Publications by authors named "Jamie P Butalewicz"

Ultraviolet photodissociation (UVPD) has been shown to be a versatile ion activation strategy for the characterization of peptides and intact proteins among other classes of biological molecules. Combining the high-performance mass spectrometry (MS/MS) capabilities of UVPD with the high-resolution separation of trapped ion mobility spectrometry (TIMS) presents an opportunity for enhanced structural elucidation of biological molecules. In the present work, we integrate a 193 nm excimer laser in a TIMS-time-of-flight (TIMS-TOF) mass spectrometer for UVPD in the collision cell and use it for the analysis of several mass-mobility-selected species of ubiquitin and myoglobin.

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Article Synopsis
  • The main protease (M) of SARS-CoV-2 is crucial for the virus's maturation and is the target of the COVID-19 treatment Paxlovid, but there's a pressing need to find new inhibitors due to potential viral resistance.
  • The study utilized advanced techniques like native mass spectrometry and UV photodissociation to analyze the structure of M and how it interacts with potential covalent inhibitors.
  • Results indicated that certain inhibitors enhance the stability of M by creating dimeric complexes with higher melting temperatures and lower charge states, providing valuable insights into how these inhibitors work.
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Here we used native mass spectrometry (native MS) to probe a SARS-CoV protease, PLpro, which plays critical roles in coronavirus disease by affecting viral protein production and antagonizing host antiviral responses. Ultraviolet photodissociation (UVPD) and variable temperature electrospray ionization (vT ESI) were used to localize binding sites of PLpro inhibitors and revealed the stabilizing effects of inhibitors on protein tertiary structure. We compared PLpro from SARS-CoV-1 and SARS-CoV-2 in terms of inhibitor and ISG15 interactions to discern possible differences in protease function.

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Owing to its ability to generate extensive fragmentation of proteins, ultraviolet photodissociation (UVPD) mass spectrometry (MS) has emerged as a versatile ion activation technique for the structural characterization of native proteins and protein complexes. Interpreting these fragmentation patterns provides insight into the secondary and tertiary structures of protein ions. However, the inherent complexity and diversity of proteins often pose challenges in resolving their numerous conformations.

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Article Synopsis
  • RNA polymerase II (Pol II) undergoes important post-translational modifications on its C-terminal domain (CTD), which help regulate transcription by attracting different proteins at various stages of the transcription process.
  • *The phosphorylation of specific serine residues (Ser5 and Ser2) on the CTD is crucial, as it occurs in relation to the transcription phases and influences which transcriptional regulators bind to Pol II.
  • *The study identified calcium homeostasis endoplasmic reticulum protein (CHERP) as a significant regulatory protein that associates with the phosphorylated CTD, impacting alternative splicing when disrupted.
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Coupling 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.

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The 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.

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Article Synopsis
  • The discovery of asymmetric trimers within the tautomerase superfamily introduces new structural diversity and enhances the understanding of this complex protein group.
  • By using native mass spectrometry and ultraviolet photodissociation, researchers can quickly and sensitively distinguish between symmetric and asymmetric trimers based on their structural behavior under certain conditions.
  • The findings reveal that asymmetric trimers are more stable and show unique unfolding pathways compared to symmetric trimers, which may have implications for the evolutionary development and classification of the tautomerase superfamily.
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The 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 (C═C) 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.

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Article Synopsis
  • Fourier transform multiplexing enhances drift tube ion mobility coupling to mass spectrometers, leading to better ion utilization and duty cycles compared to older methods.
  • Presenting data in absorption mode rather than magnitude mode, along with phase correction, significantly improves resolution and signal-to-noise ratio (SNR) without changing hardware or procedures.
  • Techniques like apodization and zero padding further reduce acquisition times by up to 80%, while boosting resolution by 1.6 times and SNR by 1.2 times overall.
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The phosphorylation states of RNA polymerase II coordinate the process of eukaryotic transcription by recruitment of transcription regulators. The individual residues of the repetitive heptad of the C-terminal domain (CTD) of the biggest subunit of RNA polymerase II are phosphorylated temporally at different stages of transcription. Intriguingly, despite similar flanking residues, phosphorylation of Ser2 and Ser5 in CTD heptads play dramatically different roles.

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