Optimized plasma sample preparation and LC-MS analysis to support large-scale proteomic analysis of clinical trial specimens: Application to the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial.

O'Rourke, MB; Januszewski, AS; Sullivan, DR; Lengyel, I; Stewart, AJ; Arya, S; Ma, R; Galande, S; Hardikar, AA; Joglekar, MV; Keech, AC; Jenkins, AJ; Molloy, MP

Optimized plasma sample preparation and LC-MS analysis to support large-scale proteomic analysis of clinical trial specimens: Application to the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial.

O'Rourke, MB Januszewski, AS Sullivan, DR Lengyel, I Stewart, AJ Arya, S Ma, R Galande, S Hardikar, AA Joglekar, MV Keech, AC Jenkins, AJ Molloy, MP

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Publisher:: Wiley
Publication Type:: Journal Article
Citation:: Proteomics Clin Appl, 2023, pp. e2200106
Issue Date:: 2023-03-08

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Field	Value	Language
dc.contributor.author	O'Rourke, MB
dc.contributor.author	Januszewski, AS
dc.contributor.author	Sullivan, DR
dc.contributor.author	Lengyel, I
dc.contributor.author	Stewart, AJ
dc.contributor.author	Arya, S
dc.contributor.author	Ma, R
dc.contributor.author	Galande, S
dc.contributor.author	Hardikar, AA
dc.contributor.author	Joglekar, MV
dc.contributor.author	Keech, AC
dc.contributor.author	Jenkins, AJ
dc.contributor.author	Molloy, MP
dc.date.accessioned	2023-03-14T14:06:20Z
dc.date.available	2023-02-28
dc.date.available	2023-03-14T14:06:20Z
dc.date.issued	2023-03-08
dc.identifier.citation	Proteomics Clin Appl, 2023, pp. e2200106
dc.identifier.issn	1862-8346
dc.identifier.issn	1862-8354
dc.identifier.uri	http://hdl.handle.net/10453/167322
dc.description.abstract	PURPOSE: Robust, affordable plasma proteomic biomarker workflows are needed for large-scale clinical studies. We evaluated aspects of sample preparation to allow LC-MS analysis of more than 1500 samples from the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial of adults with type 2 diabetes. METHODS: Using LC-MS with data-independent acquisition we evaluated four variables: plasma protein depletion, EDTA or citrated anti-coagulant blood collection tubes, plasma lipid depletion strategies and plasma freeze-thaw cycles. Optimised methods were applied in a pilot study of FIELD participants. RESULTS: LC-MS of undepleted plasma conducted over a 45 min gradient yielded 172 proteins after excluding immunoglobulin isoforms. Cibachrome-blue-based depletion yielded additional proteins but with cost and time expenses, while immunodepleting albumin and IgG provided few additional identifications. Only minor variations were associated with blood collection tube type, delipidation methods and freeze-thaw cycles. From 65 batches involving over 1500 injections, the median intra-batch quantitative differences in the top 100 proteins of the plasma external standard was less than 2%. Fenofibrate altered seven plasma proteins. CONCLUSIONS AND CLINICAL RELEVANCE: A robust plasma handling and LC-MS proteomics workflow for abundant plasma proteins has been developed for large-scale biomarker studies that balances proteomic depth with time and resource costs. This article is protected by copyright. All rights reserved.
dc.format	Print-Electronic
dc.language	eng
dc.publisher	Wiley
dc.relation.ispartof	Proteomics Clin Appl
dc.relation.isbasedon	10.1002/prca.202200106
dc.rights	info:eu-repo/semantics/embargoedAccess
dc.subject	1101 Medical Biochemistry and Metabolomics
dc.subject.classification	Biochemistry & Molecular Biology
dc.title	Optimized plasma sample preparation and LC-MS analysis to support large-scale proteomic analysis of clinical trial specimens: Application to the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial.
dc.type	Journal Article
utslib.location.activity	Germany
utslib.for	1101 Medical Biochemistry and Metabolomics
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Faculty of Science/School of Life Sciences
pubs.organisational-group	/University of Technology Sydney/Strength - CFI - Centre for Inflammation
utslib.copyright.status	open_access	*
utslib.copyright.embargo	2024-03-08T00:00:00+1000Z
dc.date.updated	2023-03-14T14:06:18Z
pubs.publication-status	Published online

Abstract:

PURPOSE: Robust, affordable plasma proteomic biomarker workflows are needed for large-scale clinical studies. We evaluated aspects of sample preparation to allow LC-MS analysis of more than 1500 samples from the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial of adults with type 2 diabetes. METHODS: Using LC-MS with data-independent acquisition we evaluated four variables: plasma protein depletion, EDTA or citrated anti-coagulant blood collection tubes, plasma lipid depletion strategies and plasma freeze-thaw cycles. Optimised methods were applied in a pilot study of FIELD participants. RESULTS: LC-MS of undepleted plasma conducted over a 45 min gradient yielded 172 proteins after excluding immunoglobulin isoforms. Cibachrome-blue-based depletion yielded additional proteins but with cost and time expenses, while immunodepleting albumin and IgG provided few additional identifications. Only minor variations were associated with blood collection tube type, delipidation methods and freeze-thaw cycles. From 65 batches involving over 1500 injections, the median intra-batch quantitative differences in the top 100 proteins of the plasma external standard was less than 2%. Fenofibrate altered seven plasma proteins. CONCLUSIONS AND CLINICAL RELEVANCE: A robust plasma handling and LC-MS proteomics workflow for abundant plasma proteins has been developed for large-scale biomarker studies that balances proteomic depth with time and resource costs. This article is protected by copyright. All rights reserved.

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http://hdl.handle.net/10453/167322