An Automated and Fast Sample Preparation Workflow for Laser Microdissection Guided Ultrasensitive Proteomics
Spatial tissue proteomics integrating whole-slide imaging, laser microdissection, and ultrasensitive mass spectrometry is a powerful approach to link cellular phenotypes to functional proteome states in (patho)physiology. To be applicable to large patient cohorts and low sample input amounts, includ...
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Published in | Molecular & cellular proteomics Vol. 23; no. 5; p. 100750 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
01.05.2024
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Subjects | |
Online Access | Get full text |
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Summary: | Spatial tissue proteomics integrating whole-slide imaging, laser microdissection, and ultrasensitive mass spectrometry is a powerful approach to link cellular phenotypes to functional proteome states in (patho)physiology. To be applicable to large patient cohorts and low sample input amounts, including single-cell applications, loss-minimized and streamlined end-to-end workflows are key. We here introduce an automated sample preparation protocol for laser microdissected samples utilizing the cellenONE robotic system, which has the capacity to process 192 samples in 3 h. Following laser microdissection collection directly into the proteoCHIP LF 48 or EVO 96 chip, our optimized protocol facilitates lysis, formalin de-crosslinking, and tryptic digest of low-input archival tissue samples. The seamless integration with the Evosep ONE LC system by centrifugation allows ‘on-the-fly’ sample clean-up, particularly pertinent for laser microdissection workflows. We validate our method in human tonsil archival tissue, where we profile proteomes of spatially-defined B-cell, T-cell, and epithelial microregions of 4000 μm2 to a depth of ∼2000 proteins and with high cell type specificity. We finally provide detailed equipment templates and experimental guidelines for broad accessibility.
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•A scalable sample preparation workflow for spatial tissue proteomics.•Pipeline coupling laser microdissection to the cellenONE robotic system.•Optimized protocol for the processing of up to 192 samples in 3 to 4 h.•Loss-reduced sample transfer by centrifugation for peptide clean-up.
Spatial tissue proteomics integrating whole-slide imaging, laser microdissection, and ultrasensitive mass spectrometry is a powerful approach to link cellular phenotypes to functional proteome states but relies on streamlined and robust sample preparation protocols. This work introduces an automated sample preparation protocol for laser microdissected samples utilizing the cellenONE robotic system. The optimized protocol was finally validated in the archival tissue of murine liver and human tonsil. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1535-9476 1535-9484 |
DOI: | 10.1016/j.mcpro.2024.100750 |