A Microdissection Protocol for Proteogenomic Analysis of Histological Sections to Advance Drug Development.
Methods Mol Biol
; 2823: 55-75, 2024.
Article
in En
| MEDLINE
| ID: mdl-39052214
ABSTRACT
Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the different microenvironment components. This is especially important for immuno-oncology (IO) research where improvements in the predictability of IO-based drugs are sorely needed, and depends on a better understanding of the spatial relationships involving the tumor, blood supply, and immune cell interactions, in the context of their associated microenvironments. LCM is used to isolate and obtain distinct histological cell types, which may be routinely performed on complex and heterogeneous solid tumor specimens. Once cells have been captured, nucleic acids and proteins may be extracted for in-depth multimodality molecular profiling assays. Optimizing the minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, methylation profiles, etc. However, there remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of this protocol is an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. Included is a detailed in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Described is a proteogenomic approach leveraged from microdissected fresh frozen tissue. The protocols may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.
Key words
1D SDS-PAGE; Biomarker discovery; Drug discovery; Fresh frozen tissue; Genomics; Immuno-oncology; Laser capture microdissection (LCM); Liquid chromatography mass spectrometry (LC-MS)-based proteomics; Parenchyma; Proteogenomics; Proteomics; Silver stain; Solid tumor heterogeneity; Stroma; Tumor microenvironment
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Laser Capture Microdissection
/
Proteogenomics
Limits:
Humans
Language:
En
Journal:
Methods Mol Biol
Journal subject:
BIOLOGIA MOLECULAR
Year:
2024
Document type:
Article
Affiliation country: