Development and evaluation of data analysis approaches to increase the specificity and performance of thermal shift assays for assessment of protein-small molecule interactions DOI Open Access

Amanda M. Figueroa‐Navedo

Published: Jan. 1, 2023

Thermal proteome profiling effectively investigates protein-protein, protein-nucleic acid, or protein-drug interactions, and the impact of metabolite binding post-translational modifications on these interactions. The experiments quantitatively characterize biological samples treated with small molecules versus controls subjected to timed exposures multiple temperatures. Typically, each enzymatically digested sample is labeled a tandem mass tag (TMT), where TMT channel corresponds specific temperature treatment profiled using liquid chromatography coupled spectrometry in data-dependent data acquisition mode. resulting spectra are processed computational tools identify quantify proteins filter out noise. Protein interactions detected by fitting curves protein abundances across Interacting identified shifts fitted between controls.This dissertation emphasizes importance processing curve thermal profiling. We demonstrate that employing different techniques for steps can yield substantially results. review existing methods advocate use open-source R package MSstatsTMT. MSstatsTMT-based minimizes subjective filtering. Its statistical appropriately represent structure variation do not require fitting, making results numerically stable. MSstatsTMT supports experimental designs trade off temperatures larger number replicates, thus increasing sensitivity increased stability as compared currently used alternatives series simulated datasets. These include comparison conventional its OnePot counterpart pools at into one sample. workflow documented publicly available vignettes.--Author's abstract

Language: Английский

Current Advances in CETSA DOI Creative Commons
Tuomas Tolvanen

Frontiers in Molecular Biosciences, Journal Year: 2022, Volume and Issue: 9

Published: June 9, 2022

Knowing that the drug candidate binds to its intended target is a vital part of discovery. Thus, several labeled and label-free methods have been developed study engagement. In recent years, cellular thermal shift assay (CETSA) with variations has widely adapted discovery workflows. Western blot-based CETSA used primarily validate binding molecule protein whereas based on bead chemistry detection (CETSA HT) screen molecular libraries find novel molecules pre-determined target. Mass spectrometry-based also known as proteome profiling (TPP) emerged powerful tool for deconvolution finding partners old molecules. With this technology, it possible probe shifts among over 7,000 proteins from one sample identify wanted but unwanted off-targets cause adverse effects. addition, proteome-wide method can provide information biological process initiated by ligand binding. The continued development mass spectrometry labeling reagents, such isobaric tandem tag technology (TMT) continues increase throughput MS, allowing use structure-activity relationship (SAR) studies limited number review, we discussed differences between different engagement, our focus was advances in method.

Language: Английский

Citations

51
Small-molecule probes from bench to bedside: advancing molecular analysis of drug–target interactions toward precision medicine DOI
Sijun Pan, Aixiang Ding, Yisi Li

et al.

Chemical Society Reviews, Journal Year: 2023, Volume and Issue: 52(16), P. 5706 - 5743

Published: Jan. 1, 2023

Integrated development of designer chemical tools and complementary analytical technologies empowers new scientific clinical opportunities.

Language: Английский

Citations

31
Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling DOI Creative Commons
Amy L. George, Frances R. Sidgwick, Jessica E. Watt

et al.

Journal of Proteome Research, Journal Year: 2023, Volume and Issue: 22(8), P. 2629 - 2640

Published: July 13, 2023

Thermal proteome profiling (TPP) provides a powerful approach to studying proteome-wide interactions of small therapeutic molecules and their target off-target proteins, complementing phenotypic-based drug screens. Detecting differences in thermal stability due engagement requires high quantitative accuracy consistent detection. Isobaric tandem mass tags (TMTs) are used multiplex samples increase quantification precision TPP analysis by data-dependent acquisition (DDA). However, advances data-independent (DIA) can provide higher sensitivity protein coverage with reduced costs sample preparation steps. Herein, we explored the performance different DIA-based label-free approaches compared TMT-DDA for shift quantitation. Acute myeloid leukemia cells were treated losmapimod, known inhibitor MAPK14 (p38α). Label-free DIA approaches, particularly library-free mode DIA-NN, comparable ability detect losmapimod one its downstream targets, MAPKAPK3. Using quantitation is cost-effective alternative labeled pipeline.

Language: Английский

Citations

26
Relative quantification of proteins and post-translational modifications in proteomic experiments with shared peptides: a weight-based approach DOI Creative Commons
Mateusz Staniak, Ting Huang,

Amanda M. Figueroa‐Navedo

et al.

Bioinformatics, Journal Year: 2025, Volume and Issue: 41(3)

Published: Jan. 31, 2025

Bottom-up mass spectrometry-based proteomics studies changes in protein abundance and structure across conditions. Since the currency of these experiments are peptides, i.e. subsets sequences that carry quantitative information, conclusions at a different level must be computationally inferred. The inference is particularly challenging situations where peptides shared by multiple proteins or post-translational modifications. While many approaches infer underlying abundances from unique there need to distinguish patterns when shared. We propose statistical approach for estimating abundances, as well site occupancies modifications, based on information peptides. treats convex combinations individual modification sites, estimates each source sample together with weights combination. In simulation-based evaluations, proposed improved precision estimated fold between further demonstrated practical utility diverse biological objectives, ranging degradation thermal proteome stability, implemented an open-source R package MSstatsWeightedSummary. currently available https://github.com/Vitek-Lab/MSstatsWeightedSummary (doi: 10.5281/zenodo.14662989). Code required reproduce results presented this article can found repository https://github.com/mstaniak/MWS_reproduction 10.5281/zenodo.14656053).

Language: Английский

Citations

0
Comparative Analysis of Protein Folding Stability-Based Profiling Methods for Characterization of Biological Phenotypes DOI

Morgan A. Bailey,

Yun Tang, Hye-Jin Park

et al.

Journal of the American Society for Mass Spectrometry, Journal Year: 2023, Volume and Issue: 34(3), P. 383 - 393

Published: Feb. 20, 2023

Recently, a new suite of mass spectrometry-based proteomic methods has been developed that enables evaluation protein folding stability on the scale. These utilize chemical and thermal denaturation approaches (SPROX TPP, respectively) as well proteolysis strategies (DARTS, LiP, PP) to assess stability. The analytical capabilities these technique have well-established for target discovery applications. However, less is known about relative advantages disadvantages using different characterize biological phenotypes. Reported here comparative study SPROX, conventional expression level measurements both mouse model aging mammalian cell culture breast cancer. Analyses proteins in brain tissue lysates derived from 1- 18-month-old mice (n = 4–5 at each time point) MCF-7 MCF-10A lines revealed majority differentially stabilized hits phenotype analysis had unchanged levels. In analyses, TPP generated largest number fraction hits. Only quarter all identified differential was detected multiple techniques. This work also reports first peptide-level data, which required correct interpretation analyses performed here. Studies selected uncovered phenotype-related functional changes.

Language: Английский

Citations

8
Toward the analysis of functional proteoforms using mass spectrometry-based stability proteomics DOI Creative Commons

Ji Hye Kang,

Meena Seshadri,

Kellye A. Cupp‐Sutton

et al.

Frontiers in Analytical Science, Journal Year: 2023, Volume and Issue: 3

Published: June 22, 2023

Functional proteomics aims to elucidate biological functions, mechanisms, and pathways of proteins proteoforms at the molecular level examine complex cellular systems disease states. A series stability methods have been developed protein functionality by measuring resistance a chemical or thermal denaturation proteolysis. These can be applied measure thousands in samples such as cell lysate, intact cells, tissues, other fluids proteome stability. Stability popularly observe shifts upon ligand binding for drug target identification. More recently, these characterize effect structural changes those caused post-translational modifications (PTMs) mutations, which affect structures interactions diversify functions. Here, we discussed current application suite methods, including profiling (TPP), from rates oxidation (SPROX), limited proteolysis (LiP) PTM-induced on We also discuss future perspectives highlighting integration top-down mass spectrometry proteoform understand function variable modifications.

Language: Английский

Citations

7
Label-free target protein characterization for small molecule drugs: recent advances in methods and applications DOI

Fei Feng,

Weiyue Zhang, Yifeng Chai

et al.

Journal of Pharmaceutical and Biomedical Analysis, Journal Year: 2022, Volume and Issue: 223, P. 115107 - 115107

Published: Oct. 19, 2022

Language: Английский

Citations

12
Experimental and data analysis advances in thermal proteome profiling DOI Creative Commons

Amanda M. Figueroa‐Navedo,

Alexander R. Ivanov

Cell Reports Methods, Journal Year: 2024, Volume and Issue: 4(2), P. 100717 - 100717

Published: Feb. 1, 2024

Method development for mass spectrometry (MS)-based thermal shift proteomic assays have advanced to probe small molecules with known and unknown protein-ligand interaction mechanisms specificity, which is predominantly used in characterization of drug-protein interactions. In the discovery target off-target interactions, a thorough investigation method their impact on sensitivity accuracy protein-small molecule protein-protein interactions warranted. this review, we discuss areas improvement at each stage proteome profiling data analysis that includes processing MS-based data, development, effect overall quality profiles. We also overview optimization experimental strategies prioritization an increased number independent biological replicates over evaluated temperatures.

Language: Английский

Citations

2
Chemical Biology Approaches Confirm MCT4 as the Therapeutic Target of a Cellular Optimized Hit DOI
Aarti Kawatkar, Roger Clark, Lorna Hopcroft

et al.

ACS Chemical Biology, Journal Year: 2023, Volume and Issue: 18(2), P. 296 - 303

Published: Jan. 5, 2023

Lactic acid transport is a key process maintaining glycolytic flux in tumors. Inhibition of this will result shutdown, impacting on cell growth and survival thus has been pursued as therapeutic approach for cancers. Using cell-based screen MCT4-dependent line, we identified optimized compounds their ability to inhibit the efflux intracellular lactic with good physical pharmacokinetic properties. To deconvolute mechanism inhibition, have developed three assays measure cellular target engagement. Specifically, synthesized biologically active photoaffinity probe (IC50 < 10 nM), using probe, demonstrated selective engagement MCT4 our parent molecule through combination confocal microscopy in-cell chemoproteomics. As an orthogonal assay, thermal shift assay (CETSA) confirmed binding system. Comparisons potencies cells differential expression MCT family members further that inhibition MCT4. Taken together, these data demonstrate power chemical biology methods determine engagement, particularly proteins not readily amenable traditional biophysical methods.

Language: Английский

Citations

6
Profiling protein targets of cellular toxicant exposure DOI
Joseph C. Genereux

Molecular Omics, Journal Year: 2023, Volume and Issue: 19(3), P. 191 - 204

Published: Jan. 1, 2023

Protein profiling technologies enable the identification of proteins that are damaged by exposure to environmental toxins.

Language: Английский

Citations

6