Mobility-Assisted Pseudo-MS3 Sequencing of Protein Ions.

The sequencing of intact proteins within a mass spectrometer has many benefits but is frequently limited by the fact that tandem mass spectrometry (MS/MS) techniques often generate poor sequence coverages when applied to protein ions. To overcome this limitation, exotic MS/MS techniques that rely on lasers and radical chemistry have been developed. These techniques generate high sequence coverages, but they require specialized instrumentation, create products through multiple dissociation mechanisms, and often require long acquisition times. Recently, we demonstrated that protein ions can be dissociated in a trapped ion mobility spectrometry (TIMS) device prior to mobility separation in a commercial timsTOF. All generated product ions were distributed throughout the mobility dimension, and this separation enabled deconvolution of complex tandem mass spectra and could enable facile pseudo-MS3 interrogation of generated product ions with the downstream quadrupole and collision cell. A second activation step improves sequence coverage because the most labile bonds have been depleted during the first dissociation and subsequent dissociation events are more evenly distributed throughout the product ion backbone. In this work, we explore the potential of this mobility-assisted pseudo-MS3 (MAP) method on a commercial timsTOF and timsTOF Pro 2. We demonstrate that while MAP only generates 92% of the sequence coverage of the most effective MS/MS technique, it accomplished this feat in 1.5 min and could be facilely integrated with liquid chromatographic separations.

Characterizing the top-down sequencing of protein ions prior to mobility separation in a timsTOF.

Mass spectrometry (MS)-based proteomics workflows of intact protein ions have increasingly been utilized to study biological systems. These workflows, however, frequently result in convoluted and difficult to analyze mass spectra. Ion mobility spectrometry (IMS) is a promising tool to overcome these limitations by separating ions by their mass- and size-to-charge ratios. In this work, we further characterize a newly developed method to collisionally dissociate intact protein ions in a trapped ion mobility spectrometry (TIMS) device. Dissociation occurs prior to ion mobility separation and thus, all product ions are distributed throughout the mobility dimension, enabling facile assignment of near isobaric product ions. We demonstrate that collisional activation within a TIMS device is capable of dissociating protein ions up to 66 kDa. We also demonstrate that the ion population size within the TIMS device significantly influences the efficiency of fragmentation. Lastly, we compare CIDtims to the other modes of collisional activation available on the Bruker timsTOF and demonstrate that the mobility resolution in CIDtims enables the annotation of overlapping fragment ions and improves sequence coverage.

Collision-Induced Unfolding of Native-like Protein Ions Within a Trapped Ion Mobility Spectrometry Device.

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 has been increasingly adopted due to its inherently high resolution and reduced instrumental footprint. In currently deployed commercial instruments, however, typical modes of collisional activation do not precede IMS analysis, and thus, the instruments are incapable of performing CIU. 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 native-like protein ions. We demonstrate the unfolding of native-like ions of ubiquitin, cytochrome C, ß-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.

Fragmentation and Mobility Separation of Peptide and Protein Ions in a Trapped-Ion Mobility Device.

Ion mobility separations (IMS) have increasingly been coupled with mass spectrometry to increase peak capacity and deconvolute complex mass spectra in proteomics workflows. IMS separations can be integrated prior to or following the collisional activation step. Post-activation IMS separations have demonstrated many advantages, yet few instrument platforms are capable of this feat. Here, we present the fragmentation of peptide ions within a commercially available trapped-ion mobility spectrometry device. Fragmentation is initiated prior to mobility analysis enabling the separation of generated product ions. The added separation step deconvolutes product ion spectra and permits improved annotation of product ions. Furthermore, we demonstrate the isolation and fragmentation of mobility separated product ions with the downstream quadrupole and collisional cell. When applied to melittin and ubiquitin, this ion mobility assisted pseudo-MS3 fragmentation approach generates sequence coverage ∼50% greater than that of typical MS2 analyses. We envision this ion-mobility-assisted fragmentation technique as the foundation of a powerful new pseudo-MS3 workflow for application toward middle- or top-down proteomics.

Is Hay for the Birds? Investigating Landowner Willingness to Time Hay Harvests for Grassland Bird Conservation.

Birds in agricultural environments have exhibited steep global population declines in recent decades, and effective conservation strategies targeting their populations are urgently needed. In grasslands used for hay production, breeding birds' nest success improves substantially if hay harvests are delayed until after mid-July. However, few studies have investigated private hay producers' willingness to alter their harvesting practices, which is a critical factor for bird conservation where most land is privately owned, such as in the North American Great Plains. We surveyed Nebraska hay producers to examine whether livestock production, wildlife knowledge, and hunting activity affects their willingness to alter haying practices for bird conservation. The majority (60%) of respondents expressed willingness to delay harvesting hay to allow birds time to nest successfully. Livestock producers and those more knowledgeable about wildlife were more willing to delay hay harvests, whereas active hunters were less willing to do so. Our findings suggest that a majority of private producers show a high potential for engaging in grassland bird conservation activities. Landowners' willingness to participate in bird conservation programs and actions could be further encouraged through extension and education efforts connecting hay producers with information, support, and funding for bird conservation.

Optimizing the fold stability of the circularly permuted Trp-cage motif.

Through optimization of the linker region and key stabilizing mutations, it has been possible to improve the stability of the circularly permuted (cp) Trp-cage miniprotein. However, even the most stable Trp-cage circular permutants are still less stable than the analogous standard topology (std) Trp-cages. Extending mutational studies of Trp-cage fold stability to cp-species, including analogs lacking chain terminal charges, has uncovered and quantitated some additional stabilizing and destabilizing interactions. Upon protonation, the circular permutants are destabilized to a much greater extent than the standard topology series. End effects, particularly Coulombic interactions, appear to be more important for the cp-series while the Y10/P4 interaction in the cp-series is not as significant a stabilizing feature as the corresponding Y3/P19 in the standard topology series.

Reversing the typical pH stability profile of the Trp-cage.

The Trp-cage, an 18-20 residue miniprotein, has emerged as a primary test system for evaluating computational fold prediction and folding rate determination efforts. As it turns out, a number of stabilizing interactions in the Trp-cage folded state have a strong pH dependence; all prior Trp-cage mutants have been destabilized under carboxylate-protonating conditions. Notable among the pH dependent stabilizing interactions within the Trp-cage are: (1) an Asp as the helix N-cap, (2) an H-bonded Asp9/Arg16 salt bridge, (3) an interaction between the chain termini which are in close spatial proximity, and (4) additional side chain interactions with Asp9. In the present study, we have prepared Trp-cage species that are significantly more stable at pH 2.5 (rather than 7) and quantitated the contribution of each interaction listed above. The Trp-cage structure remains constant with the pH change. The study has also provided measures of the stabilizing contribution of indole ring shielding from surface exposure and the destabilizing effects of an ionized Asp at the C-terminus of an α-helix.