I am interested to purify a mixture of proteins using HPLC and LC/MSMS. the analysis of HPLC fractions showed the occurrence of the same protein (but different set of peptides) in many distinct HPLC fractions that present different retention times and molecular mass in sdspage. Any explanations?
Different sets of peptides?? So u mean to say isozymes.. In that case its normal as in RP columns the retention of the peptides depends upon the hydrophobicity of the analyte.. likewise for the mass difference in SDS which can be due to either change in aa"s or the other reason might be due to PTM's! :)
difference in retention time of protein can also be due to PTM's
There can be many forms of the same polypeptide with post translational modification, proteolytic cleavages and also isozymes. N terminal or full sequencing can help to sort it out.
Thanks Kathirvel and Peter for your comments this is really helpful.
What i mean by a "set of peptides" is that we identified different peptides from the same protein (same accession number) in different HPLC fractions. its likely that we have the same protein identified everywhere. which can't be correct since they show
with diffrents molecular masses in SDS page.
It is not surprising that you identify different peptides, from the same protein, in different HPLC fractions. I'd be worried if this wasn't the case. Just because you see different masses on SDS-PAGE, this doesn't mean the proteins are different. As has been said, there are many PTMs that can lead to a range of masses in SDS-PAGE.
Mass spec doesn't lie, it identifies what is in your sample. So you either have issues with the sample itself, ie. carryover, contamination, etc, or you are seeing the same protein (modified in some way) in different mass bands on SDS-PAGE. You just need to work out which case you have.
Thank you Peter Hains for you comment
the truth is i don't know what to expect from the results its a work that is done for the first time. what i know is that these proteins are structuraly very conserved. the identities between homologues are up to 85%. identical peptides can correpond to more than one protein but in my case the same protein was found in many samples.
Hey Ibtissam,
Easy way is to run trough the SDS-PAGE and check for potential PTM's.
If u think ur protein might be glycosylated, check out this paper for more information on how to characterize it!
Determination of site-specific glycan heterogeneity on glycoproteins
Kolarich D, Jensen PH, Altmann F, Packer NH
Nature Protocols. 06/2012; 7(7):1285-98.
Thanks kathirvel
I am not sure if my protein is glycosylated i need to check this out! thanks for the reference.
To check for glycosylation in proteins, Periodic Acid Staining (PAS) can be done. This staining specifically detects glycosylation/sugar groups in proteins. Else, the protein can be treated with PNGase and then SDS-PAGE needs to be carried out. PNGase is an enzyme that specifically cleaves N- glycosylation groups. So, if the protein(s) that you have is/are heavily glycosylated, after PNGase treatment, in SDS-PAGE, the band will appear at lower molecular mass than the earlier position without PNGase treatment. There are other proteases as well that are known to remove glycosylation, which may be attempted along with SDS-PAGE to check for the presence of glycosylation.
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