Proteomics Strategies to Analyze HPV-Transformed Cells: Relevance to Cervical Cancer

Fabio Di Domenico; Federico De Marco; Marzia Perluigi

Disclosures

Expert Rev Proteomics. 2013;10(5):461-472. 

In This Article

Protein–Protein Interactions: Proteome Fractionation

Yeast-two-hybrid (Y2H) and affinity purification-mass spectrometry (AP/MS) are the two essential strategies usually used to define protein interaction, including proteins localized in different cellular compartments.[40] Both methods aim to determine the target protein (prey) of the 'bait' protein (E6 and E7 in HPV proteomics studies) and collect data in order to build a map with the whole set of bait/prey interactions (interactome). MS is used to identify prey-bound proteins. Initially, this approach was coupled with protein separation on gel and identification of proteins present in different individual bands. The recent use of LC-MS/MS made possible the high-throughput sequencing of complex protein mixtures and the identification of hundreds of potential interacting proteins from a single sample.[41]

In reverse-phase protein arrays (RPPAs) multiple samples in small amounts are probed with a single antibody to establish the expression pattern of a specific target in all the samples at the same time. RPPA allows surveying the expression of specific proteins or phosphoproteins for which specific antibodies exist but does not approach the comprehensive proteome analysis.[42] This antibody-based proteomics approach allow to explore the human functional proteome and enable the generation of a comprehensive proteomic network.[43]

Aptamer arrays exploit affinity reagents, designed to mimic antibodies, which recognize and bind target proteins and allow the analysis of specific biological processes. Peptide aptamers are constituted of peptide sequences, usually from 6–20 amino acids-long, constrained into a scaffold protein that contribute in increasing the aptamer affinity for the target protein.[44] The built of peptide aptamers array allow the separation of a protein mixture according with the sequences/properties targeted by the aptamers employed.

Tissue microarray and automated quantitative assessment of immunofluorescence (TMA-AQUA) have been recently developed and applied with success to various types of human cancer studies including HPV-related carcinogenesis. This is a powerful high-throughput technique that couple the TMA platform that principally uses immunohistochemistry method with AQUA platform that uses an immunofluorescence-based method for in situ assessment of protein concentration.[43]

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