Data-Independent Acquisition (DIA) Strategy for Measuring Protein Stability Using Stability of Proteins from Rates of Oxidation (SPROX).
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| Title: | Data-Independent Acquisition (DIA) Strategy for Measuring Protein Stability Using Stability of Proteins from Rates of Oxidation (SPROX). |
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| Authors: | Labbe, Natalie M.1 (AUTHOR), Virani, Meghan K.1 (AUTHOR), Fitzgerald, Michael C.1 (AUTHOR) michael.c.fitzgerald@duke.edu |
| Source: | Journal of the American Society for Mass Spectrometry. Apr2026, Vol. 37 Issue 4, p836-845. 10p. |
| Abstract: | The stability of proteins from the rates of oxidation (SPROX) technique is a mass spectrometry-based approach for making protein folding stability measurements on the proteomic scale. The development and application of SPROX, to date, have primarily relied on the use of quantitative bottom-up proteomics and data-dependent acquisition (DDA) strategies using isobaric mass tags. Use of isobaric mass tags is attractive, as it enables the mass spectrometry readout in SPROX to be highly multiplexed. However, the use of such isobaric mass tags is restricted to DDA strategies, which can be limited in their proteomic coverage compared with data-independent acquisition (DIA) strategies. Reported here is a new "one-pot" SPROX workflow that employs a DIA readout and a label-free quantification strategy. Analysis of the proteins in an E. coli cell lysate using the DIA-SPROX strategy allowed for the calculation of transition midpoints with reasonable accuracy. The proteins from a S. cerevisiae cell lysate were also assessed for ligand-induced changes in their transition midpoints upon the introduction of cyclosporine A (CsA) to identify the protein targets of this well-studied ligand. The DIA-SPROX strategy developed here successfully identified known protein targets of CsA with a low false positive rate using a combination of two different software, Spectronaut and DIA-NN, for DIA data processing. We also find that the proteomic coverage obtained using DIA-SPROX is comparable to the coverage obtained in conventional DDA-SPROX experiments. Significantly, this comparable coverage can be achieved without a fractionation strategy (e.g., methionine-containing peptide enrichment) in DIA-SPROX. [ABSTRACT FROM AUTHOR] |
| Copyright of Journal of the American Society for Mass Spectrometry is the property of American Chemical Society and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) | |
| Database: | Engineering Source |
| FullText | Text: Availability: 0 |
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| Header | DbId: egs DbLabel: Engineering Source An: 192727633 AccessLevel: 6 PubType: Academic Journal PubTypeId: academicJournal PreciseRelevancyScore: 0 |
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| Items | – Name: Title Label: Title Group: Ti Data: Data-Independent Acquisition (DIA) Strategy for Measuring Protein Stability Using Stability of Proteins from Rates of Oxidation (SPROX). – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AR" term="%22Labbe%2C+Natalie+M%2E%22">Labbe, Natalie M.</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Virani%2C+Meghan+K%2E%22">Virani, Meghan K.</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Fitzgerald%2C+Michael+C%2E%22">Fitzgerald, Michael C.</searchLink><relatesTo>1</relatesTo> (AUTHOR)<i> michael.c.fitzgerald@duke.edu</i> – Name: TitleSource Label: Source Group: Src Data: <searchLink fieldCode="JN" term="%22Journal+of+the+American+Society+for+Mass+Spectrometry%22">Journal of the American Society for Mass Spectrometry</searchLink>. Apr2026, Vol. 37 Issue 4, p836-845. 10p. – Name: Abstract Label: Abstract Group: Ab Data: The stability of proteins from the rates of oxidation (SPROX) technique is a mass spectrometry-based approach for making protein folding stability measurements on the proteomic scale. The development and application of SPROX, to date, have primarily relied on the use of quantitative bottom-up proteomics and data-dependent acquisition (DDA) strategies using isobaric mass tags. Use of isobaric mass tags is attractive, as it enables the mass spectrometry readout in SPROX to be highly multiplexed. However, the use of such isobaric mass tags is restricted to DDA strategies, which can be limited in their proteomic coverage compared with data-independent acquisition (DIA) strategies. Reported here is a new "one-pot" SPROX workflow that employs a DIA readout and a label-free quantification strategy. Analysis of the proteins in an E. coli cell lysate using the DIA-SPROX strategy allowed for the calculation of transition midpoints with reasonable accuracy. The proteins from a S. cerevisiae cell lysate were also assessed for ligand-induced changes in their transition midpoints upon the introduction of cyclosporine A (CsA) to identify the protein targets of this well-studied ligand. The DIA-SPROX strategy developed here successfully identified known protein targets of CsA with a low false positive rate using a combination of two different software, Spectronaut and DIA-NN, for DIA data processing. We also find that the proteomic coverage obtained using DIA-SPROX is comparable to the coverage obtained in conventional DDA-SPROX experiments. Significantly, this comparable coverage can be achieved without a fractionation strategy (e.g., methionine-containing peptide enrichment) in DIA-SPROX. [ABSTRACT FROM AUTHOR] – Name: AbstractSuppliedCopyright Label: Group: Ab Data: <i>Copyright of Journal of the American Society for Mass Spectrometry is the property of American Chemical Society and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.) |
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| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.1021/jasms.5c00316 Languages: – Code: eng Text: English PhysicalDescription: Pagination: PageCount: 10 StartPage: 836 Titles: – TitleFull: Data-Independent Acquisition (DIA) Strategy for Measuring Protein Stability Using Stability of Proteins from Rates of Oxidation (SPROX). Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: Labbe, Natalie M. – PersonEntity: Name: NameFull: Virani, Meghan K. – PersonEntity: Name: NameFull: Fitzgerald, Michael C. IsPartOfRelationships: – BibEntity: Dates: – D: 01 M: 04 Text: Apr2026 Type: published Y: 2026 Identifiers: – Type: issn-print Value: 10440305 Numbering: – Type: volume Value: 37 – Type: issue Value: 4 Titles: – TitleFull: Journal of the American Society for Mass Spectrometry Type: main |
| ResultId | 1 |
