Simon Mysling

Utilizing Ion-Pairing Hydrophilic Interaction Chromatography Solid Phase Extraction for Efficient Glycopeptide Enrichment in Glycoproteomics

Glycopeptide enrichment is a prerequisite to enable structural characterization of protein glycosylation in glycoproteomics. Here we present an improved method for glycopeptide enrichment based on zwitter-ionic hydrophilic interaction chromatography solid phase extraction (ZIC-HILIC SPE) in a microcolumn format. The method involves TFA ion pairing (IP) to increase the hydrophilicity difference between glycopeptides and nonglycosylated peptides. Three mobile phases were investigated, i.e., 2% formic acid (defined as IP(2% FA) ZIC-HILIC SPE), 0.1% TFA and 1% TFA (defined as IP(0.1% TFA) and IP(1% TFA) ZIC-HILIC SPE) all containing 80% acetonitrile. Samples of increasing complexities, i.e., digests of single glycoproteins, a five-glycoprotein mixture, and depleted plasma, were used in the study. The presence of TFA in the mobile phase significantly improved the glycopeptide enrichment for all complexities, as evaluated by enhanced glycopeptide detection using MALDI-TOF MS and RP-LC-ESI-MS/MS, e.g., the glycopeptide ion signals were increased by up to 3.7-fold compared to IP(2% FA) conditions. The enhanced glycopeptide detection was promoted by a substantial depletion of nonglycosylated peptides, offering an almost complete isolation of IgG glycopeptides using a single SPE enrichment step and a reduction from 711 nonglycosylated peptides observed in the IP(2% FA) ZIC-HILIC SPE retained plasma fraction, to only 157 and 97 when 0.1% and 1% TFA was used in the mobile phase. In conclusion, this systematic study has shown that TFA-containing mobile phases increase glycopeptide enrichment efficiency considerably for a broad range of sample complexities when using ZIC-HILIC SPE.

Site-Specific Glycoprofiling of N-Linked Glycopeptides Using MALDI-TOF MS: Strong Correlation between Signal Strength and Glycoform Quantities

Site-specific glycoprofiling of N-linked glycopeptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique, but its quantitative accuracy lacks documentation. Thus, a systematic study of widely different glycopeptides was performed to determine the relationship between the relative abundances of the individual glycoforms and the MALDI-TOF MS signal strength. Glycopeptides derived from glycoproteins containing neutral glycans (ribonuclease B, IgG, and ovalbumin) were initially profiled and yielded excellent and reproducible quantitation (correlation coefficient r = 0.9958, n = 5) when evaluated against a normal phase HPLC 2-AB glycan profile. Similarly, precise quantitation was observed for various forms of N-glycans (free, permethylated, and fluorescence-labeled) using MS. In addition, three different sialoglycopeptides from fetuin were site-specifically profiled, and good correlation between peak intensities and relative abundances was found with only a minor loss of sialic acids (r = 0.9664, n = 5). For glycopeptide purification, a range of hydrophilic and graphite materials packed in microcolumn format proved capable of performing desalting without loss of quantitative information, but highlighted the column capacity as a critical parameter. In conclusion, MALDI-TOF MS signal strength of glycopeptides has been found to accurately reflect the relative quantities of glycoforms, providing that certain technical issues are considered, i.e., nonbiased sample handling, matrix choice, and instrumental settings. This enables rapid and sensitive site-specific glycoprofiling of N-glycan populations to promote biomarker discovery and elucidation of glycan structure/function relationships.

Co‐existence of Two Different α‐Synuclein Oligomers with Different Core Structures Determined by Hydrogen/Deuterium Exchange Mass Spectrometry

Neurodegenerative disorders are characterized by the formation of protein oligomers and amyloid fibrils, which in the case of Parkinsons disease involves the protein α-synuclein (αSN). Cytotoxicity is mainly associated with the oligomeric species, but we still know little about their assembly and structure. Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is used to analyze oligomers formed by wild-type (wt) αSN and also three familial αSN mutants (A30P, E46K, and A53T). All four variants show co-existence of two different oligomers. The backbone amides of oligomer type I are protected from exchange with D2 O until they dissociate into monomeric αSN by EX1 exchange kinetics. Fewer residues are protected against exchange in oligomer type II, but this type does not revert to αSN monomers. Both oligomers are protected in the core sequence Y39-A89. Based on incubation studies, oligomer type I appears to form straight fibrils, while oligomer type II forms amorphous clusters that do not directly contribute to the fibrillation process.

Activity-regulating structural changes and autoantibody epitopes in transglutaminase 2 assessed by hydrogen/deuterium exchange

Significance The enzyme transglutaminase 2 (TG2) is the target of autoantibodies characteristic of the gluten-sensitive enteropathy celiac disease, and intact enzyme activity seems to be required for the disease-causing immune response. TG2 activity is regulated through conformational changes. Ca2+ binding is required for enzyme activity, whereas oxidation inactivates the enzyme. Using hydrogen/deuterium exchange monitored by mass spectrometry, we have studied differences between active and inactive forms of TG2 in solution and found that oxidation prevents Ca2+-induced structural changes. Further, we have characterized the TG2 binding of a panel of monoclonal autoantibodies derived from disease lesion plasma cells. Autoantibody binding affected the structure of TG2, and mapping of the targeted epitopes suggests a possible mechanism for the induction of the autoimmune response. The multifunctional enzyme transglutaminase 2 (TG2) is the target of autoantibodies in the gluten-sensitive enteropathy celiac disease. In addition, the enzyme is responsible for deamidation of gluten peptides, which are subsequently targeted by T cells. To understand the regulation of TG2 activity and the enzyme’s role as an autoantigen in celiac disease, we have addressed structural properties of TG2 in solution by using hydrogen/deuterium exchange monitored by mass spectrometry. We demonstrate that Ca2+ binding, which is necessary for TG2 activity, induces structural changes in the catalytic core domain of the enzyme. Cysteine oxidation was found to abolish these changes, suggesting a mechanism whereby disulfide bond formation inactivates the enzyme. Further, by using TG2-specific human monoclonal antibodies generated from intestinal plasma cells of celiac disease patients, we observed that binding of TG2 by autoantibodies can induce structural changes that could be relevant for the pathogenesis. Detailed mapping of two of the main epitopes targeted by celiac disease autoantibodies revealed that they are located adjacent to each other in the N-terminal part of the TG2 molecule.

Characterizing the Dynamics of α-Synuclein Oligomers Using Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

Soluble oligomers formed by α-synuclein (αSN) are suspected to play a central role in neuronal cell death during Parkinsons disease. While studies have probed the surface structure of these oligomers, little is known about the backbone dynamics of αSN when they form soluble oligomers. Using hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS), we have analyzed the structural dynamics of soluble αSN oligomers. The analyzed oligomers were metastable, slowly dissociating to monomers over a period of 21 days, after excess monomer had been removed. The C-terminal region of αSN (residues 94-140) underwent isotopic exchange very rapidly, demonstrating a highly dynamic region in the oligomeric state. Three regions (residues 4-17, 39-54, and 70-89) were strongly protected against isotopic exchange in the oligomers, indicating the presence of a stable hydrogen-bonded or solvent-shielded structure. The protected regions were interspersed by two somewhat more dynamic regions (residues 18-38 and 55-70). In the oligomeric state, the isotopic exchange pattern of the region of residues 35-95 of αSN corresponded well with previous nuclear magnetic resonance and electron paramagnetic resonance analyses performed on αSN fibrils and indicated a possible zipperlike maturation mechanism for αSN aggregates. We find the protected N-terminus (residues 4-17) to be of particular interest, as this region has previously been observed to be highly dynamic for both monomeric and fibrillar αSN. This region has mainly been described in relation to membrane binding of αSN, and structuring may be important in relation to disease.

Electrochemical Reduction of Disulfide-Containing Proteins for Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

Characterization of disulfide bond-containing proteins by hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) requires reduction of the disulfide bonds under acidic and cold conditions, where the amide hydrogen exchange reaction is quenched (pH 2.5, 0 °C). The reduction typically requires a high concentration (>200 mM) of the chemical reducing agent Tris(2-carboxyethyl)phosphine (TCEP) as its reduction rate constant is decreased at low pH and temperature. Serious adverse effects on chromatographic and mass spectrometric performances have been reported when using high concentrations of TCEP. In the present study, we explore the feasibility of using electrochemical reduction as a substitute for TCEP in HDX-MS analyses. Our results demonstrate that efficient disulfide bond reduction is readily achieved by implementing an electrochemical cell into the HDX-MS workflow. We also identify some challenges in using electrochemical reduction in HDX-MS analyses and provide possible conditions to attenuate these limitations. For example, high salt concentrations hamper disulfide bond reduction, necessitating additional dilution of the sample with aqueous acidic solution at quench conditions.

A flexible multidomain structure drives the function of the urokinase-type plasminogen activator receptor (uPAR)

Background: The urokinase receptor (uPAR) is a modular receptor containing three LU domains. Results: Ligand-free uPAR is inherently flexible with a detached N-terminal domain (DI). Conclusion: Allosteric regulation of uPAR is driven by uPA-induced compaction of the intact receptor and a concomitant stabilization of DI. Significance: This flexibility and ligand-induced allostery are expected to impact future studies on uPAR function and targeted intervention. The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin-mediated adhesion to vitronectin. These processes are, however, tightly linked because the high affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes, it is evident that the dynamic property of uPAR plays a decisive role in its function. In the present study, we combine small angle x-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research, including targeted intervention therapy and non-invasive tumor imaging in vivo.

The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain

GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPLs catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia. DOI: http://dx.doi.org/10.7554/eLife.12095.001

Conformational analysis of large and highly disulfide-stabilized proteins by integrating online electrochemical reduction into an optimized H/D exchange mass spectrometry workflow.

Analysis of disulfide-bonded proteins by hydrogen/deuterium exchange mass spectrometry (HDX-MS) requires effective and rapid reduction of disulfide bonds before enzymatic digestion in order to increase sequence coverage. In a conventional HDX-MS workflow, disulfide bonds are reduced chemically by addition of a reducing agent to the quench solution (e.g., tris(2-carboxyethyl)phosphine (TCEP)). The chemical reduction, however, is severely limited under quenched conditions due to a narrow time window as well as low pH and temperature. Here, we demonstrate the real-world applicability of integrating electrochemical reduction into an online HDX-MS workflow. We have optimized the electrochemical reduction efficiency during HDX-MS analysis of two particularly challenging disulfide stabilized proteins: a therapeutic IgG1-antibody and nerve growth factor-β (NGF). Several different parameters (flow rate and applied square wave potential, as well as the type of labeling and quench buffer) were investigated, and the optimized workflow increased the sequence coverage of NGF from 46% with chemical reduction to 99%, when electrochemical reduction was applied. Additionally, the optimized workflow also enabled a similar high sequence coverage of 96% and 87% for the heavy and light chain of the IgG1-antibody, respectively. The presented results demonstrate the successful electrochemical reduction during HDX-MS analysis of both a small exceptional tightly disulfide-bonded protein (NGF) as well as the largest protein attempted to date (IgG1-antibody). We envision that online electrochemical reduction is poised to decrease the complexity of sample handling and increase the versatility of the HDX-MS technique.

Glycopeptide Enrichment for MALDI-TOF Mass Spectrometry Analysis by Hydrophilic Interaction Liquid Chromatography Solid Phase Extraction (HILIC SPE)

Glycoproteins, and in particular glycopeptides, are highly hydrophilic and are often not retained by reversed phase (RP) chromatography. The separation principle of normal phase (NP) is based on hydrophilic interactions, which in many aspects is complementary to RP separations. Hydrophilic interaction liquid chromatography (HILIC) is a fairly new variation of the NP separations used in the 1970s, the major difference being the use of aqueous solvents. HILIC provides a versatile tool for enrichment of glycopeptides before mass spectrometric (MS) analysis, particularly when used for solid phase extraction (SPE), or in combination with other chromatographic resins or ion-pairing reagents. HILIC SPE can be used for glyco-profiling, i.e., for determining the glycan heterogeneity at one specific glycosylation site, for enrichment of glycopeptides from a complex mixture of peptides, as well as for pre-fractionation of complex samples at the protein or peptide level. In this chapter we present a straightforward HILIC SPE enrichment technique and then combine C18 RP and HILIC enrichment for analysis of glycopeptides. Finally, we demonstrate HILIC enrichment using trifluoroacetic acid as an ion-pairing reagent for the enrichment of glycopeptides prior to mass spectrometry analysis.