Analysis of laser capture microdissected cells by 2-dimensional gel electrophoresis.
Laser capture microdissection (LCM) is a powerful tool for procuring near-pure populations of targeted cell types from specific microscopic regions of tissue sections, by overcoming problems due to tissue heterogeneity and minimizing intermixture and contamination by other cell types. The combination of LCM with various proteomic technologies has enabled high-throughput molecular analysis of human tumors, and provided critical tools in the search for novel disease markers and therapeutic targets. As an example, we describe the application of LCM in dissecting the tumor cells in breast cancer for macromolecular extraction and subsequent protein separation by 2-dimensional gel electrophoresis (2-D GE). The protocols and the key issues involved in preparing ethanol-fixed paraffin-embedded tissue blocks and microscopic sections, microdissecting the cells of interest using the PixCell II LCM system, extracting and separating the cellular proteins by 2-D GE, and preparing selective proteins for peptide mass analysis by mass spectrometry, are discussed. The aim is to provide a practical guide in performing high-throughput microdissection of target cells and gel-based proteomics, which can be adapted to research in cancer formation and growth.
Zhang
Daohai
D
Molecular Diagnosis Center, Department of Laboratory Medicine, National University Hospital, Singapore.
Koay
Evelyn Siew-Chuan
ES
D014408:Biomarkers, Tumor / Q000302:isolation & purification; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D002469:Cell Separation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005260:Female; D006801:Humans; D007525:Isoelectric Focusing; D007834:Lasers; D042282:Microdissection / Q000379:methods*; D009363:Neoplasm Proteins / Q000302:isolation & purification; D016612:Paraffin Embedding; D020543:Proteome / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling; D014357:Trypsin
D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D020543:Proteome; D018719:Erb-b2 Receptor Tyrosine Kinases; D014357:Trypsin; C508053:ERBB2 protein, human