Top-down proteomics: Enhancing 2D gel electrophoresis from tissue processing to high-sensitivity protein detection

Wright, EP; Partridge, MA; Padula, MP; Gauci, VJ; Malladi, CS; Coorssen, JR

Top-down proteomics: Enhancing 2D gel electrophoresis from tissue processing to high-sensitivity protein detection

Wright, EP Partridge, MA Padula, MP

Gauci, VJ Malladi, CS Coorssen, JR

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Publication Type:: Journal Article
Citation:: Proteomics, 2014, 14 (7-8), pp. 872 - 889
Issue Date:: 2014-01-01

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Field	Value	Language
dc.contributor.author	Wright, EP	en_US
dc.contributor.author	Partridge, MA	en_US
dc.contributor.author	Padula, MP https://orcid.org/0000-0002-8283-0643	en_US
dc.contributor.author	Gauci, VJ	en_US
dc.contributor.author	Malladi, CS	en_US
dc.contributor.author	Coorssen, JR	en_US
dc.date.available	2013-12-16	en_US
dc.date.issued	2014-01-01	en_US
dc.identifier.citation	Proteomics, 2014, 14 (7-8), pp. 872 - 889	en_US
dc.identifier.issn	1615-9853	en_US
dc.identifier.uri	http://hdl.handle.net/10453/37792
dc.description.abstract	The large-scale resolution and detection of proteins from complex native mixtures is fundamental to quantitative proteomic analyses. Comprehensive analyses depend on careful tissue handling and quantitative protein extraction and assessment. To most effectively link these analyses with an understanding of underlying molecular mechanisms, it is critical that all protein types - isoforms, splice variants and those with functionally important PTMs - are quantitatively extracted with high reproducibility. Methodological details concerning protein extraction and resolution using 2DE are discussed with reference to current in-gel protein detection limits. We confirm a significant increase in total protein, and establish that extraction, resolution and detection of phospho- and glycoproteins are improved following automated frozen disruption relative to manual homogenisation. The quality of 2DE protein resolution is established using third-dimension separations and 'deep imaging'; substantially more proteins/protein species than previously realised are actually resolved by 2DE. Thus, the key issue for effective proteome analyses is most likely to be detection, not resolution. Thus, these systematic methodological and technical advances further solidify the role of 2DE in top-down proteomics. By routinely assessing as much proteomic data from a sample as possible, 2DE enables more detailed and critical insights into molecular mechanisms underlying different physiological states. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.	en_US
dc.relation.ispartof	Proteomics	en_US
dc.relation.isbasedon	10.1002/pmic.201300424	en_US
dc.subject.classification	Biochemistry & Molecular Biology	en_US
dc.subject.mesh	Proteins	en_US
dc.subject.mesh	Complex Mixtures	en_US
dc.subject.mesh	Electrophoresis, Gel, Two-Dimensional	en_US
dc.subject.mesh	Proteomics	en_US
dc.title	Top-down proteomics: Enhancing 2D gel electrophoresis from tissue processing to high-sensitivity protein detection	en_US
dc.type	Journal Article
utslib.citation.volume	7-8	en_US
utslib.citation.volume	14	en_US
utslib.for	110106 Medical Biochemistry: Proteins and Peptides (incl. Medical Proteomics)	en_US
utslib.for	060109 Proteomics and Intermolecular Interactions (excl. Medical Proteomics)	en_US
utslib.for	06 Biological Sciences	en_US
utslib.for	08 Information and Computing Sciences	en_US
utslib.for	11 Medical and Health Sciences	en_US
pubs.embargo.period	Not known	en_US
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
utslib.copyright.status	closed_access
pubs.issue	7-8	en_US
pubs.publication-status	Published	en_US
pubs.volume	14	en_US

Abstract:

The large-scale resolution and detection of proteins from complex native mixtures is fundamental to quantitative proteomic analyses. Comprehensive analyses depend on careful tissue handling and quantitative protein extraction and assessment. To most effectively link these analyses with an understanding of underlying molecular mechanisms, it is critical that all protein types - isoforms, splice variants and those with functionally important PTMs - are quantitatively extracted with high reproducibility. Methodological details concerning protein extraction and resolution using 2DE are discussed with reference to current in-gel protein detection limits. We confirm a significant increase in total protein, and establish that extraction, resolution and detection of phospho- and glycoproteins are improved following automated frozen disruption relative to manual homogenisation. The quality of 2DE protein resolution is established using third-dimension separations and 'deep imaging'; substantially more proteins/protein species than previously realised are actually resolved by 2DE. Thus, the key issue for effective proteome analyses is most likely to be detection, not resolution. Thus, these systematic methodological and technical advances further solidify the role of 2DE in top-down proteomics. By routinely assessing as much proteomic data from a sample as possible, 2DE enables more detailed and critical insights into molecular mechanisms underlying different physiological states. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Please use this identifier to cite or link to this item:

http://hdl.handle.net/10453/37792