Native mass spectrometry and collision-induced unfolding (CIU) workflows continue to grow in utilization due to their ability to rapidly characterize protein conformation and stability. To perform these experiments, the instrument must be capable of collisionally activating ions prior to ion mobility spectrometry (IMS) analyses. Trapped ion mobility spectrometry (TIMS) is an ion mobility implementation that continues to grow in utilization due to its inherently high resolution and reduced instrumental footprint. In currently deployed instruments, however, typical modes of collisional activation do not precede IMS analysis and thus, the instruments are incapable of performing CIU workflows. In this work, we expand on a recently developed method of activating protein ions within the TIMS device and explore its analytical utility toward the unfolding of protein ions. We demonstrate the unfolding of native-like ions of ubiquitin, cytochrome C, β-lactoglobulin, and carbonic anhydrase. These ions undergo extensive unfolding upon collisional activation. Additionally, the improved resolution provided by the TIMS separation uncovers previously obscured unfolding complexity.

Native mass spectrometry and collision-induced unfolding (CIU) workflows continue to grow in utilization due to their ability to rapidly characterize protein conformation and stability. To perform these experiments, the instrument must be capable of collisionally activating ions prior to ion mobility spectrometry (IMS) analyses. Trapped ion mobility spectrometry (TIMS) is an ion mobility implementation that continues to grow in utilization due to its inherently high resolution and reduced instrumental footprint. In currently deployed instruments, however, typical modes of collisional activation do not precede IMS analysis and thus, the instruments are incapable of performing CIU workflows. In this work, we expand on a recently developed method of activating protein ions within the TIMS device and explore its analytical utility toward the unfolding of protein ions. We demonstrate the unfolding of native-like ions of ubiquitin, cytochrome C, β-lactoglobulin, and carbonic anhydrase. These ions undergo extensive unfolding upon collisional activation. Additionally, the improved resolution provided by the TIMS separation uncovers previously obscured unfolding complexity.

Native mass spectrometry and collision-induced unfolding (CIU) workflows continue to grow in utilization due to their abil-ity to rapidly characterize protein conformation and stability. To perform these experiments, the instrument must be capa-ble of collisionally activating ions prior to ion mobility spectrometry (IMS) analyses. Trapped ion mobility spectrometry (TIMS) is an ion mobility implementation that continues to grow in utilization due to its inherently high resolution and re-duced instrumental footprint. In currently deployed instruments, however, typical modes of collisional activation do not precede IMS analysis and thus, the instruments are incapable of performing CIU workflows. In this work, we expand on a recently developed method of activating protein ions within the TIMS device and explore its analytical utility toward the unfolding of protein ions. We demonstrate the unfolding of native-like ions of ubiquitin, cytochrome C, β-lactoglobulin, and carbonic anhydrase. These ions undergo extensive unfolding upon collisional activation. Additionally, the improved resolu-tion provided by the TIMS separation uncovers previously obscured unfolding complexity.

Free radical-initiated peptide sequencing (FRIPS) is a tandem mass spectrometry (MS/MS) technique that generates sequence informative ions via collisionally-initiated radical chemistry. Collision activation homolytically cleaves an installed radical precursor, initiates radical formation, extensive hydrogen atom transfer, and peptide backbone dissociation. While the FRIPS technique shows great promise, when applied to multiply charged derivatized peptide ions, a series of high abundance mass losses are observed which syphon ion abundance from radically generated sequence ions. This loss of ion abundance reduces the sequence coverage generated by FRIPS fragmentation. In this work, we hypothesized that these mass losses were assisted by the ortho-orientation of the radical precursor undergoing facile conversion into five- or six-membered intermediates or products and that when combined with the lower bond dissociation energy of the para-precursor, conjugated peptides would not undergo this chemistry. To test this assertion, we synthesized para-TEMPO-Bz, conjugated it to these peptides, and collisionally activated each. And indeed, we see dramatic attenuation of these undesired collisional processes and the significant increase in radical precursor ion abundance. The increase in ion abundance leads to a significant increase in the sequence coverage generated. These results demonstrate that p-TEMPO-Bz significantly improves the performance of positive-ion mode FRIPS and may be a compelling alternative to the currently utilized ortho-TEMPO-Bz-based FRIPS.

Free radical-initiated peptide sequencing (FRIPS) is a tandem mass spectrometry technique that generates sequence informative ions via collisionally initiated radical chemistry. Collision activation homolytically cleaves an installed radical precursor and initiates radical formation, extensive hydrogen atom transfer, and peptide backbone dissociation. While the FRIPS technique shows great promise, when applied to multiply charged derivatized peptide ions, a series of high-abundance mass losses are observed which siphon ion abundance from radically generated sequence ions. This loss of ion abundance reduces the sequence coverage generated by FRIPS fragmentation. In this work, we hypothesized that these mass losses were assisted by the ortho-orientation of the radical precursor undergoing facile conversion into five-or six-membered intermediates or products and that when combined with the lower bond dissociation energy of the para-precursor, conjugated peptides would not undergo this chemistry. To test this assertion, we synthesized p-TEMPO-Bz, conjugated it to these peptides, and collisionally activated each. Indeed, we see dramatic attenuation of these undesired collisional processes and a significant increase in radical precursor ion abundance. The increase in ion abundance leads to a significant increase in the sequence coverage generated. These results demonstrate that p-TEMPO-Bz significantly improves the performance of positive-ion mode FRIPS and may be a compelling alternative to the currently utilized o-TEMPO-Bz-based FRIPS.

Tandem mass spectrometry (MS/MS) is often employed for the sequencing of protein ions and the annotation of protein post-translational modification. Collision-induced dissociation (CID) and electron transfer dissociation (ETD) are the most frequently utilized MS/MS techniques, but both have significant limitations. CID is often funneled into a few kinet-ically labile bonds limiting the effectiveness of sequencing and ETD, is only applicable to multiply charged ions and re-quires specialized instrumentation. Free radical-initiated peptide sequencing (FRIPS) is an alternative fragmentation technique that generates sequence informative ions via collisionally-initiated radical chemistry. Its main advantages over the previously mentioned fragmentation techniques include charge state independence, improved efficiency of sequence ion generation, and applicability to most MS instrumentation. Collision activation (CA) homolytically cleaves the radical precursor, initiates radical formation, extensive hydrogen atom transfer, and peptide backbone dissociation. Although the FRIPS technique shows great promise when applied to cations, radical initiation competes directly with typical CID path-ways. When multiply charged ions of conjugated ACTH 1-14, b-amyloid 10-20, substance p, and melittin are collisionally activated, a series of high abundance mass losses are observed which syphon ion abundance from radically generated sequence ions. This loss of ion abundance reduces the sequence coverage generated by FRIPS fragmentation. In this work, we hypothesized that these mass losses were instigated by the ortho-orientation of the radical precursor undergoing fac-ile conversion into five- or six-membered intermediates and that the para-precursor would not undergo this chemistry. To test this assertion, we synthesized para-TEMPO-Bz, conjugated it to these peptides, and collisionally activated each. And indeed, we see the complete elimination of these undesired collisional processes and the significant increase in radi-cal precursor ion abundance. Moreover, we see a significant increase in the sequence coverage generated when the p-TEMPO-Bz is utilized. From these results, p-TEMPO-Bz significantly improves the performance of positive-ion mode FRIPS and may be a suitable alternative to the currently utilized ortho-TEMPO-Bz-based FRIPS.

Native mass spectrometry and collision-induced unfolding (CIU) workflows continue to grow in utilization due to their abil-ity to rapidly characterize protein conformation and stability. To perform these experiments, the instrument must be capa-ble of collisionally activating ions prior to ion mobility spectrometry (IMS) analyses. Trapped ion mobility spectrometry (TIMS) is an ion mobility implementation that continues to grow in utilization due to its inherently high resolution and re-duced instrumental footprint. In currently deployed instruments, however, typical modes of collisional activation do not precede IMS analysis and thus, the instruments are incapable of performing CIU workflows. In this work, we expand on a recently developed method of activating protein ions within the TIMS device and explore its analytical utility toward the unfolding of protein ions. We demonstrate the unfolding of native-like ions of ubiquitin, cytochrome C, β-lactoglobulin, and carbonic anhydrase. These ions undergo extensive unfolding upon collisional activation. Additionally, the improved resolu-tion provided by the TIMS separation uncovers previously obscured unfolding complexity.

Native mass spectrometry and collision-induced unfolding (CIU) workflows continue to grow in utilization due to their ability to rapidly characterize protein conformation and stability. To perform these experiments, the instrument must be capable of collisionally activating ions prior to ion mobility spectrometry (IMS) analyses. Trapped ion mobility spectrometry (TIMS) is an ion mobility implementation that continues to grow in utilization due to its inherently high resolution and reduced instrumental footprint. In currently deployed instruments, however, typical modes of collisional activation do not precede IMS analysis and thus, the instruments are incapable of performing CIU workflows. In this work, we expand on a recently developed method of activating protein ions within the TIMS device and explore its analytical utility toward the unfolding of protein ions. We demonstrate the unfolding of native-like ions of ubiquitin, cytochrome C, β-lactoglobulin, and carbonic anhydrase. These ions undergo extensive unfolding upon collisional activation. Additionally, the improved resolution provided by the TIMS separation uncovers previously obscured unfolding complexity.