Conformational Characteristics and Phase Behavior of Intrinsically Disordered Proteins─Where Physical Chemistry Meets Biology

Arora, Lisha; Mukhopadhyay, Samrat

doi:10.1021/acs.jpcb.2c04017

Cited by 3 publications

(3 citation statements)

References 33 publications

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“…This consideration is particularly relevant for low abundance signals, such as those of XLs, amidst intense background. Therefore, the XL-MS community has developed further validation approaches that leverage available protein models to use XL distance constraints as false positive discriminators. , Although this approach proves its usefulness for large proteome-wide data sets, it is limited by the availability and accuracy of protein models and is ineffective for intrinsically disordered proteins or regions with flexible conformation ensembles that defy standard modeling and distance analysis …”

Section: Resultsmentioning

confidence: 99%

“…22,23 Although this approach proves its usefulness for large proteome-wide data sets, it is limited by the availability and accuracy of protein models and is ineffective for intrinsically disordered proteins or regions with flexible conformation ensembles that defy standard modeling and distance analysis. 24 This technical note proposes a complementary validation approach for XLs identified with data acquired in DDA mode, adopting strategies from "peptide-centric" methods 20 used in DIA and targeted data analysis. Although the list of XLs that are tested for detection still depends on spectrum-centric search engines such as MeroX, here we show how to use the complete LC-(TIMS)-MS/MS data collected to test detection and alignment of identifications proposed from any replicate measurement.…”

Section: ■ Introductionmentioning

confidence: 99%

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A Workflow for Improved Analysis of Cross-Linking Mass Spectrometry Data Integrating Parallel Accumulation-Serial Fragmentation with MeroX and Skyline

Rojas Echeverri,

Hause,

Iacobucci

et al. 2024

Anal. Chem.

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Cross-linking mass spectrometry (XL-MS) has evolved into a pivotal technique for probing protein interactions. This study describes the implementation of Parallel Accumulation-Serial Fragmentation (PASEF) on timsTOF instruments, enhancing the detection and analysis of protein interactions by XL-MS. Addressing the challenges in XL-MS, such as the interpretation of complex spectra, low abundant cross-linked peptides, and a data acquisition bias, our current study integrates a peptide-centric approach for the analysis of XL-MS data and presents the foundation for integrating data-independent acquisition (DIA) in XL-MS with a vendor-neutral and open-source platform. A novel workflow is described for processing data-dependent acquisition (DDA) of PASEF-derived information. For this, software by Bruker Daltonics is used, enabling the conversion of these data into a format that is compatible with MeroX and Skyline software tools. Our approach significantly improves the identification of cross-linked products from complex mixtures, allowing the XL-MS community to overcome current analytical limitations.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

A Workflow for Improved Analysis of Cross-Linking Mass Spectrometry Data Integrating Parallel Accumulation-Serial Fragmentation with MeroX and Skyline

Rojas Echeverri,

Hause,

Iacobucci

et al. 2024

Anal. Chem.

View full text Add to dashboard Cite

show abstract

“…Therefore, the XL-MS community developed further validation approaches that leverage available protein models to use cross-link distance constraints as false positive discriminators 21,22 . Although this approach proves its usefulness for large proteome-wide datasets, it is limited by the availability and accuracy of protein models and is ineffective for intrinsically disordered proteins or regions with flexible conformation ensembles that defy standard modeling and distance analysis 23 . This technical note proposes a complementary validation approach for XLs identified with data acquired in DDA mode, adopting strategies from 'peptide-centric' methods 19 used in DIA and targeted data analysis.…”

Section: Resultsmentioning

confidence: 99%

A Workflow for Improved Analysis of Cross-linking Mass Spectrometry Data Integrating Parallel Accumulation-Serial Fragmentation with MeroX and Skyline

Rojas Echeverri,

Hause,

Iacobucci

et al. 2024

Preprint

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Cross-linking mass spectrometry (XL-MS) has evolved into a pivotal technique for probing protein interactions. This study describes the implementation of Parallel Accumulation-Serial Fragmentation (PASEF) on timsTOF instruments, enhancing the detection and analysis of protein interactions by XL-MS. Addressing the challenges in XL-MS, such as the interpretation of complex spectra, low abundant cross-linked peptides, and a data acquisition bias, our current study integrates a peptide-centric approach for the analysis of XL-MS data and presents the foundation for integrating data-independent acquisition (DIA) in XL-MS with a vendor-neutral and open-source platform. A novel workflow is described for processing data-dependent analysis (DDA) of PASEF-derived information. For this, software by Bruker Daltonics is used, enabling the conversion of these data into a format that is compatible with MeroX and Skyline software tools. Our approach significantly improves the identification of cross-linked products from complex mixtures, allowing the XL-MS community to overcome current analytical limitations.

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Unravelling the microscopic characteristics of intrinsically disordered proteins upon liquid–liquid phase separation

Wen

Perrett

2022

Essays in Biochemistry

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Biomolecular condensate formation via liquid–liquid phase separation (LLPS) has emerged as a ubiquitous mechanism underlying the spatiotemporal organization of biomolecules in the cell. These membraneless condensates form and disperse dynamically in response to environmental stimuli. Growing evidence indicates that the liquid-like condensates not only play functional physiological roles but are also implicated in a wide range of human diseases. As a major component of biomolecular condensates, intrinsically disordered proteins (IDPs) are intimately involved in the LLPS process. During the last decade, great efforts have been made on the macroscopic characterization of the physicochemical properties and biological functions of liquid condensates both in vitro and in the cellular context. However, characterization of the conformations and interactions at the molecular level within phase-separated condensates is still at an early stage. In the present review, we summarize recent biophysical studies investigating the intramolecular conformational changes of IDPs upon LLPS and the intermolecular clustering of proteins undergoing LLPS, with a particular focus on single-molecule fluorescence detection. We also discuss how these microscopic features are linked to the macroscopic phase transitions that are relevant to the physiological and pathological roles of the condensates.

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Conformational Characteristics and Phase Behavior of Intrinsically Disordered Proteins─Where Physical Chemistry Meets Biology

Cited by 3 publications

References 33 publications

A Workflow for Improved Analysis of Cross-Linking Mass Spectrometry Data Integrating Parallel Accumulation-Serial Fragmentation with MeroX and Skyline

A Workflow for Improved Analysis of Cross-Linking Mass Spectrometry Data Integrating Parallel Accumulation-Serial Fragmentation with MeroX and Skyline

A Workflow for Improved Analysis of Cross-linking Mass Spectrometry Data Integrating Parallel Accumulation-Serial Fragmentation with MeroX and Skyline

Unravelling the microscopic characteristics of intrinsically disordered proteins upon liquid–liquid phase separation

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