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

Gel-free

Shotgun proteomics include bottom-up proteomics techniques, which allow the identification of proteins in complex mixtures using a combination of HPLC and MS. Shotgun analyses are commonly used on a standardized platform to compare proteome differences corresponding to disease phenotypes, molecular characteristics and responses to stimuli.[34] Recently, shotgun proteomics was applied to human thinprep cervical smear using isobaric tag for relative and absolute quantitation (iTRAQ) mass-tagging and 2D LC-FT-Orbitrap-MS.[35]

In LC-MS/MS-based approaches, whole protein extract is digested to generate peptides, using proteolytic enzyme and separated through fractionation steps and analyzed using MS/MS analysis (usually ESI). Liquid chromatography by HPLC and usually reverse-phase (RP) chromatography allows the separation according to hydrophobicity. The peptides trapped in the stationary phase are then eluted and directly submitted to MS analyses.[36]

Multidimensional protein identification technology (MudiPIT) consists of a 2D-chromatography separation, prior to ESI. This procedure allows greater separation of peptides that can be directly interfaced with the ion source of a MS, which maximizes sensitivity. The first dimension is normally a strong cation exchange and the second dimension is a RP chromatography.[37] However, several orthogonal combinations including size exclusion chromatography and affinity chromatography can be performed.[36,38]

Quantitative Approaches

SILAC is one of the metabolic labeling techniques in quantitative proteomics. In this technique two populations are grown in culture media that are identical except that one of them contains a 'light' and the other a 'heavy' form of a particular amino acid, usually arginine and/or lysine that are labeled with incorporation of stable isotopic nuclei (e.g.,13C atoms and/or 15N atoms).[38,39] Digestion of these proteins from the mixtures leads to peptides with a heavy or light amino acid at their C terminus. In MS spectra, each peptide appears twice with light and heavy amino acids labeling but identical physicochemical properties. The ratio of the two identical peak intensities yields the protein abundance in the sample.

One of the most common methods used in quantitative proteomics, to evaluate the abundance of expressed proteins, is based on the use of isotope-coded affinity tag (ICAT) reagents. The ICAT approach consists of tagging samples populations with chemical reagents, which consists of a thio-reactive group, an isotopic linker region and an acid cleavable biotin moiety, with different mass. Samples labeled with heavy or light mass reagents are then pooled, proteolytically cleaved and subjected to cation and then avidin chromatography purification to reduce their complexity prior to MS analysis. Then samples are analyzed by MS/MS analysis with an ESI-tandem mass spectrometer and proteins are identified by automated database searching.[30,38]

iTRAQ represents an improvement of quantitative proteomic analyses using amine-specific stable isotope reagents for peptide labeling and allowing to perform a multiplexed analysis (up to eight biological samples). In this approach the N-terminus of peptides, obtained by protein mixture digestion, is chemically modified with isobaric tags. Labeled peptides are then combined and separated by LC to obtain reporter ions evident at low m/z values in MS/MS analysis.[38] Then, the identification of labeled peptides, by database searching, allows the identification of corresponding protein. iTRAQ technique was recently applied to human thin prep cervical smear HPV infected and non-providing a global whole new set of qualitative and quantitative data[35]

SELDI is a variation of MALDI that uses a target modified to achieve biochemical affinity with the analyte compound. The analytical procedure begins with chromatographic separation where the sample is pre-fractionated by protein chip arrays with specific chromatographic features (cationic, anionic, hydrophobic, hydrophilic and ion metal chelating).[30] The proteins actively interact with the chromatographic array surface, and become sequestered according to their surface interaction potential. SELDI is typically used with time-TOF-MS and is used to detect proteins in tissue samples, blood, urine or other clinical samples.[24]

processing....