Site-Specific Analysis of Gas-Phase Hydrogen/Deuterium Exchange of Peptides and Proteins by Electron Transfer Dissociation

To interpret the wealth of information contained in the hydrogen/deuterium exchange (HDX) behavior of peptides and proteins in the gas-phase, analytical tools are needed to resolve the HDX of individual exchanging sites. Here we show that ETD can be combined with fast gas-phase HDX in ND₃ gas and used to monitor the exchange of side-chain hydrogens of individual residues in both small peptide ions and larger protein ions a few milliseconds after electrospray. By employing consecutive traveling wave ion guides in a mass spectrometer, peptide and protein ions were labeled on-the-fly (0.1–10 ms) in ND₃ gas and subsequently fragmented by ETD. Fragment ions were separated using ion mobility and mass analysis enabled the determination of the gas-phase deuterium uptake of individual side-chain sites in a range of model peptides of different size and sequence as well as two proteins; cytochrome C and ubiquitin. Gas-phase HDX-ETD experiments on ubiquitin ions ionized from both denaturing and native solution conditions suggest that residue-specific HDX of side-chain hydrogens is sensitive to secondary and tertiary structural features occurring in both near-native and unfolded gas-phase conformers present shortly after electrospray. The described approach for online gas-phase HDX and ETD paves the way for making mass spectrometry techniques based on gas-phase HDX more applicable in bioanalytical research

Simple Setup for Gas-Phase H/D Exchange Mass Spectrometry Coupled to Electron Transfer Dissociation and Ion Mobility for Analysis of Polypeptide Structure on a Liquid Chromatographic Time Scale

Gas-phase hydrogen/deuterium exchange (HDX) is a fast and sensitive, yet unharnessed analytical approach for providing information on the structural properties of biomolecules, in a complementary manner to mass analysis. Here, we describe a simple setup for ND₃-mediated millisecond gas-phase HDX inside a mass spectrometer immediately after ESI (gas-phase HDX-MS) and show utility for studying the primary and higher-order structure of peptides and proteins. HDX was achieved by passing N₂-gas through a container filled with aqueous deuterated ammonia reagent (ND₃/D₂O) and admitting the saturated gas immediately upstream or downstream of the primary skimmer cone. The approach was implemented on three commercially available mass spectrometers and required no or minor fully reversible reconfiguration of gas-inlets of the ion source. Results from gas-phase HDX-MS of peptides using the aqueous ND₃/D₂O as HDX reagent indicate that labeling is facilitated exclusively through gaseous ND₃, yielding similar results to the infusion of purified ND₃-gas, while circumventing the complications associated with the use of hazardous purified gases. Comparison of the solution-phase- and gas-phase deuterium uptake of Leu-Enkephalin and Glu-Fibrinopeptide <i>B</i>, confirmed that this gas-phase HDX-MS approach allows for labeling of sites (heteroatom-bound non-amide hydrogens located on side-chains, N-terminus and C-terminus) not accessed by classical solution-phase HDX-MS. The simple setup is compatible with liquid chromatography and a chip-based automated nanoESI interface, allowing for online gas-phase HDX-MS analysis of peptides and proteins separated on a liquid chromatographic time scale at increased throughput. Furthermore, online gas-phase HDX-MS could be performed in tandem with ion mobility separation or electron transfer dissociation, thus enabling multiple orthogonal analyses of the structural properties of peptides and proteins in a single automated LC-MS workflow

Enlarged fragment of a two dimensional IM-MS spectrum of WT Aβ 1–40 after exchange (flow 40 mL/min) focusing on 2164–2185 <i>m/z</i> range.

<p>Vertical axis in the colored panel shows m/z whereas horizontal axis the ion mobility drift time. The colored spots indicate MS peaks with amplitude increasing from purple to yellow. Signals corresponding to MON²⁺, DIM⁴⁺, TRI⁶⁺, TET⁸⁺ were identified based on the analysis of signal spacing in the isotopic envelopes, as shown previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201761#pone.0201761.ref043" target="_blank">43</a>]. Cross sections of isotopic envelopes at two drift times indicated by arrows, corresponding to two alternative TRI⁶⁺ structural forms (insets) show the difference in the distribution of signals after exchange between a more compact form of shorter drift time (upper inset) and a more extended one, characterized by a longer drift time (lower inset). For the extended form the slow exchanging species are minor in contrast to compact form. Projection of this region on the drift time axis (lower panel—vertical axis: signal intensity) shows relative amplitudes of the five signal groups.</p

Side-chain moieties from the N-terminal region of Aβ are Involved in an oligomer-stabilizing network of interactions - Fig 3

<p>Difference in deuterium uptake per monomer between dimers and monomers (circles) and trimers and monomers (tringles) in WT Aβ 1–40 (A), NSCR Aβ 1–40 (B) and SCR Aβ 1–40 (C), based on the analysis of the m/z region 2164–2169 which contains well resolved signals from MON²⁺, DIM⁴⁺, TRI⁶⁺ oligomeric forms bearing the same charge per monomer. The difference in deuterium uptake (D) is shown at the vertical axis and HDX reagent gas flow rate (20–50 mL/min) at the horizontal axis. Four batch-to-batch replicates are represented on the scatter. Dashes refer to the mean difference. Error bars indicate the standard deviation calculated from replicate measurements (number of replicates n was 4).</p

Deuterium content per monomer (HDX/M) of individual charge states of monomers and oligomers of WT Aβ 1–40, induced by gas-phase HDX-MS as a function of make-up gas flow rate (20–50 mL/min) averaged over all signals of the appropriate isotopic envelope.

<p>Deuterium content per monomer (HDX/M) of individual charge states of monomers and oligomers of WT Aβ 1–40, induced by gas-phase HDX-MS as a function of make-up gas flow rate (20–50 mL/min) averaged over all signals of the appropriate isotopic envelope.</p

DLS experiment of WT Aβ 1–40 and SCR Aβ 1–40.

<p>Representative autocorrelation function (ACF), directly adapted from dynamic light scattering data obtained for WT Aβ 1–40 (line) and SCR Aβ 1–40 (dashes). The autocorrelation function decays in the range of 1 down to 0, and the gap remaining to 1 observed at the shortest correlation time is indicative for the existence of monomeric or low-order oligomers, which remain undetectable. The short-correlation-time asymptote of ACF is marked by thin solid lines. The mid-point of an ACF decay is marked by the cross.</p

Fully Automated Electro Membrane Extraction Autosampler for LC–MS Systems Allowing Soft Extractions for High-Throughput Applications

The current work describes the implementation of electro membrane extraction (EME) into an autosampler for high-throughput analysis of samples by EME-LC–MS. The extraction probe was built into a luer lock adapter connected to a HTC PAL autosampler syringe. As the autosampler drew sample solution, analytes were extracted into the lumen of the extraction probe and transferred to a LC–MS system for further analysis. Various parameters affecting extraction efficacy were investigated including syringe fill strokes, syringe pull up volume, pull up delay and volume in the sample vial. The system was optimized for soft extraction of analytes and high sample throughput. Further, it was demonstrated that by flushing the EME-syringe with acidic wash buffer and reverting the applied electric potential, carry-over between samples can be reduced to below 1%. Performance of the system was characterized (RSD, <10%; <i>R</i>², 0.994) and finally, the EME-autosampler was used to analyze <i>in vitro</i> conversion of methadone into its main metabolite by rat liver microsomes and for demonstrating the potential of known CYP3A4 inhibitors to prevent metabolism of methadone. By making use of the high extraction speed of EME, a complete analytical workflow of purification, separation, and analysis of sample could be achieved within only 5.5 min. With the developed system large sequences of samples could be analyzed in a completely automated manner. This high degree of automation makes the developed EME-autosampler a powerful tool for a wide range of applications where high-throughput extractions are required before sample analysis

Fully Automated Electro Membrane Extraction Autosampler for LC–MS Systems Allowing Soft Extractions for High-Throughput Applications

The current work describes the implementation of electro membrane extraction (EME) into an autosampler for high-throughput analysis of samples by EME-LC–MS. The extraction probe was built into a luer lock adapter connected to a HTC PAL autosampler syringe. As the autosampler drew sample solution, analytes were extracted into the lumen of the extraction probe and transferred to a LC–MS system for further analysis. Various parameters affecting extraction efficacy were investigated including syringe fill strokes, syringe pull up volume, pull up delay and volume in the sample vial. The system was optimized for soft extraction of analytes and high sample throughput. Further, it was demonstrated that by flushing the EME-syringe with acidic wash buffer and reverting the applied electric potential, carry-over between samples can be reduced to below 1%. Performance of the system was characterized (RSD, <10%; <i>R</i>², 0.994) and finally, the EME-autosampler was used to analyze <i>in vitro</i> conversion of methadone into its main metabolite by rat liver microsomes and for demonstrating the potential of known CYP3A4 inhibitors to prevent metabolism of methadone. By making use of the high extraction speed of EME, a complete analytical workflow of purification, separation, and analysis of sample could be achieved within only 5.5 min. With the developed system large sequences of samples could be analyzed in a completely automated manner. This high degree of automation makes the developed EME-autosampler a powerful tool for a wide range of applications where high-throughput extractions are required before sample analysis

Probing the Conformational and Functional Consequences of Disulfide Bond Engineering in Growth Hormone by Hydrogen–Deuterium Exchange Mass Spectrometry Coupled to Electron Transfer Dissociation

Human growth hormone (hGH), and its receptor interaction, is essential for cell growth. To stabilize a flexible loop between helices 3 and 4, while retaining affinity for the hGH receptor, we have engineered a new hGH variant (Q84C/Y143C). Here, we employ hydrogen–deuterium exchange mass spectrometry (HDX-MS) to map the impact of the new disulfide bond on the conformational dynamics of this new hGH variant. Compared to wild type hGH, the variant exhibits reduced loop dynamics, indicating a stabilizing effect of the introduced disulfide bond. Furthermore, the disulfide bond exhibits longer ranging effects, stabilizing a short α-helix quite distant from the mutation sites, but also rendering a part of the α-helical hGH core slightly more dynamic. In the regions where the hGH variant exhibits a different deuterium uptake than the wild type protein, electron transfer dissociation (ETD) fragmentation has been used to pinpoint the residues responsible for the observed differences (HDX-ETD). Finally, by use of surface plasmon resonance (SPR) measurements, we show that the new disulfide bond does not compromise receptor affinity. Our work highlight the analytical potential of HDX-ETD combined with functional assays to guide protein engineering

A Conformational Investigation of Propeptide Binding to the Integral Membrane Protein γ‑Glutamyl Carboxylase Using Nanodisc Hydrogen Exchange Mass Spectrometry

Gamma (γ)-glutamyl carboxylase (GGCX) is an integral membrane protein responsible for the post-translational catalytic conversion of select glutamic acid (Glu) residues to γ-carboxy glutamic acid (Gla) in vitamin K-dependent (VKD) proteins. Understanding the mechanism of carboxylation and the role of GGCX in the vitamin K cycle is of biological interest in the development of therapeutics for blood coagulation disorders. Historically, biophysical investigations and structural characterizations of GGCX have been limited due to complexities involving the availability of an appropriate model membrane system. In previous work, a hydrogen exchange mass spectrometry (HX MS) platform was developed to study the structural configuration of GGCX in a near-native nanodisc phospholipid environment. Here we have applied the nanodisc–HX MS approach to characterize specific domains of GGCX that exhibit structural rearrangements upon binding the high-affinity consensus propeptide (pCon; AVFLS­REQAN­QVLQ­RRRR). pCon binding was shown to be specific for monomeric GGCX-nanodiscs and promoted enhanced structural stability to the nanodisc-integrated complex while maintaining catalytic activity in the presence of carboxylation co-substrates. Noteworthy modifications in HX of GGCX were prominently observed in GGCX peptides 491–507 and 395–401 upon pCon association, consistent with regions previously identified as sites for propeptide and glutamate binding. Several additional protein regions exhibited minor gains in solvent protection upon propeptide incorporation, providing evidence for a structural reorientation of the GGCX complex in association with VKD carboxylation. The results herein demonstrate that nanodisc–HX MS can be utilized to study molecular interactions of membrane-bound enzymes in the absence of a complete three-dimensional structure and to map dynamic rearrangements induced upon ligand binding