Web Release Date: September 29,
Proteomic Analysis of High-Grade Dysplastic Cervical Cells Obtained from ThinPrep Slides Using Laser Capture Microdissection and Mass Spectrometry
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Northeastern University, Boston, Massachusetts 02115, Cytyc Corporation, Marlborough, Massachusetts 01752, and University of Nebraska Medical Center, Omaha, Nebraska 68132
Received May 25, 2007
Abstract:
The purpose of this discovery phase study was to identify candidate protein biomarkers for high-grade dysplastic cervical cells using mass spectrometry. Laser Capture Microdissection (LCM) was utilized to isolate high-grade dysplastic and normal cells from ThinPrep slides prepared from cervical cytological specimens. Following cell capture, samples were solubilized and proteins separated by gel electrophoresis in preparation for enzymatic digestion and liquid chromatography mass spectrometry analysis (LC-MS). Processed samples were subsequently analyzed using a linear ion trap coupled with a Fourier transform mass spectrometer (LTQ-FT MS). It was determined that both PreservCyt Solution and ThinPrep Pap Stain (Cytyc Corporation) were compatible with the sample processing and LC-MS analysis. In total, from 9 normal and 9 abnormal cervical cytological specimens, more than 1000 unique proteins were identified with high confidence, based on approximately 12 000 captured cells per specimen. Quantitative protein differences between HSIL (High-Grade Squamous Intraepithelial Lesion) and NILM (Negative for Intraepithelial Lesions or Malignancy) samples were determined by comparing the intensities of the representative (label-free) peptide ions. More than 200 proteins were found to exhibit a 3-fold difference in protein level. Interestingly, significant up-regulation of nuclear and mitochondrial proteins in HSIL specimens was noted. In several cases, the increased protein abundance observed in high-grade cells, as determined by quantitative LC-MS, was validated by immunocytochemical methods using ThinPrep cervical specimens. With the study of additional clinical specimens, the differential abundance of proteins in high-grade dysplastic cells versus morphologically normal cervical cells may lead to validated novel biomarkers for cervical disease.
Keywords: laser capture microdissection 
mass spectrometry cervical cancer tissue 10 000 cells label-free
quatitation LC-MS HSIL HPV CIN
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