8 research outputs found
Recommended from our members
Advancements in characterizing protein structures and dynamics through innovative mass spectrometry instrumentation
No full textUnderstanding the structural heterogeneity of proteins and protein complexes in the gas phase remains a challenge even with the advanced stage of tandem mass spectrometry, ion mobility, and computational programs. Modification to common commercial instrumentation as well as custom lab-built instruments provides an avenue for specialized analysis of these complex systems that could not be achieved otherwise. Through the modification of commercial mass spectrometers, a 193nm laser can be installed to generate numerous protein fragments that can be used to better investigate protein sequence, secondary and tertiary structure, as well as interactions. To investigate the size and shape of proteins, ion mobility mass spectrometry is typically employed. A custom atmospheric pressure ion mobility drift tube was optimized to interface with a commercial high resolution Orbitrap mass spectrometer for enhanced protein conformational analysis. In addition, a low pressure one-meter-long drift tube was built to improve the ion transmission and sensitivity for protein conformational analysis. Beyond the factors of protein shape, size, structure, and conformation, the mechanism of protein unfolding and dissociation is also of great interest. A variable temperature electrospray ionization source that interfaces directly to a commercial mass spectrometer was built. This source allows the investigation of the unfolding and dissociation mechanisms of protein complexes and measurement of thermodynamic properties such as entropy and enthalpy. The instrumentation developed, built, or modified in-house has enhanced the characterization of proteins.Chemistr
Improving Ion Mobility Mass Spectrometry of Proteins through Tristate Gating and Optimization of Multiplexing Parameters
No full textCoupling drift tube
ion mobility (IM) to Fourier transform mass
spectrometry (FT-MS) affords the opportunity for gas-phase separation
of ions based on size and conformation with high-resolution mass analysis.
However, combining IM and FT-MS is challenging because ions exit the
drift tube on a much faster time scale than the rate of mass analysis.
Fourier transform (FT) and Hadamard transform multiplexing methods
have been implemented to overcome the duty-cycle mismatch, offering
new avenues for obtaining high-resolution, high-mass-accuracy analysis
of mobility-selected ions. The gating methods used to integrate the
drift tube with the FT mass analyzer discriminate against the transmission
of large, low-mobility ions owing to the well-known gate depletion
effect. Tristate gating strategies have been shown to increase ion
transmission for drift tube IM-FT-MS systems through implementation
of dual ion gating, controlling the quantity and timing of ions through
the drift tube to reduce losses of slow-moving ions. Here we present
an optimized set of multiplexing parameters for tristate gating ion
mobility of several proteins on an Orbitrap mass spectrometer and
further report parameters for increased ion transmission and mobility
resolution as well as decreased experimental times from 15 min down
to 30 s. On average, peak intensities in the arrival time distributions
(ATDs) for ubiquitin increased 2.1× on average, while those of
myoglobin increased by 1.5× with a resolving power increase on
average of 11%
Insights into the Main Protease of SARS-CoV-2: Thermodynamic Analysis, Structural Characterization, and the Impact of Inhibitors
No full textThe main protease of SARS-CoV-2 (Mpro) is an essential enzyme for coronaviral maturation and is the target of Paxlovid, which is currently the standard-of-care treatment for COVID-19. There remains a need to identify new inhibitors of Mpro as viral resistance to Paxlovid emerges. Here, we report the use of native mass spectrometry coupled with 193-nm ultraviolet photodissociation (UVPD) to structurally characterize Mpro and its interactions with potential covalent inhibitors. Melting temperatures and the overall energy landscape were obtained using variable temperature nano-electrospray ionization (vT-nESI), thus providing quantitative evaluation of inhibitor binding on the stability of Mpro. The melting temperature was determined to be approximately 30°C for the dimer and 36°C for the monomer, suggesting an initial thermal dissociation pathway before subsequent unfolding of the monomer species. Thermodynamic parameters extracted from Van’t Hoff plots revealed that the dimeric complexes containing each inhibitor showed enhanced stability through increased melting temperatures as well as overall lower average charge states, giving insight into the basis for potential inhibition mechanisms
Symmetry of 4‑Oxalocrotonate Tautomerase Trimers Influences Unfolding and Fragmentation in the Gas Phase
No full textThe
recent discovery of asymmetric arrangements of trimers in the
tautomerase superfamily (TSF) adds structural diversity to this already
mechanistically diverse superfamily. Classification of asymmetric
trimers has previously been determined using X-ray crystallography.
Here, native mass spectrometry (MS) and ultraviolet photodissociation
(UVPD) are employed as an integrated strategy for more rapid and sensitive
differentiation of symmetric and asymmetric trimers. Specifically,
the unfolding of symmetric and asymmetric trimers initiated by collisional
heating was probed using UVPD, which revealed unique gas-phase unfolding
pathways. Variations in UVPD patterns from native-like, compact trimeric
structures to unfolded, extended conformations indicate a rearrangement
of higher-order structure in the asymmetric trimers that are believed
to be stabilized by salt-bridge triads, which are absent from the
symmetric trimers. Consequently, the symmetric trimers were found
to be less stable in the gas phase, resulting in enhanced UVPD fragmentation
overall and a notable difference in higher-order re-structuring based
on the extent of hydrogen migration of protein fragments. The increased
stability of the asymmetric trimers may justify their evolution and
concomitant diversification of the TSF. Facilitating the classification
of TSF members as symmetric or asymmetric trimers assists in delineating
the evolutionary history of the TSF
Distinctive interactomes of RNA polymerase II phosphorylation during different stages of transcription
No full textSummary: During eukaryotic transcription, RNA polymerase II undergoes dynamic post-translational modifications on the C-terminal domain (CTD) of the largest subunit, generating an information-rich PTM landscape that transcriptional regulators bind. The phosphorylation of Ser5 and Ser2 of CTD heptad occurs spatiotemporally with the transcriptional stages, recruiting different transcriptional regulators to Pol II. To delineate the protein interactomes at different transcriptional stages, we reconstructed phosphorylation patterns of the CTD at Ser5 and Ser2 in vitro. Our results showed that distinct protein interactomes are recruited to RNA polymerase II at different stages of transcription by the phosphorylation of Ser2 and Ser5 of the CTD heptads. In particular, we characterized calcium homeostasis endoplasmic reticulum protein (CHERP) as a regulator bound by phospho-Ser2 heptad. Pol II association with CHERP recruits an accessory splicing complex whose loss results in broad changes in alternative splicing events. Our results shed light on the PTM-coded recruitment process that coordinates transcription
Enhanced Ion Mobility Separation and Characterization of Isomeric Phosphatidylcholines Using Absorption Mode Fourier Transform Multiplexing and Ultraviolet Photodissociation Mass Spectrometry
No full textThe structural diversity of phospholipids
plays a critical role
in cellular membrane dynamics, energy storage, and cellular signaling.
Despite its importance, the extent of this diversity has only recently
come into focus, largely owing to advances in separation science and
mass spectrometry methodology and instrumentation. Characterization
of glycerophospholipid (GP) isomers differing only in their acyl chain
configurations and locations of carbon–carbon double bonds
(CC) remains challenging due to the need for both effective
separation of isomers and advanced tandem mass spectrometry (MS/MS)
technologies capable of double-bond localization. Drift tube ion mobility
spectrometry (DTIMS) coupled with MS can provide both fast separation
and accurate determination of collision cross section (CCS) of molecules
but typically lacks the resolving power needed to separate phospholipid
isomers. Ultraviolet photodissociation (UVPD) can provide unambiguous
double-bond localization but is challenging to implement on the timescales
of modern commercial drift tube time-of-flight mass spectrometers.
Here, we present a novel method for coupling DTIMS with a UVPD-enabled
Orbitrap mass spectrometer using absorption mode Fourier transform
multiplexing that affords simultaneous localization of double bonds
and accurate CCS measurements even when isomers cannot be fully resolved
in the mobility dimension. This method is demonstrated on two- and
three-component mixtures and shown to provide CCS measurements that
differ from those obtained by individual analysis of each component
by less than 1%
Nanohydrophobic Interaction Chromatography Coupled to Ultraviolet Photodissociation Mass Spectrometry for the Analysis of Intact Proteins in Low Charge States
No full textThe direct correlation between proteoforms
and biological phenotype necessitates the exploration of mass spectrometry
(MS)-based methods more suitable for proteoform detection and characterization.
Here, we couple nano-hydrophobic interaction chromatography (nano-HIC)
to ultraviolet photodissociation MS (UVPD-MS) for separation and characterization
of intact proteins and proteoforms. High linearity, sensitivity, and
sequence coverage are obtained with this method for a variety of proteins.
Investigation of collisional cross sections of intact proteins during
nano-HIC indicates semifolded conformations in low charge states,
enabling a different dimension of separation in comparison to traditional,
fully denaturing reversed-phase separations. This method is demonstrated
for a mixture of intact proteins from Escherichia coli ribosomes; high sequence coverage is obtained for a variety of modified
and unmodified proteoforms
