Chromatography Forum

Hi,

I have some old samples that I want analyze by LC-MS for differenal analysis. These samples are peptide digests and represent different experimental conditions. The total amounts of peptides from these samples are known, however since they are sitting in a freeze for a long time (several months), I am skeptical about the peptide assays.
My question is, what is the best way to normalize the sample-loading amounts on the LC column so that any varioations in the peptide abundance at the end of the LC-MS experiment can be atributed to experimental conditions rather than sample-loading differences?

Here is what I think : Run initial LC-MS runs on the samples with a fixed injection volume. Compare either BPC/MS spectra of some of the commonly observed peaks (either peak intensities or peak areas?).
Based on these differences, apply appropriate dilution factor to the concentrated sample and run LC-MS analysis.

Any suggestions/comments are greatly appreciated!

Thanks!
Arti

Your approach is valid. Most people would perform a BCA assay and normalize accordingly.

Thank you for your reply.

Does anyone know if using peak area is better than peak intensity while doing the normalization?

Thanks again!
Arti

Generally speaking peak area will be better. That said, have a look at your peak shapes for high and low abundant peptides as the integration might be off depending on your electrospray stability... If this is the case, you might have to do some additional post-processing...