A goldilocks computational protocol for inhibitor discovery targeting DNA damage responses including replication-repair functions DOI Creative Commons
Davide Moiani, John A. Tainer

Frontiers in Molecular Biosciences, Journal Year: 2024, Volume and Issue: 11

Published: Nov. 28, 2024

While many researchers can design knockdown and knockout methodologies to remove a gene product, this is mainly untrue for new chemical inhibitor designs that empower multifunctional DNA Damage Response (DDR) networks. Here, we present robust Goldilocks (GL) computational discovery protocol efficiently innovate tools preclinical drug candidates cellular structural biologists without requiring extensive virtual screen (VS) synthesis expertise. By computationally targeting DDR replication repair proteins, exemplify the identification of target sites compounds probe cancer biology. Our GL pipeline integrates experimental predicted structures discover leads, allowing early-structure early-testing (ESET) experiments by laboratories. employing an efficient VS examine protein-protein interfaces (PPIs) allosteric interactions, identify ligand binding beyond active sites, leveraging

Language: Английский

Hydrogen/Deuterium Exchange Mass Spectrometry: Fundamentals, Limitations, and Opportunities DOI Creative Commons
Lars Konermann, Pablo M. Scrosati

Molecular & Cellular Proteomics, Journal Year: 2024, Volume and Issue: 23(11), P. 100853 - 100853

Published: Oct. 9, 2024

Highlights•Bottom-up HDX-MS yields qualitative insights into protein dynamics.•Amide back exchange is an under-appreciated problem.•The (m–m0)/(m100–m0) correction can produce large errors.•Future analysis strategies will have to go beyond the Linderstrøm-Lang model.•Enhanced workflows and computational modeling might soon be able generate atomically resolved motions.Graphical abstract

Language: Английский

Citations

6
Neurofilament Light Chain under the Lens of Structural Mass Spectrometry DOI Creative Commons
Salomé Coppens, Dea Gogishvili,

Valentina Faustinelli

et al.

ACS Chemical Neuroscience, Journal Year: 2025, Volume and Issue: 16(2), P. 141 - 151

Published: Jan. 2, 2025

Neurofilament light chain (NfL) is an early nonspecific biomarker in neurodegenerative diseases and traumatic brain injury, indicating axonal damage. This work describes the detailed structural characterization of a selected primary calibrator with potential to be used future reference measurement procedure (RMP) development for accurate quantification NfL. As part described workflow, sequence, higher-order structure as well solvent accessibility, hydrogen-bonding profile were assessed under three different conditions KPBS, artificial cerebrospinal fluid, fluid presence human serum albumin. The results revealed that NfL structurally heterogeneous protein, eliciting large conformational flexibility. Its ensemble changed when it was diluted aqueous buffer versus surrogate matrix, (aCSF), and/or aCSF Various regions protection deprotection protein head, central helical, tail domains experienced altered accessibility changes caused by identified. Moreover, interfacial residues, which may play role direct interaction between albumin, emerged from hydrogen–deuterium exchange mass spectrometry (HDX-MS). These data pinpointed distinct participate such interaction. Overall, critical quality attributes measurements are provided. findings will ultimately inform ongoing biochemical clinical assay procedures manufacturing practices, giving careful consideration during sample handling method development.

Language: Английский

Citations

0
Recent Advances in Mass Spectrometry-Based Bottom-Up Proteomics DOI
Cameron S. Movassaghi, Jie Sun, Yuming Jiang

et al.

Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 25, 2025

Mass spectrometry-based proteomics is about 35 years old, and recent progress appears to be speeding up across all subfields. In this review, we focus on advances over the last two in select areas within bottom-up proteomics, including approaches high-throughput experiments, data analysis using machine learning, drug discovery, glycoproteomics, extracellular vesicle structural proteomics.

Language: Английский

Citations

0
HRaDeX: R Package and Web Server for Computing High-Resolution Deuterium Uptake Rates for HDX–MS Data DOI Creative Commons
Weronika Puchała, Michał Kistowski,

Liliya Zhukova

et al.

Journal of Proteome Research, Journal Year: 2025, Volume and Issue: unknown

Published: March 19, 2025

Hydrogen–deuterium exchange monitored by mass spectrometry (HDX–MS) is a well-established and powerful technique used to study protein dynamics stability capturing local global unfolding events in structures. However, this technique, obtaining region-specific information requires proteolytic digestion that breaks the into peptide fragments, causing HDX data reflect averages over these fragments rather than individual amino acids. We propose new computational method provides deuterium uptake kinetic parameters with high resolution, considering trajectories of superimposed peptides. Our algorithm, HRaDeX, available as web server an R package capable processing from single-state comparative HDX–MS studies. Utilizing eight benchmark sets, we demonstrate HRaDeX reaches average root-mean-square error 7.15% reconstitution experimental normalized curves.

Language: Английский

Citations

0
Examining Instrumental Factors Influencing the Performance of Data-Independent Acquisition Methods in Hydrogen/Deuterium Exchange Mass Spectrometry DOI
František Filandr, Morgan Hepburn,

Vladimir Sarpe

et al.

Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: March 31, 2025

Hydrogen/deuterium exchange mass spectrometry (HX-MS) is a method used to study solution-phase protein structure and dynamics. Despite its many applications, HX-MS limited in throughput because manual data analysis still the norm. We previously developed HX-MS2 technology add second dimension of deuteration promote automated processing. Data-independent acquisition (DIA) techniques enable this approach, but we require optimized methods for best performance. Using an Orbitrap Eclipse illustration, show that ion optics collision energy settings typical proteomics DIA experiment generate maximal peptide retrieval from library. As few as three MS2 sequence ions are sufficient measurement with precision exceeds what possible traditional HX-MS. window sizes based on chromatographic resolution method. An inter-scan offset recommended default configuration most HX-DIA applications butan intra-scan overlap can be tuned highest performance when maximum desired. demonstrate robustness one (consisting Trajan HDX automation technology, spectrometer AutoHX software) extensive time-course phosphorylase B epitope single-domain antibodies (VHHs, nanobodies) specific receptor binding domain SARS-CoV-2 spike protein.

Language: Английский

Citations

0
Efficient, Zero Scrambling Fragmentation of Deuterium Labeled Peptides on the ZenoToF 7600 Electron Activated Dissociation Platform DOI

Joseph F. Anacleto,

Ebadullah Kabir,

M. Mar Blanco

et al.

Journal of the American Society for Mass Spectrometry, Journal Year: 2025, Volume and Issue: unknown

Published: April 15, 2025

Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) has become an increasingly important tool in protein research, with large-scale applications biopharmaceutical development and manufacturing. One of the limitations classical bottom-up HDX is that it usually provides a "peptide-averaged" picture structure dynamics, rather than site-specific (i.e., individual amino acid-level) information. A major challenge for HDX-MS analyses been fragmentation techniques such as CAD invariably cause random redistribution deuterium labels across peptide backbone, known scrambling. Several groups have demonstrated this problem can be overcome using nonergodic "cool" ion flight conditions. hurdle to widespread adoption approach, however, exceedingly low efficiency electron capture dissociation (ECD) combined lower transmission conditions impose very strong attenuation on sensitivity, point where method becomes impractical many "real-world" applications. Here, we introduce workflow instrument Sciex 7600 ZenoToF activated (EAD) platform allow zero scrambling ECD limited (and some cases no) sensitivity loss. We expect will ideal broadly applicable, middle-down workflow.

Language: Английский

Citations

0
Computational Tools for Hydrogen–Deuterium Exchange Mass Spectrometry Data Analysis DOI Creative Commons
Michele Stofella, Anna Grimaldi, Jochem H. Smit

et al.

Chemical Reviews, Journal Year: 2024, Volume and Issue: 124(21), P. 12242 - 12263

Published: Oct. 31, 2024

Hydrogen–deuterium exchange (HDX) has become a pivotal method for investigating the structural and dynamic properties of proteins. The versatility sensitivity mass spectrometry (MS) made technique ideal companion HDX, today HDX-MS is addressing growing number applications in both academic research industrial settings. prolific generation experimental data spurred concurrent development numerous computational tools, designed to automate parts workflow while employing different strategies achieve common objectives. Various methods are available perform automated peptide searches identification; statistical tests have been implemented quantify differences pattern between two or more conditions; alternative developed deconvolve analyze peptides showing multimodal behavior; algorithms proposed computationally increase resolution data, with ultimate aim provide information at level single residue. This review delves into comprehensive examination merits drawbacks associated diverse by software tools analysis data.

Language: Английский

Citations

2
A goldilocks computational protocol for inhibitor discovery targeting DNA damage responses including replication-repair functions DOI Creative Commons
Davide Moiani, John A. Tainer

Frontiers in Molecular Biosciences, Journal Year: 2024, Volume and Issue: 11

Published: Nov. 28, 2024

While many researchers can design knockdown and knockout methodologies to remove a gene product, this is mainly untrue for new chemical inhibitor designs that empower multifunctional DNA Damage Response (DDR) networks. Here, we present robust Goldilocks (GL) computational discovery protocol efficiently innovate tools preclinical drug candidates cellular structural biologists without requiring extensive virtual screen (VS) synthesis expertise. By computationally targeting DDR replication repair proteins, exemplify the identification of target sites compounds probe cancer biology. Our GL pipeline integrates experimental predicted structures discover leads, allowing early-structure early-testing (ESET) experiments by laboratories. employing an efficient VS examine protein-protein interfaces (PPIs) allosteric interactions, identify ligand binding beyond active sites, leveraging

Language: Английский

Citations

0