Rationale:The function of a protein or the binding affinity of an antibody can be substantially altered by the replacement of leucine (Leu) with isoleucine (Ile), and vice versa, so the ability to identify the correct isomer using mass spectrometry can help resolve important biological questions. Tandem mass spectrometry approaches for Leu/Ile (Xle) discrimination have been developed, but they all have certain limitations.Methods: Four model peptides and two wild-type peptide sequences containing either Leu or Ile residues were subjected to charge transfer dissociation (CTD) mass spectrometry on a modified three-dimensional ion trap. The peptides were analyzed in both the 1+ and 2+ charge states, and the results were compared to conventional collision-induced dissociation spectra of the same peptides obtained using the same instrument.Results: CTD resulted in 100% sequence coverage for each of the studied peptides and provided a variety of side-chain cleavages, including d, w and v ions. Using CTD, reliable d and w ions of Xle residues were observed more than 80% of the time.

The ability to understand the function of a protein often relies on knowledge about its detailed structure. Sometimes, seemingly insignificant changes in the primary structure of a protein, like an...

Research in natural products (NPs) has gained interest as drug developers turn to nature to combat problems with drug resistance, drug delivery, and emerging diseases. Whereas NPs offer a tantalizing source of new pharmacologically active compounds, their structural complexity presents a challenge for analytical characterization and organic synthesis. Of particular concern is the characterization of cyclic-, polycyclic-, or macrocyclic compounds. One example of endogenous compounds as inspiration for NP development are cobalamins, like vitamin B12. An example of exogenous NPs is the class of macrolides that includes erythromycin. Both classes of macrocycles feature analogues with a range of modifications on their macrocyclic cores, but because of their cyclic nature, they are generally resistant to fragmentation by collision-induced dissociation (CID). In the present work, charge-transfer dissociation (CTD) was employed, with or without supplemental collisional activation, to produce radical-driven, high-energy fragmentation products of different macrocyclic precursors. With the assistance of collisional activation of CTnoD products, CTD frequently cleaved two covalent bonds within the macrocycle cores to reveal rich, informative spectra that helped identify sites of modification and resolve structural analogues. In a third example of macrocycle fragmentation, CTD enabled an impurity in a biological sample to be characterized as a cyclic polymer of nylon-6,6. In each example, CTD spectra are starkly different from CID and are highly reminiscent of other high-energy fragmentation techniques like extreme ultraviolet dissociative photoionization (XUV-DPI) and electron ionization-induced dissociation (EID). The results indicate that CTD-MS is a useful tool for the characterization of natural and synthetic macrocycles.

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