Development of improved drug-target profiling methods using mass spectrometry-based thermal stability assays DOI Open Access
Clifford G. Phaneuf

Опубликована: Янв. 1, 2023

Most drug candidates in the development pipeline fail during Phase II clinical trials because of inefficacy or unacceptable safety profiles. The costly financial burden failed late-stage may be transferred to few drugs that successfully navigate approval process. efficient discovery and therapeutic require deep profiling interactions between their biological targets for gaining a critical understanding mechanisms while limiting unwanted binding other (off-target) proteins, which can lead adverse reactions toxic effects. Thermal proteome (TPP) mass spectrometry-based cellular thermal shift assay (CETSA MS) are adaptations CETSA technique, exploit phenomenon ligand-induced stabilization proteins. Collectively referred as stability assays (MS-TSA), they have refined process target profiling. While rapidly evolving MS-TSA techniques provided remarkable insights into drug-protein interactome, challenges remain, particularly obtaining melting curves from low-abundance poorly soluble objective this dissertation is innovate conventional approaches with novel combinations MS-enabling technologies enhanced performance. We specifically focused on improving characterization profiles proteins low abundance soluble. Chapter 1 presents historical overview tools preceded current drug-target techniques, provides context insight about modern setting. A thorough review discussing evolution impact pharmaceutical research also included 1. Chapters 2 3 assess integration tequnique. In 2, we introduced improved MS-based acquisition (iMAATSA), combine several technologies. combination demonstrated synergistic effect performance by increased protein identifications high-quality using model system. Aspects iMAATSA were further developed 3, where membrane-enriched stable isotope isobaric-labeled carrier channel (meSIILCC) approach was detection membrane MS-TSA. After testing four protein-enrichment protocols, commercially available kit selected improve meSIILCC workflow. separate DMSO-only control experiment optimized conducted. significant increase number identified unique quantified peptides per annotated "plasma membrane" approach. Also, acquired results unsuitable isobaric tag channels due interference. final Chapter, focus how could utilized future highlight aspects need developed. --Author's abstract

Язык: Английский

Diversity of post-translational modifications and cell signaling revealed by single cell and single organelle mass spectrometry DOI Creative Commons
Dong‐Gi Mun, Firdous Ahmad Bhat, Neha Joshi

и другие.

Communications Biology, Год журнала: 2024, Номер 7(1)

Опубликована: Июль 19, 2024

Abstract The rapid evolution of mass spectrometry-based single-cell proteomics now enables the cataloging several thousand proteins from single cells. We investigated whether we could discover cellular heterogeneity beyond proteome, encompassing post-translational modifications (PTM), protein-protein interaction, and variants. By optimizing spectrometry data interpretation strategy to enable detection PTMs variants, have generated a high-definition dataset nuclear proteomic-states. demonstrate cell-states signaling dependencies at level reveal epigenetic drug-induced changes in nuclei. This approach exploration previously uncharted organellar proteomes revealing molecular characteristics that are inaccessible through RNA profiling.

Язык: Английский

Процитировано

3
Boosting the Sensitivity of Quantitative Single-Cell Proteomics with Activated lon-Tandem Mass Tags (AI-TMT) DOI Creative Commons
Trenton M. Peters-Clarke, Yiran Liang, Keaton L. Mertz

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Фев. 25, 2024

ABSTRACT Single-cell proteomics is a powerful approach to precisely profile protein landscapes within individual cells toward comprehensive understanding of proteomic functions and tissue cellular states. The inherent challenges associated with limited starting material in single-cell analyses demands heightened analytical sensitivity. Just as advances sample preparation maximize the amount that makes it from cell mass spectrometer, we strive number ions make ion source detector. In isobaric tagging experiments, reporter generation limits quantitative accuracy precision. combination infrared photoactivation parking circumvents m/z dependence HCD, maximizing avoiding unintended degradation TMT molecules method term activated ion-tandem tags (AI-TMT). was applied human proteomes using 18-plex TMTpro, resulting 4-5-fold increase signal on average compared conventional SPS-MS 3 approaches. AI-TMT enables faster duty cycles, higher throughput, increased peptide identification quantification. Comparative experiments showcase lower injection times for AI-TMT, providing superior sensitivity without compromising accuracy. all, enhances dynamic range compatible other techniques, including gas-phase fractionation real-time searching, promising gains study heterogeneity disease mechanisms.

Язык: Английский

Процитировано

2
Review and Practical Guide for Getting Started With Single‐Cell Proteomics DOI
Hsien‐Jung L. Lin,

Kei G. I. Webber,

Andikan J. Nwosu

и другие.

PROTEOMICS, Год журнала: 2024, Номер unknown

Опубликована: Ноя. 16, 2024

ABSTRACT Single‐cell proteomics (SCP) has advanced significantly in recent years, with new tools specifically designed for the preparation and analysis of single cells now commercially available to researchers. The field is sufficiently mature be broadly accessible any lab capable isolating performing bulk‐scale proteomic analyses. In this review, we highlight work SCP that lowered barrier entry, thus providing a practical guide those who are newly entering field. We outline fundamental principles report multiple paths accomplish key steps successful experiment including sample preparation, separation, mass spectrometry data acquisition analysis. recommend researchers start label‐free workflow, as achieving high‐quality quantitatively accurate results more straightforward than label‐based multiplexed strategies. By leveraging these means, can confidently perform experiments make meaningful discoveries at single‐cell level.

Язык: Английский

Процитировано

2
Increasing the Throughput and Reproducibility of Activity-Based Proteome Profiling Studies with Hyperplexing and Intelligent Data Acquisition DOI Creative Commons
Hanna G. Budayeva, P. Taylur, Shuai Wang

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Сен. 13, 2023

Abstract Intelligent data acquisition (IDA) strategies, such as real-time database search (RTS), have improved the depth of proteome coverage for experiments that utilize isobaric labels and gas phase purification techniques (i.e., SPS-MS3). While most applications IDA been focused on analysis protein abundance, these approaches recently applied to activity-based profiling (ABPP) studies aimed at characterization site engagement by small molecules. In this work, we extend capabilities offered vendor software through a program called InSeqAPI. First, demonstrate robust performance InSeqAPI in biotinylated cysteine peptides from ABPP experiments. Then, describe PairQuant, method within designed hyperplexing approach utilizes protein-level isotopic labeling peptide-level TMT labeling. PairQuant allows 36 conditions single sample achieves ∼98% both peptide pair partners hyperplexed experiment well 40% improvement number quantified sites compared non-RTS acquisition. We study ligandable nucleus leading an identification additional druggable protein-and DNA-interaction domains transcription regulators nuclear ubiquitin ligases.

Язык: Английский

Процитировано

6
Toward Single Bacterium Proteomics DOI Creative Commons
Ákos Végvári, Xuepei Zhang, Roman A. Zubarev

и другие.

Journal of the American Society for Mass Spectrometry, Год журнала: 2023, Номер 34(10), С. 2098 - 2106

Опубликована: Сен. 15, 2023

Bacteria are orders of magnitude smaller than mammalian cells, and while single cell proteomics (SCP) currently detects quantifies several thousands proteins per cell, it is not clear whether conventional SCP methods will be suitable for bacteria. Here we report on the first successful attempt to detect from individual Escherichia coli bacteria, with validation our findings by comparison two bacteria samples bulk data. Data available via ProteomeXchange identifier PXD043473.

Язык: Английский

Процитировано

4
Are We There Yet? Assessing the Readiness of Single-Cell Proteomics to Answer Biological Hypotheses DOI Creative Commons

Alyssa A Nitz,

Jose Humberto Giraldez Chavez,

Zachary G. Eliason

и другие.

Journal of Proteome Research, Год журнала: 2024, Номер unknown

Опубликована: Июль 9, 2024

Single-cell analysis is an active area of research in many fields biology. Measurements at single-cell resolution allow researchers to study diverse populations without losing biologically meaningful information sample averages. Many technologies have been used single cells, including mass spectrometry-based proteomics (SCP). SCP has seen a lot growth over the past couple years through improvements data acquisition and analysis, leading greater proteomic depth. Because method development main focus SCP, biological applications sprinkled only as proof-of-concept. However, methods now provide significant coverage proteome implemented laboratories. Thus, primary question address our community whether current state technology ready for widespread adoption inquiry. In this Perspective, we examine potential three thematic areas investigation: cell annotation, developmental trajectories, spatial mapping. We identify that limitation throughput. As depth target date, advocate change facilitate measuring tens thousands proteomes enable beyond

Язык: Английский

Процитировано

1
Ultralow flow liquid chromatography and related approaches: A focus on recent bioanalytical applications DOI Creative Commons
Michal Greguš, Alexander R. Ivanov, Steven Ray Wilson

и другие.

Journal of Separation Science, Год журнала: 2023, Номер 46(18)

Опубликована: Авг. 1, 2023

Ultralow flow LC employs ultra‐narrow bore columns and mid‐range pL/min to low nL/min rates (i.e., ≤20 nL/min). The separation that are used under these conditions typically 2–30 μm in inner diameter. systems allow for exceptionally high sensitivity frequently resolution. There has been an increasing interest the analysis of scarce biological samples, example, circulating tumor cells, extracellular vesicles, organelles, single ultralow was efficiently applied such samples. Hence, advances towards dedicated instrumentation, technical approaches, higher throughput (e.g., tens‐to‐hundreds cells analyzed per day) were recently made. Here, we review types technology, followed by a discussion selected representative applications, focusing on progress made bioanalysis amount‐limited samples during last 10 years. We also discuss several reported high‐sensitivity applications utilizing up 100 nL/min, which below commonly nanoLC rates. Finally, path forward future developments LC.

Язык: Английский

Процитировано

2
Stable Isotope Labeling-Based Nontargeted Strategy for Characterization of the In Vitro Metabolic Profile of a Novel Doping BPC-157 in Doping Control by UHPLC-HRMS DOI Creative Commons
Tian Tian, Jing Jing, Yuanyuan Li

и другие.

Molecules, Год журнала: 2023, Номер 28(21), С. 7345 - 7345

Опубликована: Окт. 30, 2023

Traditional strategies for the metabolic profiling of doping are limited by unpredictable pathways and numerous proportions background chemical noise that lead to inadequate metabolism knowledge, thereby affecting selection optimal detection targets. Thus, a stable isotope labeling-based nontargeted strategy combined with ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) was first proposed effective rapid analysis small-molecule agents demonstrated via its application novel BPC-157. Using 13C/15N-labeled BPC-157, complete workflow including automatic 13C0,15N0-13C6,15N2m/z pair picking based on characteristic behaviors pairs constructed, one metabolite produced pathway plus eight metabolites conventional amide-bond breaking were successfully discovered from two incubation models. Furthermore, specific method BPC-157 five main in human urine developed validated satisfactory limits (0.01~0.11 ng/mL) excellent quantitative ability (linearity: 0.02~50 ng/mL R2 > 0.999; relative error (RE)% < 10% standard deviation (RSD)% 5%; recovery 90%). The vitro profile could provide new insights into biotransformation improved targets control.

Язык: Английский

Процитировано

2
Comprehensive Overview of Bottom-up Proteomics using Mass Spectrometry DOI Creative Commons
Yuming Jiang, Rex Devasahayam Arokia Balaya, Dina Schuster

и другие.

arXiv (Cornell University), Год журнала: 2023, Номер unknown

Опубликована: Янв. 1, 2023

Proteomics is the large scale study of protein structure and function from biological systems through identification quantification. "Shotgun proteomics" or "bottom-up prevailing strategy, in which proteins are hydrolyzed into peptides that analyzed by mass spectrometry. studies can be applied to diverse ranging simple proteoforms, protein-protein interactions, structural alterations, absolute relative quantification, post-translational modifications, stability. To enable this range different experiments, there strategies for proteome analysis. The nuances how proteomic workflows differ may challenging understand new practitioners. Here, we provide a comprehensive overview proteomics methods aid novice experienced researcher. We cover biochemistry basics extraction interpretation orthogonal validation. expect work serve as basic resource practitioners field shotgun bottom-up proteomics.

Язык: Английский

Процитировано

2
Efficient and Sensitive Sample Preparation, Separations, and Data Acquisition for Label-Free Single-Cell Proteomics DOI
Thy Truong,

Ximena Sanchez-Avila,

Kei G. I. Webber

и другие.

Methods in molecular biology, Год журнала: 2024, Номер unknown, С. 67 - 84

Опубликована: Янв. 1, 2024

Язык: Английский

Процитировано

0