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Proteomics-based Approaches for the Isolation, Detection and Analysis of the Blood Plasma Proteome with Specific Interest in Cytokine Activity Involved in SAPHO.
The study of proteomics is essential for our understanding of how cells function. By learning how the proteins within cells interact and respond to certain conditions, allows us to discover diagnostic protein biomarkers and create new therapies for specific diseases and other conditions. One group of diseases which often have no cure and require lifelong treatment are autoimmune diseases. These diseases cause an ‘immune-mediated attack by the body on its own organs, tissue or cells’ (Rose 2008) which can have serious health consequences for those affected, especially when gone untreated or diagnosis is difficult to reach. Synovitis Acne Pustulosis Hyperostosis and Osteitis (SAPHO), is a very rare autoimmune disease that carries many health implications, making it an extremely difficult disease to diagnose and treat. The effects of SAPHO are chronic bone and joint inflammation, increased bone density (often in particular the sternum or clavicle) and pus blisters (acne or palmo-plantar pustulosis).
Study of diseases such as SAPHO through proteomic-based research methods allow for the analysis of the entire proteome, which is essential to obtain a comprehensive view of the cellular interactions caused by the disease. There are also proteomic-based methods which are able to target specific proteins of interest, for example biomarkers such as cytokines. Cytokines are small secreted immunoregulatory proteins that play crucial role in pro and anti-inflammatory responses. Proteomic analysis of cytokines is important because they ‘possess diagnostic and prognostic potential and cytokine production may reflect effects of immunotherapies’ (Skalnikova et al. 2017).
There are two proteomic-based approaches when analysing a sample, top-down and bottom-up. A study by Castagnola et al. (2017) found that a top-down HPLC-ESIMS and MS/MS approach for the analysis of salivary biomarkers involved in SAPHO showed a pattern in individuals with the disease. Taking into account their research, a similar approach will be taken in this experimental design to analyse the whole plasma proteome. This study also focuses on blood plasma cytokines, but previous research on the analysis of plasma cytokines involved in SAPHO is not readily available. Therefore, a more specific approach has been created to isolate the albumin and cytokines to detect differences between inflammatory biomarkers between healthy and SAPHO affected individuals through a bottom-up approach.
2. Experimental Design
2.1. Sample Collection
In this experiment blood samples will be used because although the analysis of saliva samples have lower cost and saliva is easier to obtain and transport, they also carry higher risk of bacterial contamination. Additionally, as indicated by Gauri & Desai (2014), ‘research is still needed to validate salivary biomarkers, establish reference ranges and characterise the influence of diet and drug treatments’ in the results obtained from saliva and Bozdon-Kulakowska et al. (2007) stated that plasma is recommended instead of serum. As this experiment uses human samples, all ethical procedures will be adhered to and approval by the Human Ethics Committee will be acquired prior to the commencement of the experiment. The samples will comprise two groups, control (healthy patients) and treatment (SAPHO positive patients).
2.2. Sample Preparation
Sample preparation is essential to ensure high quality results that are an accurate representation of the disease being studied. Samples of blood plasma will be used instead of serum, plasma is ‘the soluble fraction of anticoagulated blood’ (Burska et al. 2014) and investigations showed that it produces ‘the most consistent results for many cytokines’ (Zhou et al. 2010). Samples need to be centrifuged immediately in anticoagulant EDTA-treated tubes for 15 minutes at 5000 rpm to separate the whole blood into red blood cells (RBCs) and plasma, to prevent the coagulation process. Although there are other anticoagulant treated tubes available such as heparin-treated tubes, heparin ‘can often become contaminated with endotoxin, which can stimulate white blood cells to release cytokines’ (ThermoFisher 2007) and citrate produces the ‘reduction of total protein concentration due to dilution’ (Burska et al. 2014). If this occurred, the results could not be confidently relied on as being indicative of inflammatory biomarkers present in SAPHO.
2.2.1. Removal of Contaminants
Working with blood samples can be difficult due to the complexity and presence of high-abundant proteins such as albumin. Removal of these contaminants is essential, however, studies by Granger et al. (2005) and Yasuda et al. (2012) have shown that the removal of albumin also decreases the number of cytokines in the sample affecting the results. Albumin is a carrier protein that has the ability ‘to bind biologically significant molecules such as cytokines’ (Granger et al. 2005). Due to this, it is important that albumin is removed and analysed as a separate sample. This will show which cytokines are reduced due to albumin depletion and possibly provide information for new target therapies.
Other contaminants such as lipids are readily removed by processes such as acetone precipitation. While this method is effective it has a few disadvantages such as loss of low-abundant proteins, addition of ions that interfere with IEF (Isoelectric focusing) and can require overnight incubation. To combat these issues the use of a 2-D Clean-up Kit (GE Healthcare) will remove detergents, salts, lipids, phenolics and nucleic acids without the use of chaotropes and other common reagents. As protein recovery is >90% and the procedure can be completed within an hour, this method is more efficient for IEF preparation.
2.2.2. Total protein quantification
Assay techniques are ‘crucial for quantification in basic research and clinical settings’ as was indicated by Skalnikova et al. (2017), specifically to determine the total protein in a sample after clean-up and prior to fractionation. The two main protein assays are the Lowry bicinchoninic acid assay (BCA assay) and Bradford protein assay, however the BCA assay is more sensitive therefore it will be used here to quantify the protein.
The ‘cleaned’ samples from healthy and SAPHO affected individuals require fractionation to reduce complexity and analyse a higher amount of information from the proteins of interest. By eluting the samples through an affinity column, it is possible to separate the albumin and cytokines from each sample. Affinity chromatography, a type of liquid chromatography, is a diverse chromatographic method for protein purification. Extracting the albumin and cytokines makes it possible to stain the samples obtained using fluorescence, (1) pooled sample (standard), (2) albumin and (3) cytokines and further analyse them through a 2D-DIGE.
In this design a boronate affinity column which uses ligands to bind the target protein will be used. Another possible method for albumin extraction is simulated bed moving chromatography (SMB), while this method has high efficiency and produces highly pure albumin ‘the limitation of isocratic elution mode is a major problem’ (Raoufinia et al. 2016).
For cytokine purification, an assay will be used to select for pro- and anti-inflammatory molecules while the other proteins are washed out.
2.3.1. Two-dimensional difference gel electrophoresis (2D-DIGE)
After albumin and cytokines have been extracted from the samples, two 2D-DIGE tests will be done to assess the differences in protein expression between the healthy and SAPHO affected individuals. In one 2D-DIGE multiple protein samples are able to be simultaneously detected due to co-migration through the use of cyanine fluorescent dyes (Cy2, Cy3 and Cy5). Compared to traditional post-staining in 2D-PAGE e.g. colloidal Coomassie, 2D-DIGE ‘provides faster and more reliable gel matching, limiting the impact of gel to gel variation, and also allows a good dynamic range for quantitative comparisons’ (Pasquali et al. 2017). In turn, it also reduces experimental and analytical time. This method will be used to analyse the albumin and cytokine samples from both groups.
Alternatively, liquid chromatography (as above) can be used directly before mass spectrometry analysis without the use of a gel. Although, by using a 2D-DIGE a fluorescent representation of differential protein expression allows for the selection of a specific analysis.
2.3.2. Trypsin Digestion
Spots on the gel that do not overlap (are different between the two groups) when overlaying the fluorescent images, will be cut from the gel and digested with trypsin. Post digestion, the gel pieces need to be recovered by rehydration to extract the peptide fragments which will be analysed by mass spectrometry in a bottom-up approach to determine which pro- and anti-inflammatory biomarkers are present and/or absent in SAPHO.
The difficulty of finding an appropriate analytical method for a given samples is made easier with the large range of proteomic instruments available for both top-down and bottom-up approaches. Ferguson & Smith (2003) discuss that while the bottom-up approach has ‘higher mass measurement accuracy’ when analysing low-abundant proteins, the pre-digestion of the proteins prior to mass spectrometry analysis ‘result in greatly increased sample complexity’ while ‘complete sequence coverage of proteins is rarely achieved’. This disadvantages decreas ‘the ability to examine site-specific mutations and post-translational modifications of individual proteins’ which is not ideal as they are important in biological functions. The major advantage to the top-down approach is its ‘ability to observe modified proteins in their intact state, enabling the identification of modifications that would otherwise be transparent to peptide-level analysis’.
2.4.1. Whole Plasma Proteome and Albumin
As Castagnola et al. (2017) did in their experiment using saliva, a top-down approach for the analysis of intact proteins in the plasma proteome will be performed to provide information about the interactions within the cells from the two groups. ‘Studies have largely been implemented using ESI coupled to either Fourier transform ion cyclotron resonance (FT-ICR) or Orbitrap mass analysers’ (Catherman et al. 2014), however a study by Compton et al. showed that Orbitrap instruments were better than ion cyclotron instruments as they ‘provide higher resolution at high m/z’. Therefore, this analysis will be done through a LC-MS Orbitrap Fusion Mass Spectrometer (ThermoFisher).
As albumin is a carrier protein a similar top-down approach may be taken to analyse the large molecules and observe the post-translational modifications between the two groups. However, the tripsin-digested spots from the 2D-DIGE should be analysed in a bottom-up approach through the use of a Q Exactive HF hybrid Quadrupole-Orbitrap Mass Spectrometer (ThermoFisher).
Cytokines can be analysed by mass spectrometry, although it should be noted that mass spectrometry can ‘only analyse ionised peptides and not all peptides are ionised with the same efficiency’ (Skalnikova et al. 2017). However, it should still be utilised because although despite being less sensitive than immunoassays, Skalnikova et al. (2017) state that ‘mass spectrometry approaches provide relatively easy multiplexing capability and higher specificity’. In this case, an LC-MS/MS approach utilising the trypsin-digested cytokine sample from the 2D-DIGE will be used in a bottom-up approach due to increased mass accuracy compared to top-down. This will be completed with a Q Exactive HF hybrid Quadrupole-Orbitrap Mass Spectrometer (ThermoFisher).
An alternative to mass spectrometry for cytokine analysis is a bead-based multiplex immunoassay (for the detection of multiple cytokines), which uses a MACHINE to analyse the fluorescence of the samples in a 96-well plate. Other assay methods that could be used to measure cytokines are ELISAs (Enzyme-Linked Immunosorbent Assay). When comparing multiplex assays to sandwich and sequential ELISAs, multiplex assays are ‘a more rapid and cost-effective’ (Chiswick et al. 2012) and ‘allow simultaneous measurement of up to 500 proteins’ (Keustermans et al. 2013). Although multiplex assays can be affected by heterophilic and auto-antibodies, this can be easily fixed with the use of a hetero-block. This block ‘increases levels of TNF-, IL-1 and IL-6’ (Burska et al. 2014), which are biomarkers for inflammation.
3. Discussion and Conclusion
The aim of this experimental design was to assess the proteins involved in SAPHO with specific interest in inflammatory biomarkers. The methodology for each phase in the experiment was chosen to provide a reliable and indicative dataset of SAPHO. However, it should be noted that there are alternative methods for which also carry their advantages and disadvantages.
3.1. Advantages of the Chosen Methodology
Advantages of the chosen methodology include, ‘no need to remove albumin to unmask low abundance proteins’ (Granger et al. 2005) in the multiplex assay, the reduced ‘experimental and analytical time’ (Pasquali et al. 2017) by use of 2D-DIGE, and cost-effectivity of the Orbitrap as it ‘does not require expensive superconducting magnets’ (Catherman et al. 2015).
3.2. Disadvantages of the Chosen Methodology
Disadvantages of the chosen methodology include, ‘coelution of compounds with similar structure and polarity’ (Keustermans et al. 2013) through affinity chromatography, the use of an EDTA anticoagulant when acquiring plasma because this may have unknown effects on the cytokines and other low-abundant proteins, not separating the low-abundant proteins from the albumin prior to analysis as ‘nonspecific loss of cytokines in the eluted fraction may confound subsequent proteomic analysis of albumin-depleted fractions’ (Granger et al. 2005), top-down approach because it has problems with protein fractionation, protein ionization and fragmentation in the gas phase’ (Zhang et al. 2013). Another aspect of immune proteins that would be good to analyse are the immunoglobulins which were not discussed here.
Without the study of proteomics, we would not be able to create targeted drug therapies for many diseases. The methodology of this experimental design provides specific information on cytokines and whole plasma proteome, while also adding new research information about the role of albumin in SAPHO.
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