Trapped ion mobility spectra recorded for ubiquitin are consistent with structures reported the native state by NMR.

Ion mobility spectrometry–mass spectrometry methods offer the potential to correlate protein tertiary and quaternary structures variations in their amino acid sequences post-translational modifications. Because ion measures cross sections of ions gas phase, however, structure systems detected by will generally differ from native solution structures. While it is now established that does not typically detect equilibrium gas-phase systems, what remains disputed which aspects, if any, resemble...

There is currently a strong interest in the use of ion mobility spectrometry-mass spectrometry (IMS-MS) instrumentation for structural biology. In these applications, momentum transfer cross sections derived from IMS-MS measurements are used to reconstruct three-dimensional analyte structure. Recent reports indicate that additional information can be extracted measuring changes response To further this approach, we constructed tandem trapped IMS analyser (TIMS-TIMS) and incorporated it QqTOF...

Ion mobility spectrometry-mass spectrometry (IMS-MS) determines momentum transfer cross sections of ions to elucidate their structures. Recent IMS methods employ electrodynamic fields or nonstationary buffer gases separate ions. These require a calibration procedure determine ion mobilities from the experimental data. This applies in particular trapped (TIMS), novel method with reported high resolving powers. Here, we report first systematic assessment accuracy and limitations TIMS. Our data...

Glycoproteins play a central role in many biological processes including disease mechanisms. Nevertheless, because glycoproteins are heterogeneous entities, it remains unclear how glycosylation modulates the protein structure and function. Here, we assess ability of tandem-trapped ion mobility spectrometry–mass spectrometry (tandem-TIMS/MS) to characterize sequence homotetrameric glycoprotein avidin. We show that (1) tandem-TIMS/MS retains native-like avidin tetramers with deeply buried...

Native ion mobility/mass spectrometry is well-poised to structurally screen proteomes but characterizes protein structures in the absence of a solvent. This raises long-standing unanswered questions about biological significance identified through spectrometry. Using newly developed computational and experimental mobility/ion methods, we investigate unfolding ubiquitin solvent-free environment. Our data suggest that folded, observed by exists largely native fold with an intact β-grasp motif...

Top-down proteomics provides a straightforward approach to the level of proteoforms but remains technologically challenging. Using ion mobility spectrometry/mass spectrometry (IMS/MS) separate top-down fragment ions improves signal/noise and dynamic range. Such applications, however, do not yet leverage primary information obtained from IMS/MS, which is characterization structure by measured momentum transfer cross sections. Here, we perform analysis intact proteins assemblies using our...

Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized rapid analysis of peptides designed high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) platform is widely utilized proteomics and metabolomics, with mobility providing a separation dimension addition to liquid chromatography. The ability...

Protein activity depends on motional transitions between conformational states. Modifications or ligand binding can alter protein dynamics, leading to changes in activity. Established biophysical methods effectively determine structures but face challenges when investigating dynamic biological processes that involve steady states of transiently populated con-formations. Ion mobility/mass spectrometry shows promise for studying transient conformations characterizes them a solvent-free...

Ion mobility spectrometry-mass spectrometry (IMS-MS) has demonstrated the ability to characterize structures of weakly-bound peptide assemblies. However, these assemblies can potentially dissociate during IMS-MS measurement if they undergo energetic ion-neutral collisions. Here, we investigate tandem-trapped ion (TIMS-TIMS-MS) retain We assess heating and dissociaton in tandem-TIMS instrument using bradykinin its as reference systems. Our data indicate that non-covalent are largely preserved...

Tandem-ion mobility spectrometry/mass spectrometry methods have recently gained traction for the structural characterization of proteins and protein complexes. However, ion activation techniques currently coupled with tandem-ion are limited in their ability to characterize structures complexes.Here, we describe coupling separation capabilities tandem-trapped (tTIMS/MS) dissociation ultraviolet photodissociation (UVPD) structure analysis.We establish feasibility dissociating intact by UV...

"Top-down" proteomics analyzes intact proteins and identifies proteoforms by their mass as well the observed fragmentation pattern in tandem spectrometry (MS/MS) experiments. Recently, hybrid ion mobility spectrometry–mass (IM/MS) methods have gained traction for top-down experiments, either allowing analysis of individual isomers or alternatively improving signal/noise dynamic range fragment assignment. We recently described construction a tandem-trapped spectrometer/mass spectrometer...

Characterizing structures of protein complexes and their disease-related aberrations is essential to understanding molecular mechanisms many biological processes. Electrospray ionization coupled with hybrid ion mobility/mass spectrometry (ESI-IM/MS) methods offer sufficient sensitivity, sample throughput, dynamic range enable systematic structural characterization proteomes. However, because ESI-IM/MS characterizes ionized systems in the gas phase, it generally remains unclear what extent...

Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized rapid analysis of peptides designed high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time flight (TIMS-Q-TOF) platform is widely utilized proteomics and metabolomics, with mobility providing a separation dimension addition to liquid chromatography. The ability perform...

Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized rapid analysis of peptides designed high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time flight (TIMS-Q-TOF) platform is widely utilized proteomics and metabolomics, with mobility providing a separation dimension addition to liquid chromatography. The ability perform...

Ion activation methods carried out at gas pressures compatible with ion mobility separations are not yet widely established. This limits the analytical utility of emerging tandem-ion spectrometers that conduct multiple in series. The present work investigates applicability collision-induced dissociation (CID) 1 to 3 mbar a tandem-trapped spectrometer (tandem-TIMS) study architecture protein complexes. We show CID homotetrameric complexes streptavidin (53 kDa), neutravidin (60 and...

Investigating the structural heterogeneity of monoclonal antibodies is crucial to achieving optimal therapeutic outcomes. We show that tandem-trapped ion mobility spectrometry enables collision-induced unfolding measurements antibody subpopulations. Our data suggest such non-ensemble will improve characterisation complex biotherapeutics as multivalent antibodies.

The complexity of the proteome at all levels protein structure and dynamics renders systematic identification how modifications alter protein-protein interaction networks cause disease a formidable, unsolved challenge. Mass spectrometry-methods enable detection causing on proteome-wide scale but do not provide direct structural information to reveal molecular origins disease-causing events. Here, we demonstrate that (i) ultraviolet photodissociation coupled with tandem-trapped ion mobility...

Despite the significance of differentially modified proteins (proteoforms) to human health, it remains challenging identify how proteoforms alter protein structural dynamics and function. Although native ion mobility/mass spectrometry is well-suited handle proteoform heterogeneity, characterizes structures in absence solvent. This raises long-standing, unanswered questions about biological identified through spectrometry. Using newly developed computational experimental mobility/ion methods,...

Mass spectrometry-based assays in structural biology studies typically measure either intact or digested proteins. Traditionally, there are different mass spectrometers dedicated for such measurements: those focused on the rapid analysis of small molecules, as peptides, and designed high analysis. The Bruker timsTOF Pro spectrometer (ion mobility-quadrupole-time flight platform, IM-Q-TOF), has become widely utilized molecules fields proteomics metabolomics, with ion mobility spectrometry...

Investigating the structural heterogeneity of monoclonal antibodies is crucial to achieving optimal therapeutic outcomes. We show that tandem-trapped ion mobility spectrometry enables collision-induced unfolding measurements antibody subpopulations. Our data suggest such non-ensemble will improve characterisation complex biotherapeutics as multivalent antibodies.

This study addresses three unmet needs: 1) development of widely accessible native mass spectrometry (MS) methods and 2) top-down (TDMS) for high flow sources on an Agilent Q-TOF spectrometer, 3) confirming suitability structures lossless ion manipulation (SLIM) mobility (IM) MS analysis intact proteins. To decrease the cost training barrier to performing MS, TDMS, IM-MS proteins, we have developed nanospray, microspray, heated electrospray ionization sources. SLIM offers highest full-scan...