Proteomic Analysis of High-Grade Dysplastic Cervical Cells Obtained from ThinPrep Slides Using Laser Capture Microdissection and Mass Spectrometry

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.

Differential tissue-specific protein markers of vaginal carcinoma

“…In high-grade dysplastic cervical cells filamin, vimentin and vinculin were upregulated (Gu et al, 2007) in contrast to the findings in cancer cells in our study.…”

Enhanced Interferon Signaling Pathway in Oral Cancer Revealed by Quantitative Proteome Analysis of Microdissected Specimens Using 16O/18O Labeling and Integrated Two-dimensional LC-ESI-MALDI Tandem MS

“…Laser capture microdissection is often used in conjunction with MS-based protein identification technology to assist with the discovery of tumor-associated molecules in tissue specimens containing various types of cells (27,31,(61)(62)(63)(64). However, relatively few studies have used these techniques to identify OSCC/HNSCC-associated proteins in tissue specimens from patients with OSCC/HNSCC (15,17,29).…”

In Situ Proteomic Analysis of Human Breast Cancer Epithelial Cells Using Laser Capture Microdissection: Annotation by Protein Set Enrichment Analysis and Gene Ontology

“…Each gel section was separately subjected to in-gel tryptic digestion as described previously (24). Briefly, proteins were reduced and alkylated, and incubated with trypsin for 18 h at 37°C.…”

“…Using only the limited amount of material collected by LCM (60,000 cells), a proteomic profile of each sample was separately acquired by label-free proteomics using our previously developed platform for analysis of LCM samples (24). To the best of our knowledge, this is the most comprehensive global study that compares proteomic signatures of phenotypically normal and ERϩ invasive malignant breast epithelial cells in multiple individual samples.…”