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

Proteomics Approaches Employed in HPV-Related Studies

The advent of proteomics in the last decade has provided new insights in the discovery of molecular pathways influenced by HPV infection, direct and indirect interactions of HPV oncoproteins with cellular proteins and novel biological markers for the screening, early diagnosis and prediction of cervical cancer.[23]

Proteomics is the broad-range analysis of the proteome in a cell, tissue or whole organism at a certain time under defined conditions. Proteomics aim to uncover the structure of cellular proteome in order to build a comprehensive protein map indicating protein expression and possibly localization. The proteome is influenced by alternative mRNA splicing, protein modifications (e.g., oxidation, acetylation or phosphorylation among others) and protein stability. Thus allow to decipher not only changes of protein expression levels but also post-translational modifications (PTMs) which are fundamental to regulate protein functions. The field of proteomics has largely improved, due to the advances in the accuracy, sensitivity and speed of MS and to the expansion of bioinformatics. A typical proteomics experiment involves a number of different steps and depending on the different separation methods we distinguish two main approaches: gel-based or gel-free proteomics (Figure 2).[24]

Figure 2.

Proteomics strategies employed in the studies of HPV transformed cells and cervical carcinoma. At the bottom are highlighted the merit and demerit of each proteomics technique.
Methods are described in paragraph 3.
HPV: Human papillomavirus.

Gel-based

Protein quantitation is often achieved by gel-based protein separation that allows the comparison of the intensity of protein spots among different samples before their excision and identification with MS.

Since its introduction 2DE (2D-PAGE) has evolved at different levels becoming the method of choice for differential protein expression analysis in HPV transformed cell extract as well as cervical cancer tissue.[25,26] During isoelectric focusing, proteins are separated using immobilized pH gradient (IPG) strip, while in the second dimension, proteins are separated according to their molecular weight by SDS-PAGE.[27] The post gel detection is achieved either by the use of stains such as silver and coomassie or fluorescent stains such as sypro ruby. Gels are visualized and analyzed by specialized software for image analyses that allow the matching and differential quantitation of gel spots. Target spots are excised from the gels and digested to peptides by proteolytic enzymes. Individual resolved spots are then sequentially analyzed by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS or electrospray ionization tandem mass spectrometry (ESI-MS/MS). Protein identification after MALDI-TOF analysis is achieved by peptide mass fingerprint (PMF), established by matching the experimental masses of peptide, obtained by proteolytic digestion, vs the theoretical masses reported in specific databases.[28]

2D DIGE represents a refinement of 2DE proteomics by labeling different protein mixture with specific fluorescent dyes, prior to sample separation. This method reduces variability of 2DE displaying different signals in a single 2D gel and offers a method quantitatively more reliable than colorimetric ones.[29] The 2D-DIGE pre-electrophoretic labeling of samples relies on the use of three different fluorescent CyDyes (Cy2, Cy3 and Cy5), which have an NHS-ester reactive group able to covalently attaches to the ε-amino group of protein lysines. Only about 3% of the total amount of each protein is tagged. The total protein amount is then mixed and resolved in a single 2D gel. Inter-gel comparability is achieved by the use of an internal standard labeled with Cy2 included in all gels to normalize protein abundance measurements across multiple gel experiments.[30] The protein spot patterns are visualized by alternately gel illumination with the excitation wavelengths for each of the fluorescent dyes. The 2D images achieved are then analyzed by specific software such as DeCyder (GE-Healthcare), and submitted to classic 2DE procedure for protein identification and quantitation.

Redox proteomics technique was applied in our laboratory to HPV-16 dysplastic and neoplastic tissues in order to get insights about the role of OS in HPV-driven viral carcinogenesis.[31] Redox proteomics allows the identification of oxidatively modified proteins providing new insight for the comprehension of pathologies mechanism and biomarker discovery.[32] The redox proteomics approach combines 2DE with immunochemical methods using specific antibodies that recognize proteins containing oxidized groups (protein carbonyl/3NT/HNE).[33] The membranes obtained are analyzed by image analysis and normalized on protein expression to obtain spot with significant differential oxidation. Spots are than excised from the corresponding gels, in-gel digested and submitted to MS analysis.

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