13 research outputs found
Installation, validation, and application examples of two instrumental setups for gas-phase HDX-MS analysis of peptides and proteins
Probing the Binding Interfaces of Protein Complexes Using Gas-Phase H/D Exchange Mass Spectrometry
SummaryFast gas-phase hydrogen/deuterium exchange mediated by ND3 gas and measured by mass spectrometry (gas-phase HDX-MS) is a largely unharnessed, fast, and sensitive method for probing primary- and higher-order polypeptide structure. Labeling of heteroatom-bound non-amide hydrogens in a sub-millisecond time span after electrospray ionization by ND3 gas can provide structural insights into protein conformers present in solution. Here, we have explored the use of gas-phase HDX-MS for probing the higher-order structure and binding interfaces of protein complexes originating from native solution conditions. Lysozyme ions bound by an oligosaccharide incorporated less deuterium than the unbound ion. Similarly, trypsin ions showed reduced deuterium uptake when bound by the peptide ligand vasopressin. Our results are in good agreement with crystal structures of the native protein complexes, and illustrate that gas-phase HDX-MS can provide a sensitive and simple approach to measure the number of heteroatom-bound non-amide side-chain hydrogens involved in the binding interface of biologically relevant protein complexes
Side-chain moieties from the N-terminal region of Aβ are Involved in an oligomer-stabilizing network of interactions.
Oligomeric forms of the Aβ peptide represent the most probable neurotoxic agent in Alzheimer's disease. The dynamic and heterogeneous character of these oligomers makes their structural characterization by classic methods difficult. Native mass spectrometry, when supported by additional gas phase techniques, like ion mobility separation and hydrogen-deuterium exchange (IM-HDX-MS), enable analysis of different oligomers coexisting in the sample and may provide species-specific structural information for each oligomeric form populated in the gas phase. Here, we have combined these three techniques to obtain insight into the structural properties of oligomers of Aβ1-40 and two variants with scrambled sequences. Gas-phase HDX-MS revealed a sequence-specific engagement of the side-chains of residues located at the N-terminal part of the peptide in a network of oligomer-stabilizing interactions. Oligomer-specific interactions were no longer observed in the case of the fully scrambled sequence. Also, the ability to form alternative structures, observed for WT Aβ peptide, was lost upon scrambling. Our data underscore a role for the N-terminal residues in shaping the equilibria of oligomeric forms. Although the peptide lacking the N-terminal 1-16 residues (p3 peptide) is thought to be benign, the role of the N-terminus has not been sufficiently characterized yet. We speculate that the interaction networks revealed here may be crucial for enabling structural transitions necessary to obtain mature parallel cross-β structures from smaller antiparallel oligomers. We provide a hypothetical molecular model of the trajectory that allows a gradual conversion from antiparallel to parallel oligomers without decomposition of oligomers. Oligomer-defining interactions involving the Aβ peptide N-terminus may be important in production of the neurotoxic forms and thus should not be neglected
UV Photodissociation Mass Spectrometry Accurately Localize Sites of Backbone Deuteration in Peptides
Hydrogen/deuterium
exchange mass spectrometry (HDX-MS) is now a
routinely used technique to inform on protein structure, dynamics,
and interactions. Localizing the incorporated deuterium content on
a single residue basis increases the spatial resolution of this technique
enabling detailed structural analysis. Here, we investigate the use
of ultraviolet photodissociation (UVPD) at 213 nm to measure deuterium
levels at single residue resolution in HDX-MS experiments. Using a
selectively labeled peptide, we show that UVPD occurs without H/D
scrambling as the peptide probe accurately retains its solution-phase
deuterium labeling pattern. Our results indicate that UVPD provides
an attractive alternative to electron mediated dissociation for increasing
the spatial resolution of the HDX-MS experiment, capable of yielding
high fragmentation efficiency, high fragment ion diversity, and low
precursor ion charge-state dependency
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 ND<sub>3</sub>-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 N<sub>2</sub>-gas through a container filled with aqueous
deuterated ammonia reagent (ND<sub>3</sub>/D<sub>2</sub>O) 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 ND<sub>3</sub>/D<sub>2</sub>O as HDX reagent
indicate that labeling is facilitated exclusively through gaseous
ND<sub>3</sub>, yielding similar results to the infusion of purified
ND<sub>3</sub>-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
Probing the Dissociation of Protein Complexes by Means of Gas-Phase H/D Exchange Mass Spectrometry
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
Investigating the utility of minimized sample preparation and high-resolution mass spectrometry for quantification of monoclonal antibody drugs
International audienceDetermination of the pharmacokinetic (PK) properties of therapeutic monoclonal antibodies (mAbs) is essential for their successful development as drugs. For this purpose, besides the traditional ligand binding assay (LBA), LC–MS/MS method using low resolution mass spectrometers (e.g. triple quadrupole (QqQ)) has become routinely used, however, complicated and lengthy sample pre-treatment (employing immuno-affinity) is often necessary for obtaining sufficient sensitivity and selectivity. In this study, we investigate the capabilities of high-resolution MS instruments for circumventing the complex sample preparation currently needed for sensitive LC–MS/MS-based quantification of mAbs. Employing a simple one-step sample pre-treatment workflow, we compare the ability of three different LC–MS platforms for absolute quantification of a representative monoclonal antibody Rendomab-B1 in serum and plasma. The samples are subjected to protein precipitation with methanol, followed by pellet digestion with trypsin prior to LC–MS analysis. AQUA peptides based on two surrogate mAb peptides selected from an extensive in-silico and experimental screening are used as internal standards. MS/MS acquisitions are developed and systematically examined for 1) a low-resolution QqQ operated in selected reaction monitoring (SRM) acquisition mode, 2) a high-resolution hybrid Quadrupole-Orbitrap (Q-Orbitrap) operated in parallel reaction monitoring (PRM) acquisition mode and 3) a high-resolution hybrid Quadrupole-Time-of-flight (Q-TOF) operated in SRM acquisition mode with enhanced duty cycle (EDC) function. The sensitivity of the high-resolution Q-Orbitrap and Q-TOF methods was significantly higher (LOD of 80 ng/mL) in serum/plasma samples than the low-resolution QqQ method. Finally, the real-world utility of the developed high-resolution MS method with minimized sample handling was demonstrated and validated by determining the PK profile of Rendomab-B1 in mice by a 10-point in vivo study over 15 days
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 MON<sup>2+</sup>, DIM<sup>4+</sup>, TRI<sup>6+</sup> 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