Immunolabelling perturbs the endogenous and antibody-conjugated elemental concentrations during immuno-mass spectrometry imaging.

Mello, MG; Westerhausen, MT; Lockwood, TE; Singh, P; Wanagat, J; Bishop, DP

Immunolabelling perturbs the endogenous and antibody-conjugated elemental concentrations during immuno-mass spectrometry imaging.

Mello, MG Westerhausen, MT Lockwood, TE Singh, P Wanagat, J Bishop, DP

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Publisher:: SPRINGER HEIDELBERG
Publication Type:: Journal Article
Citation:: Anal Bioanal Chem, 2023
Issue Date:: 2023-10-06

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Field	Value	Language
dc.contributor.author	Mello, MG
dc.contributor.author	Westerhausen, MT
dc.contributor.author	Lockwood, TE
dc.contributor.author	Singh, P
dc.contributor.author	Wanagat, J
dc.contributor.author	Bishop, DP
dc.date.accessioned	2023-12-05T04:19:12Z
dc.date.available	2023-09-19
dc.date.available	2023-12-05T04:19:12Z
dc.date.issued	2023-10-06
dc.identifier.citation	Anal Bioanal Chem, 2023
dc.identifier.issn	1618-2642
dc.identifier.issn	1618-2650
dc.identifier.uri	http://hdl.handle.net/10453/173696
dc.description.abstract	Immuno-mass spectrometry imaging uses lanthanide-conjugated antibodies to spatially quantify biomolecules via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The multi-element capabilities allow for highly multiplexed analyses that may include both conjugated antibodies and endogenous metals to reveal relationships between disease and chemical composition. Sample handling is known to perturb the composition of the endogenous elements, but there has been little investigation into the effects of immunolabelling and coverslipping. Here, we used cryofixed muscle sections to examine the impact of immunolabelling steps on the concentrations of a Gd-conjugated anti-dystrophin primary antibody, and the endogenous metals Cu and Zn. Primary antibody incubation resulted in a decrease in Zn, and an increase in Cu. Zn was removed from the cytoplasm where it was hypothesised to be more labile, whereas concentrated locations of Zn remained in the cell membrane in all samples that underwent the immunostaining process. Cu increased in concentration and was found mostly in the cell membrane. The concentration of the Gd-conjugated antibody when compared to the standard air-dried sample was not significantly different when coverslipped using an organic mounting medium, whereas use of an aqueous mounting medium significantly reduced the concentration of Gd. These results build on the knowledge of how certain sample handling techniques change elemental concentrations and distributions in tissue sections. Immunolabelling steps impact the concentration of endogenous elements, and separate histological sections are required for the quantitative analysis of endogenous elements and biomolecules. Additionally, coverslipping tissue sections for complementary immunohistochemical/immunofluorescent imaging may compromise the integrity of the elemental label, and organic mounting media are recommended over aqueous mounting media.
dc.format	Print-Electronic
dc.language	eng
dc.publisher	SPRINGER HEIDELBERG
dc.relation.ispartof	Anal Bioanal Chem
dc.relation.isbasedon	10.1007/s00216-023-04967-2
dc.rights	info:eu-repo/semantics/openAccess
dc.subject	03 Chemical Sciences, 06 Biological Sciences, 09 Engineering
dc.subject.classification	Analytical Chemistry
dc.subject.classification	31 Biological sciences
dc.subject.classification	34 Chemical sciences
dc.subject.classification	40 Engineering
dc.title	Immunolabelling perturbs the endogenous and antibody-conjugated elemental concentrations during immuno-mass spectrometry imaging.
dc.type	Journal Article
utslib.location.activity	Germany
utslib.for	03 Chemical Sciences
utslib.for	06 Biological Sciences
utslib.for	09 Engineering
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 Mathematical and Physical Sciences
utslib.copyright.status	open_access	*
dc.date.updated	2023-12-05T04:19:10Z
pubs.publication-status	Published online

Abstract:

Immuno-mass spectrometry imaging uses lanthanide-conjugated antibodies to spatially quantify biomolecules via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The multi-element capabilities allow for highly multiplexed analyses that may include both conjugated antibodies and endogenous metals to reveal relationships between disease and chemical composition. Sample handling is known to perturb the composition of the endogenous elements, but there has been little investigation into the effects of immunolabelling and coverslipping. Here, we used cryofixed muscle sections to examine the impact of immunolabelling steps on the concentrations of a Gd-conjugated anti-dystrophin primary antibody, and the endogenous metals Cu and Zn. Primary antibody incubation resulted in a decrease in Zn, and an increase in Cu. Zn was removed from the cytoplasm where it was hypothesised to be more labile, whereas concentrated locations of Zn remained in the cell membrane in all samples that underwent the immunostaining process. Cu increased in concentration and was found mostly in the cell membrane. The concentration of the Gd-conjugated antibody when compared to the standard air-dried sample was not significantly different when coverslipped using an organic mounting medium, whereas use of an aqueous mounting medium significantly reduced the concentration of Gd. These results build on the knowledge of how certain sample handling techniques change elemental concentrations and distributions in tissue sections. Immunolabelling steps impact the concentration of endogenous elements, and separate histological sections are required for the quantitative analysis of endogenous elements and biomolecules. Additionally, coverslipping tissue sections for complementary immunohistochemical/immunofluorescent imaging may compromise the integrity of the elemental label, and organic mounting media are recommended over aqueous mounting media.

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http://hdl.handle.net/10453/173696