide b-Ions

Performing collisionally activated dissociation (CAD) and electron capture dissociation (ECD) in tandem has shown great promise in providing comprehensive sequence information that was otherwise unobtainable by using either fragmentation method alone or in duet. However, the general applicability of this MS3 approach in peptide sequencing may be undermined by the formation of non-direct sequence ions, as sometimes observed under CAD, particularly when multiple stages of CAD are involved. In this study, varied-sized doubly-charged b-ions from three tachykinin peptides were investigated by ECD. Sequence scrambling was observed in ECD of all b-ions from neurokinin A (HKTDSFVGLM-NH2), suggesting the presence of N- and C-termini linked macro-cyclic conformers. On the contrary, none of the b-ions from eledoisin (pEPSKDAFIGLM-NH2) produced non-direct sequence ions under ECD, as it does not contain a free N-terminal amino group. ECD of several b-ions from Substance P (RPKPQQFFGLM-NH2) showed series of cm-Lys fragment ions which suggested that the macro-cyclic structure may also be formed by connecting the C-terminal carbonyl group and the ε-amino group of the lysine side chain. Theoretical investigation of selected Substance P b-ions revealed several low energy conformers, including both linear oxazolones and macro-ring structures, in corroboration with the experimental observation. This study showed that a b-ion may exist as a mixture of several forms, with their propensities influenced by its N-terminus, length, and certain side-chain groups. Further, the presence of several macro-cyclic structures may result in erroneous sequence assignment when the combined CAD and ECD methods are used in peptide sequencing