Whether the proteomic discovery phase is carried out using gel-based or gel-free MS approaches or microarray-based methodologies, the identification and differential expression of all putative biomarkers must be confirmed using further independent techniques (Paulovich et al., 2008). In addition to false discovery due to the use of a high throughput ‘omic’ technology there are now also a number of human RIDEPs (sections 3.3.3 and 3.5.1) which require careful scrutinisation (Hodgkinson et al., 2011, Petrak et al., 2008).
3.6.1 Data mining
The main aim of high-throughput technologies currently used within proteomic investigations, is to screen samples with the intent of generating hundreds of potentially interesting proteins, all of which require further confirmation and validation. Such further investigation methods are generally of higher accuracy but carried out on a smaller scale (Qian and Huang, 2005). Therefore, in order to identify and prioritise such proteins for further investigation, software is employed to interpret the data using knowledge databases. Ingenuity Pathway Analysis (IPA) (Ingenuity Systems Inc., USA) is one example of such software. Using this online facility, generated protein lists can be uploaded into the software, where they are then analysed against the Ingenuity Knowledge Base. The software then highlights all relationships (direct or indirect) between the candidate proteins
55 using different networks and canonical pathways for illustration. Through use of this software, researchers can carry out virtual investigations, helping to further understand and prioritise a selection of proteins for subsequent technical (section 3.6.2) and clinical validation (section 3.6.3).
3.6.2 Western Blotting
Semiquantitive Western blotting (also known as immunoblotting) coupled with densitometry analysis is a widely used method for the co-confirmation of differential expression and protein identification. Following protein extraction from either cell line or tissue origin, the first step of Western blotting is the separation of proteins by electrical charge using a polyacrylamide gel. A known quantity of protein extract is mixed with Laemmli buffer, containing sodium dodecyl sulphate (SDS) to unfold and reduce the proteins whilst giving them a net negative charge, and β-Mercaptoethanol to reduce disulphide bonds causing the protein to revert back to its primary conformation prior to separation. The protein sample is then loaded into the gel, separated by molecular weight and transferred onto a nitrocellulose membrane. Once proteins have transferred it is necessary to ‘block’ the free sites on the membrane using either bovine serum albumin or non-fat dried milk powder. This step ensures no non-specific binding of the probing antibody to the membrane (only to the protein of interest). A primary antibody, specific to a protein of interest, is then incubated with the membrane, enabling it to bind to its target protein if it is present. After a brief washing step to remove any unbound antibody, one commonly used method for the visualisation of protein expression is the use of chemiluminescence, employing a horseradish peroxidise (HRP) conjugated secondary. The HRP enables the production of a signal in the form of luminescence by catalysing the decomposition of the chemiluminescent reagent. Relative amount of protein can then be visualised by exposure to photographic film. The presence of an exposed band indicates the presence of the target protein within the sample, with band intensity being proportional to the amount of protein present. The photographic film can then undergo quantification using densitometry. During this process, target proteins are normalised against loading controls or anti-‘housekeeping’ antibodies (e.g anti-alpha tubulin, anti-beta actin or anti-GAPDH) which should demonstrate constant levels of expression within the protein sample. Through use of these loading controls, comparisons between band intensity produced by the primary
56 antibody can be made, enabling a quantitation of fold-change in expression to be calculated. However, Western blotting requires the availability of a reliable primary antibody specific to the precise protein identified from proteomic analysis. Where suitable antibodies do not exist, further analysis at the mRNA level using reverse transcriptase polymerase chain reaction (RT-PCR) or real time quantitative PCR (RTqPCR) can be employed for confirmation of differential transcript expression. If a quantitative or semi- quantitative method is employed, which utilises an appropriate house-keeping gene/protein as the internal control reference within each sample, then a 2-fold difference in expression between samples is commonly regarded as significant. In vitro gene silencing through the use of small interfering RNA (siRNA) molecules to cause RNA interference (RNAi) is also frequently used to confirm the cellular effects of aberrant gene knockdown. Similarly, protein function blockade via small molecule inhibitors or monoclonal antibodies can be used to demonstrate the effects in vitro on signal transduction.
3.6.3 Clinical validation
In vitro confirmation of differential expression or functional effect within experimental test
samples does not necessarily equate to clinical relevance (Paulovich et al., 2008). To validate those putative biomarkers that successfully pass through technical validation (section 3.6.2), the clinical significance must be tested using clinical samples with relevant clinical information. Frequently this is initially carried out using immunohistochemistry (IHC) on a series of retrospective archival tumour samples. IHC can be used to validate the expression and localisation of proteins in whole sections of formalin-fixed, paraffin- embedded (FFPE) clinical tissue samples mounted on glass microscope slides. Whilst this particular method is low throughput an alternative high throughput approach in the form of a suitable tissue microarray (TMA) could be employed (Hassan et al., 2008). This method involves removing cores of tissue from hundreds of different formalin-fixed paraffin- embedded (FFPE) samples and co-embedding the selected cores into a new TMA block enabling a single slide to be screened simultaneously for the expression of one particular protein using IHC. Alternative approaches for clinical validation, include the use of an ELISA, MS-based multireaction monitoring (MRM) or reverse phase assays (RPA) (Pan et al., 2009).
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