I have conducted an MRM analysis of plasma proteins using UPLC-QqQ targeted to 8 proteins without standards available. To assess sample preparation and recovery, I used yeast dehydrogenase peptides as label-free internal standard, but I am not sure of the algorithms suitable on normalising the data to correct for the internal standard. Can anyone advise if it is best to look at recovery against the average of the YADH peptides from all the samples or should it be based against a QC (pooled) sample ran in the same batch?
Depends if you're looking to do absolute or relative quantitation. If you're want to absolute quantitation then YADH peptides won't do, you will need synthetic peptides (or their isotopic heavy labeled counterparts which you can spike in and monitor simultaneously) of the exact same peptides your MRM assay is monitoring and perform a standard curve. Although I don't have experience with plasma proteomics I would recommend heavy internal standards that way you can generate a standard curve in the samples and account for the matrix effect. If you're looking to do relative (ie run to run) type quantitative experiments then YADH or any other internal peptides (not from human proteins) will do.
When I do this, I normally divide the peak area of the peptides of interest by the peak area of the internal standards, thus normalising the peak areas for the response I get for the internal standards on a run by run basis. This is a much better way to normalise, in my opinion, than to use an average.
This works well for me and I find the variation is quite low. If you are not using Skyline for your analysis, I strongly recommend you have a look, brilliant software.
https://skyline.gs.washington.edu/
Hope that helps
Thank you for your advice. It is for relative quantification purposes only so I will use the YADH peptides. The only problem is the YADH peptides often result in a much greater peak area than the peptides of interest so when I divide by this I get a very low output figure and I wasn't sure if I should then be multiplying up by another factor?
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