Laser Capture Microdissection Applications in Breast Cancer Proteomics
Breast cancer tissues are characterized by cellular heterogeneity, representing a mixture of, e.g., healthy epithelial ducts, invasive or in situ tumor cells, surrounding stroma, infiltrating immune cells, blood vessels, and capillaries. As a consequence, protein extracts from whole tissue lysates also represent a variety of cell types present in the tissues under examination. This, however, seriously hampers the analysis of tumor cell-specific signals, which is of interest when performing biomarker discovery-type of studies. Therefore, laser capture microdissection is a perfect tool to isolate a relatively pure population of cells of interest, such as tumor cells. In this chapter, we describe the use of the PALM MicroBeam system for laser microdissection and pressure catapulting. Protocols are provided for sectioning, staining, microdissection, sample preparation, and mass spectrometric analysis of snap frozen breast cancer tissue.
Key wordsBreast cancer Laser capture microdissection nLC MALDI Mass spectrometry Proteomics Biomarker discovery
The authors wish to thank Annemieke Timmermans for helpful discussions and technical assistance on tissue sectioning and staining. RBHB and AU are (partly) financially supported through the Center for Translational Molecular Medicine, CTMM BreastCARE project 030–104, and through the Netherlands Genomics Initiative.
- 6.Micke P, A Ostman, J Lundeberg et al (2005) Laser-assisted cell microdissection using the PALM system. Methods Mol Biol 293:151–66Google Scholar
- 12.Umar A, JC Dalebout, AM Timmermans et al (2005) Method optimisation for peptide profiling of microdissected breast carcinoma tissue by matrix-assisted laser desorption/ionisation-time of flight and matrix-assisted laser desorption/ionisation-time of flight/time of flight-mass spectrometry. Proteomics 5:2680–8CrossRefGoogle Scholar