Mass spectroscopy has been in use for several decades now, advancing fields like drug testing, pesticide analysis, protein assays, and carbon dating. Under current practices, mass spectroscopy is being used in tandem with designer plasmids and bacterial models to expound upon the fields of genomics and DNA therapeutics. This technique sheds new light on the genes that code for cellular machinery, and integral proteins, which in turn can help us better understand the role of proteomics in COVID-19.
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Target Proteins in COVID-19
Housekeeping machinery such as actin, ribosomes, or ubiquitin are not present within the viral capsid, as these orthornavirae use the host’s cellular machinery to replicate and divide. Instead, the virus uses its genes to code for various proteins. The envelope of the virus encompasses a network of large glycoproteins.
The target proteins researchers are interested in when trying to expound upon the nature of the disease, are spike proteins, envelope proteins, and nucleocapsid proteins. These forms of glycoproteins are difficult to research on account of their size and sensitivity. Due to this, mass spectrometry is conducted somewhat differently when assaying.
New Research Models
The stains of the virus employed in the lab should be inert, for precautionary measures. To circumvent the handling of live virus establishments such as Cambridge, the University of Kent, and the WHO have developed lentivirus pseudo-types imbued with these larger wildtype glycoproteins and envelope proteins from the original SARS virus.
Though these central proteins are present in these models, the intrusive mRNA transcripts that are ubiquitous amongst all SARS viruses are removed, making them a great standard for study.
The Prevailing Proteomic Research Approach
Typical analysis is conducted on a sample of biological fluid, most often saliva, that is enriched with viral particles and viral proteins. This is a good medium for various reasons. One is that large pathological structures that make up the COVID-19 capsid (envelope proteins, membrane proteins, spike proteins, and nucleocapsid proteins), can be separated and characterized by their respective molecular weights.
Secondly, a wash of the mucosal layer can result in the isolation of the dimer immunoglobin A, a co-precipitate that occurs as a byproduct of mRNA isolation. This dissolution buffer wash gives us a reliable IGA response, explaining just how potent the inhibition of viral adhesion is to host epithelial cells.
To concentrate this virus, a one-to-one ratio of sample to ice-cold acetone is made, followed by centrifugation at 16,000 g at 4oC for a variable amount of time (typically 30 minutes). This results in a pellet that is abundant in heavier viral analytes, while the non-target proteins are left in the supernatant which is then discarded.
Once in pellet form, to release these larger glycoproteins from the viral envelope, a slew of dissolution buffers is used to disrupt said membrane. This can be accomplished without suppressing the ionization of the material, leaving the resulting mass spectra undisturbed.
This process does more than just break down the envelope in a systematized manner, the dithiothreitol within the buffer will also disrupt disulfide bonds, segmenting the individual subunits. For example, the spike protein can be split into its S2a, S2b, and S1 subunits, so we can garner more intel on each subunit.
COVID-19 Protein Assaying Through the Use of Liquid Chromatography-Mass Spectrometry (LC-MS)
An alternative method of viral protein extraction can present itself, using liquid chromatography fitted with mass spectrometry (LC-MS). Firstly, a urea buffer is introduced to the protein sample, which will undergo a reduction reaction via Dichlorodiphenyltrichloroethane (DDT) and an alkylation using some form of alkyl halide. Followed by this protein digestion (a breaking down of protein into amino acid building blocks), trypsin or chymotrypsin is used for further digesting and isolation. This whole slurry will incubate overnight, and after a cleaning to separate the peptides, buffers, and enzymes, the sample can then be transferred to LC-MS for data analysis.
However, these are many other native COVID-19 proteins that require alternate approaches to assess. We want a methodology to capture the proteins that bind to viral RNA at different stages, mainly transcription, and translation. We yearn to identify these proteins so that we can get an idea of how the SARS-COV2 RNA is the same or different from other viruses.
Applying Viral RNA Interactome Capture (vRIC)
To capture the full scope of how COVID-19 proteins function, researchers are now trying to segregate and isolate Viral RNA from host RNA, under transcription processes. This method is dependent on distinctions between viral RNA replication, and host RNA replication. For example, host mammalian cells contain RNA polymerase II within the nucleus to synthesize mRNA, while viral RNA is dependent on RNA polymerase I that is freely roaming in the cytoplasm of the cell.
To separate and isolate the two variants of RNA, and to explicate the nature of the illness, inhibitors, photoactive nucleotides, and RNase treatments are used sequentially. One standardized methodology developed by Kamel et al. uses the specific inhibitor Flavopiridol to block RNA Pol II, and halt host transcription altogether. 4SU photoactive nucleotides are used in tandem, efficiently integrating themselves into nascent RNA on account of Flavopiridol’s inability to impede RNA pol I.
A 16-hour incubation period is performed to ensure effective labeling using the 4SU nucleotides, followed by an irradiating of the sample using 365nm Ultraviolet light. This specific wavelength is favored because wild-type nucleotides will not respond with it, only the modified nucleotides will. The 4SU will activate and crosslink the protein bound to this RNA.
At this point, the viral mature RNA is captured, and purification of the polyadenylated tail is then done under denaturing conditions. Finally, a straightening and purification of the RNA are performed with an RNase treatment, directly followed by a mass spectrometry assay. From this, sequencing readings of the vRNA bound proteome are finally achieved. Any technique that a given lab should use, must co-align with the analyte of interest, be it glycosylated proteins, spike protein subunits, vRNA, or others.
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