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Glycosylation is one of the most common types of post-translational modifications (PTMs) of proteins, which affects protein properties including conformation, stability and solubility, and plays essential roles in functions including molecular recognition, signaling and immune defense. Characterizing the higher order structures and dynamics of glycoprotein complexes at the intact level is essential for studying the biological functions of glycoproteins. Particularly, accessing structural information such as glycosylation pattern, proteoform distribution, protein interaction, and dynamic conformational evolvement requires direct measurement of intact species. Native mass spectrometry (nMS) allows preservation of non-covalent inter-protein interactions and direct analysis of topology, stoichiometry and dynamic assembly/disassembly at the intact complex level, highly complementing the conventional biophysical techniques. The combination of nMS with travelling-wave ion mobility spectrometry (TWIMS) provides not only an additional orthogonal dimension of separation, but also geometric features of the analytes through collisional cross-section (CCS) determination, thereby facilitating the structural characterization. The macro- and micro-heterogeneity caused by glycosylation, as well as binding with small molecules, induce structural changes of glycoproteins and their complexes on a scale smaller than those resulting from unfolding or macromolecular association/dissociation. However, the benefits of nMS-IMS for characterization of small-scale structural changes or differences of glycoprotein complexes remain to be further demonstrated, in aspects including proteoform-specific structural analysis, distinguishing different binding states of glycoprotein complexes in complex with small molecules, and solution-parameter-induced conformational or stability changes. In this work, aiming at characterizing the stability and small-scale structural differences of glycoprotein complexes, two formats of avidin (tetrameric biotin-binding glycoprotein) that were expressed from different species and exhibit differing carbohydrate contents and proteoform patterns were used as model systems, and the benefits of implementing ion mobility analysis in nMS for structural analysis of both intact complexes and subunits released in the gas-phase were evaluated. It was demonstrated that IMS allows separation of subpopulations of proteoforms, which facilitates not only profiling of proteoform distribution for intact glycoproteins by MS, but also investigation of correlation between glycosylation and CCS. We also demonstrated the power of IMS in measuring the geometric features of the protein complexes in complex with small molecules, and characterizing the extent of conformational changes of glycoproteins under conditions including gas-phase dissociation, supercharging, charge-reduction, and charge-manipulated dissociation. These advantages enable the stability evaluation for glycoprotein complexes.

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We present here a software suite ( ORIGAMI ) that facilitates the rapid acquisition and analysis of ion mobility data following collisional activation. ORIGAMI was developed for use on Waters Synapt instruments where data acquisition is achieved by interfacing WREnS (Waters Research Enabled Software) and MassLynx. Two components are presented, the first is ORIGAMI MS which enables the activation of ions via a sequential ramping of collision voltage prior to ion mobility analysis. We demonstrate the use of ORIGAMI on the tetrameric assemblies formed by the proteins concanavalin A (103 kDa) and alcohol dehydrogenase (143 kDa). Activation is performed in the trap collision cell of the Synapt TriWave assembly where the collision voltage can be ramped from 0 to 200 V. All of the acquired data is recorded in a single file which simplifies data acquisition while keeping the acquisition times at approx. 25 min for a single charge state exposed to a collision voltage range of 0–200 V. Following acquisition, the data are analysed in the second component, ORIGAMI ANALYSE , which allows the user to visualise the effect of activation on the mobility of the parent ion, as well as on any fragment ion. The user can export the data in the form of heat maps, waterfall or wire plots as well as interactive and shareable webpages. In addition, tools implemented in ORIGAMI enable easy data extraction from single or multiple MassLynx .raw files, in-depth interrogation of large datasets, statistical analysis and figure creation capabilities. [ABSTRACT FROM AUTHOR]

Presence of unresolved ion mobility (IM) profiles limits the efficient utilization of IM mass spectrometry (IM-MS) systems for isomer differentiation. Here, we introduce an automated ion mobility deconvolution (AIMD) computer software for streamlined deconvolution of overlapped IM-MS profiles. AIMD is based on a previously reported post-IM/collision-induced dissociation (CID) deconvolution approach [ J. Am. Soc. Mass Spectrom. 23, 1873 (2012)] and, unlike the previously reported manual approach, it does not require resampling of post-IM/CID data. A novel data preprocessing approach is utilized to improve the accuracy and efficiency of the deconvolution process. Results from AIMD analysis of overlapped IM profiles of data from (1) Waters Synapt G1 for a binary mixture of isomeric peptides (amino acid sequences: GRGDS and SDGRG) and (2) Waters Synapt G2-S for a binary mixture of isomeric trisaccharides (raffinose and isomaltotriose) are presented. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: DNA polymerase III, a decameric 420-kDa assembly, simultaneously replicates both strands of the chromosome in Escherichia coli. A subassembly of this holoenzyme, the seven-subunit clamp loader complex, is responsible for loading the sliding clamp (β2) onto DNA. Here, we use structural information derived from ion mobility mass spectrometry (IM-MS) to build three-dimensional models of one form of the full clamp loader complex, γ3δδ′ψχ (254kDa). By probing the interaction between the clamp loader and a single-stranded DNA (ssDNA) binding protein (SSB4) and by identifying two distinct conformational states, with and without ssDNA, we assemble models of ψχ–SSB4 (108kDa) and the clamp loader–SSB4 (340kDa) consistent with IM data. A significant increase in measured collision cross-section (~10%) of the clamp loader–SSB4 complex upon DNA binding suggests large conformational rearrangements. This DNA bound conformation represents the active state and, along with the presence of ψχ, stabilises the clamp loader–SSB4 complex. Overall, this study of a large heteromeric complex analysed by IM-MS, coupled with integrative modelling, highlights the potential of such an approach to reveal structural features of previously unknown complexes of high biological importance. [Copyright &y& Elsevier]

Abstract: The development and use of UHPLC-based methods for the identification, validation and analysis of biomarkers for diseases is reviewed. The currents trends in types of stationary phases and modes of detection are discussed. Afterwards, examples are provided on the use of UHPLC-MS for finding novel biomarkers in samples from in vitro or in vivo animal models of human diseases, as well as in biofluid samples (mainly urine and plasma) obtained from patients. Molecular profiling and targeted analysis are considered, providing an overview of recent experimental or clinical works carried out using UHPLC analysis of compounds from various chemical classes, such as low molecular weight metabolites, hormones, lipids, peptides and proteins. [Copyright &y& Elsevier]

Abstract: High-resolution structural information is needed in order to unveil the underlying mechanistic of biomolecular function. Due to the technical limitations or the nature of the underlying complexes, acquiring atomic resolution information is difficult for many challenging systems, while, often, low-resolution biochemical or biophysical data can still be obtained. To make best use of all the available information and shed light on these challenging systems, integrative computational tools are required that can judiciously combine and accurately translate sparse experimental data into structural information. In this review we discuss the current state of integrative approaches, the challenges they are confronting and the advances made regarding those challenges. Recent developments are underpinned by noteworthy application examples taken from the literature. Within this context, we also position our data-driven docking approach, HADDOCK that can integrate a variety of information sources to drive the modeling of biomolecular complexes. Only a synergistic combination of experiment and modeling will allow us to tackle the challenges of adding the structural dimension to interactomes, shed “atomic” light onto molecular processes and understand the underlying mechanistic of biomolecular function. The current state of integrative approaches indicates that they are poised to take those challenges. [Copyright &y& Elsevier]

We present the details of a data analysis approach for deconvolution of the ion mobility (IM) overlapped or unresolved species. This approach takes advantage of the ion fragmentation variations as a function of the IM arrival time. The data analysis involves the use of an in-house developed data preprocessing platform for the conversion of the original post-IM/collision-induced dissociation mass spectrometry (post-IM/CID MS) data to a Matlab compatible format for chemometric analysis. We show that principle component analysis (PCA) can be used to examine the post-IM/CID MS profiles for the presence of mobility-overlapped species. Subsequently, using an interactive self-modeling mixture analysis technique, we show how to calculate the total IM spectrum (TIMS) and CID mass spectrum for each component of the IM overlapped mixtures. Moreover, we show that PCA and IM deconvolution techniques provide complementary results to evaluate the validity of the calculated TIMS profiles. We use two binary mixtures with overlapping IM profiles, including (1) a mixture of two non-isobaric peptides (neurotensin (RRPYIL) and a hexapeptide (WHWLQL)), and (2) an isobaric sugar isomer mixture of raffinose and maltotriose, to demonstrate the applicability of the IM deconvolution. [ABSTRACT FROM AUTHOR]

Over the last 20 years, mass spectrometry (MS), with its ability to analyze small sample amounts with high speed and sensitivity, has more and more entered the field of structural virology, aiming to investigate the structure and dynamics of viral proteins as close to their native environment as possible. The use of non-perturbing labels in hydrogen-deuterium exchange MS allows for the analysis of interactions between viral proteins and host cell factors as well as their dynamic responses to the environment. Cross-linking MS, on the other hand, can analyze interactions in viral protein complexes and identify virus-host interactions in cells. Native MS allows transferring viral proteins, complexes and capsids into the gas phase and has broken boundaries to overcome size limitations, so that now even the analysis of intact virions is possible. Different MS approaches not only inform about size, stability, interactions and dynamics of virus assemblies, but also bridge the gap to other biophysical techniques, providing valuable constraints for integrative structural modeling of viral complex assemblies that are often inaccessible by single technique approaches. In this review, recent advances are highlighted, clearly showing that structural MS approaches in virology are moving towards systems biology and ever more experiments are performed on cellular level., (© 2019 Elsevier Inc. All rights reserved.)