Differential glycosylation does not modulate the conformational heterogeneity of a humanised IgGk NIST monoclonal antibody.

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 of subpopulations of a humanised IgGk NIST monoclonal antibody (NISTmAb). Our results indicate that differential glycosylation of NISTmAb does not modulate its conformational heterogeneity.

Evaluation of a Commercial TIMS-Q-TOF Platform for Native Mass Spectrometry.

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 for rapid analysis of peptides or those designed for high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) platform is widely utilized for proteomics and metabolomics, with ion mobility providing a separation dimension in addition to liquid chromatography.

Metastability of Protein Solution Structures in the Absence of a Solvent: Rugged Energy Landscape and Glass-like Behavior.

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 the biological significance of protein structures identified through ion mobility/mass spectrometry. Using newly developed computational and experimental ion mobility/ion mobility/mass spectrometry methods, we investigate the unfolding of the protein ubiquitin in a solvent-free environment.

Elucidating Structures of Protein Complexes by Collision-Induced Dissociation at Elevated Gas Pressures.

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 mobility spectrometers that conduct multiple ion mobility separations in series. The present work investigates the applicability of collision-induced dissociation (CID) at 1 to 3 mbar in a tandem-trapped ion mobility spectrometer (tandem-TIMS) to study the architecture of protein complexes.

Top-Down Protein Analysis by Tandem-Trapped Ion Mobility Spectrometry/Mass Spectrometry (Tandem-TIMS/MS) Coupled with Ultraviolet Photodissociation (UVPD) and Parallel Accumulation/Serial Fragmentation (PASEF) MS/MS Analysis.

"Top-down" proteomics analyzes intact proteins and identifies proteoforms by their intact mass as well as the observed fragmentation pattern in tandem mass spectrometry (MS/MS) experiments. Recently, hybrid ion mobility spectrometry-mass spectrometry (IM/MS) methods have gained traction for top-down experiments, either by allowing top-down analysis of individual isomers or alternatively by improving signal/noise and dynamic range for fragment ion assignment. We recently described the construction of a tandem-trapped ion mobility spectrometer/mass spectrometer (tandem-TIMS/MS) coupled with an ultraviolet (UV) laser and demonstrated a proof-of-principle for top-down analysis by UV photodissociation (UVPD) at 2-3 mbar.

Perspective on the potential of tandem-ion mobility /mass spectrometry methods for structural proteomics applications.

Cellular processes are usually carried out collectively by the entirety of all proteins present in a biological cell, i.e. the proteome.

Structure Relaxation Approximation (SRA) for Elucidation of Protein Structures from Ion Mobility Measurements (II). Protein Complexes.

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

Tandem Trapped Ion Mobility Spectrometry/Mass Spectrometry (tTIMS/MS) Reveals Sequence-Specific Determinants of Top-Down Protein Fragment Ion Cross Sections.

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

Tandem-trapped ion mobility spectrometry/mass spectrometry (TIMS/MS): a promising analytical method for investigating heterogenous samples.

Ion mobility spectrometry/mass spectrometry (IMS/MS) is widely used to study various levels of protein structure. Here, we review the current state of affairs in -trapped ion mobility spectrometry/mass spectrometry (TIMS/MS). Two different TIMS/MS instruments are discussed in detail: the first TIMS/MS instrument, constructed from coaxially aligning two TIMS devices; and an orthogonal TIMS/MS configuration that comprises an ion trap for irradiation of ions with UV photons.

Tandem-trapped ion mobility spectrometry/mass spectrometry coupled with ultraviolet photodissociation.

Rationale: 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 mobility spectrometry/mass spectrometry methods are limited in their ability to characterize structures of proteins and protein complexes.

Methods: Here, we describe the coupling of the separation capabilities of tandem-trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS) with the dissociation capabilities of ultraviolet photodissociation (UVPD) for protein structure analysis.

Structural Analysis of the Glycoprotein Complex Avidin by Tandem-Trapped Ion Mobility Spectrometry-Mass Spectrometry (Tandem-TIMS/MS).

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 the ability of tandem-trapped ion mobility spectrometry-mass spectrometry (tandem-TIMS/MS) to characterize the structure and sequence of the homotetrameric glycoprotein avidin.

On the Preservation of Non-covalent Peptide Assemblies in a Tandem-Trapped Ion Mobility Spectrometer-Mass Spectrometer (TIMS-TIMS-MS).

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 the IMS-MS measurement if they undergo energetic ion-neutral collisions. Here, we investigate the ability of tandem-trapped ion mobility spectrometry-mass spectrometry (TIMS-TIMS-MS) to retain weakly-bound peptide assemblies.

Structure Relaxation Approximation (SRA) for Elucidation of Protein Structures from Ion Mobility Measurements.

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

A Transferable, Sample-Independent Calibration Procedure for Trapped Ion Mobility Spectrometry (TIMS).

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 to separate ions. These methods require a calibration procedure to determine ion mobilities from the experimental data.

Tandem trapped ion mobility spectrometry.

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 the three-dimensional analyte structure. Recent reports indicate that additional structural information can be extracted from measuring changes in cross sections in response to changes of the analyte structure.

On the structural denaturation of biological analytes in trapped ion mobility spectrometry - mass spectrometry.

Key to native ion mobility/mass spectrometry is to prevent the structural denaturation of biological molecules in the gas phase. Here, we systematically assess structural changes induced in the protein ubiquitin during a trapped ion mobility spectrometry (TIMS) experiment. Our analysis shows that the extent of structural denaturation induced in ubiquitin ions is largely proportional to the amount of translational kinetic energy an ion gains from the applied electric field between two collisions with buffer gas particles.

Printing Peptide arrays with a complementary metal oxide semiconductor chip.

: In this chapter, we discuss the state-of-the-art peptide array technologies, comparing the spot technique, lithographical methods, and microelectronic chip-based approaches. Based on this analysis, we describe a novel peptide array synthesis method with a microelectronic chip printer. By means of a complementary metal oxide semiconductor chip, charged bioparticles can be patterned on its surface.