I know that cytokines are proteins, and HPLC technique employed in protein detection. But the question is can we use HPLC for cytokine detection in a dissolvable sample? Can de detect them with MS? What will be the protocol and the reagents? Cytokines are TNF-alpha, IFN-gamma and IL-12. I would really appreciate help.
Definitely maybe. HPLC can pick up some of the changes that inactivate cytokines.
Chemical modifications like oxidation, nitrosylation, glycation will often be apparent, if you digest with trypsin and resolve the peptides on a denaturing gradient (C18 water -> ACN + 0.1% TFA). You need to know what masses to look for.
Proteolysis and aggregation are going to be visible on a native SEC column (Superdex or Yarra, in PBS).
In practice, both of these together will catch many things that will happen to many cytokines, and they're both used in biopharmaceutical QC. But there can be other problems that would go unnoticed on either method, like conformational changes. To be as sure as you can be that your cytokine is OK, you would add an assay for biological activity in a cell culture model.
Article A new approach for cytokinin isolation from Arabidopsis tiss...
pls read this you may get some idea
Hi Mr Akmandor,
I agree with John Schloendorn ,
As a short answer, in case of mixture of these cytokines, both MS and LC-UV detection is capable to identify which is up-regulated or down-regulated.
For MS detection, un-targeted proteomics based studies could be performed at first in two way and these ones would be time consuming for routine testing for cytkoines at method development stage.
At first step, after top down or bottom-up approach, you should identify unique peptides that represents these cytokines than you may transfer these peptide informations based on ionization parameters, m/z values to targeted MRM (multiple reaction monitoring) acqusition for targeted proteomics.
This is the typical MS based methodology for biomarker identification. Sample pretreatment at method development like protein extraction-enrichment, fractionation, clean-up steps should be carefully conducted and validated otherwise biological samples are very complicated matrices to appropriate peptide selections.
When it comes to HPLC, chromatographic seperation much critical. You need to select high resolution LC protein column to seperate these 3 cytokines that you mentioned. Regarding their charges, moleculer weights, affinities and polarities you may accomplish to get resolution applying IEX, HIC, SEC or affinity chromatography.
At both methods, as authors declared, conformational changes and PTMs should also be in consideration and these differentiations must be charactherized prior to analysis.
Emir...
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