Abstract
Laser capture microdissection (LCM) is a powerful tool that enables the isolation of specific cell types from tissue sections, overcoming the problem of tissue heterogeneity and contamination. This study combined the LCM with isotope-coded affinity tag (ICAT) technology and two-dimensional liquid chromatography to investigate the qualitative and quantitative proteomes of hepatocellular carcinoma (HCC). The effects of three different histochemical stains on tissue sections have been compared, and toluidine blue stain was proved as the most suitable stain for LCM followed by proteomic analysis. The solubilized proteins from microdissected HCC and non-HCC hepatocytes were qualitatively and quantitatively analyzed with two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) alone or coupled with cleavable ICAT labeling technology. A total of 644 proteins were qualitative identified, and 261 proteins were unambiguously quantitated. These results show that the clinical proteomic method using LCM coupled with ICAT and 2D-LC-MS/MS can carry out not only large-scale but also accurate qualitative and quantitative analysis.
Highlights
Laser capture microdissection (LCM) is a powerful tool that enables the isolation of specific cell types from tissue sections, overcoming the problem of tissue heterogeneity and contamination
Selection of cell types of interest by dissection has received a MS, two-dimensional liquid chromatography coupled with tandem mass spectrometry; NESP, nonenzymatic sample preparation; 2DE, two-dimensional electrophoresis; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; DTT, dithiothreitol; IAA, iodoacetamide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; GO, Gene Ontology
Our results show these advantages of LCM technology
Summary
Tissues were washed several times with cold glutamine-free RPMI 1640 medium (glutamine-free, 5% fetal calf serum, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, and antibiotics: 25 g/ml oxacillin, 50 g/ml gentamycin, 100 U/ml penicillin, 100 g/ml streptomycin, 0.25 g/ml amphotericin B, 50 U/ml nistatin) and were homogenized in liquid nitrogen-cooled mortar and pestle These obtained cells were dissolved in lysis buffer (8 mol/liter urea, 4% CHAPS, 40 mmol/liter Tris, 65 mmol/liter DTT). Laser capture microdissected HCC and non-HCC hepatocytes were dissolved in lysis buffer (8 mol/liter urea, 4% CHAPS, 40 mmol/liter Tris, 65 mmol/liter DTT). CICAT Labeling of Proteins—A total of 100 g of HCC and 100 g of non-HCC soluble proteins prepared by LCM technology were reduced with tri-n-butylphosphate (final concentration 5 mmol/liter). All identified proteins were classified by their molecular function, cellular component, and biological process with the tools on www.geneontology.org
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