Effective discrimination of gas-phase peptide conformers using TIMS-ECD-ToF MS/MS.

In the present work, four, well-studied, model peptides (e.g., substance P, bradykinin, angiotensin I and AT-Hook 3) were used to correlate structural information provided by ion mobility and ECD/CID fragmentation in a TIMS-q-EMS-ToF MS/MS platform, incorporporating an electromagnetostatic cell (EMS). The structural heterogeneity of the model peptides was observed by (i) multi-component ion mobility profiles (high ion mobility resolving power, R ∼115-145), and (ii) fast online characteristic ECD fragmentation patterns per ion mobility band (∼0.2 min). Particularly, it was demonstrated that all investigated species were probably conformers, involving cis/trans-isomerizations at X-Pro peptide bond, following the same protonation schemes, in good agreement with previous ion mobility and single point mutation experiments. The comparison between ion mobility selected ECD spectra and traditional FT-ICR ECD MS/MS spectra showed comparable ECD fragmentation efficiencies but differences in the ratio of radical (˙)/prime (') fragment species (H˙ transfer), which were associated with the differences in detection time after the electron capture event. The analysis of model peptides using online TIMS-q-EMSToF MS/MS provided complementary structural information on the intramolecular interactions that stabilize the different gas-phase conformations to those obtained by ion mobility or ECD alone.

General rules of fragmentation evidencing lasso structures in CID and ETD.

Lasso peptides constitute a structurally unique class of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked structure in which the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Tandem mass spectrometry using collision induced dissociation (CID) and electron capture dissociation (ECD) have shown previously different fragmentation patterns for capistruin, microcin J25 and their corresponding branched-cyclic forms in which the C-terminal tail is unthreaded. In order to develop general rules that unambiguously discriminate the lasso and branched-cyclic topologies, this report presents experimental evidence for a set of twenty-one lasso peptides analyzed by CID and electron transfer dissociation (ETD). CID experiments on lasso peptides specifically yielded mechanically interlocked species with associated bi and yj fragments. For class II lasso peptides, these lasso-specific fragments were observed only for peptides in which the loop, located above the macrolactam ring, was strictly longer than four amino acid residues. For class I and III lasso peptides, part of the C-terminal tail remains covalently linked to the macrolactam ring by disulfide bonds; associated bi and yj fragments therefore do not clearly constitute a signature of the lasso topology. ETD experiments of lasso peptides showed a significant increase of hydrogen migration events in the loop region when compared to their branched-cyclic topoisomers, leading to the formation of specific ci˙/z'j fragments for all lasso peptides, regardless of their class and loop size. Our experiments enabled us to establish general rules for obtaining structural details from CID and ETD fragmentation patterns, obviating the need for structure determination by NMR or X-ray crystallography.