Hello,
i ordered couple of isotopic labeled peptides (13C/15N) for absolute quantification of proteins. The first measurements show that the light one from protein standard and the heavy one do not co-elute. The heavy one elutes earlier (eg. Heavy elutes at 8.2 min and light 8.4 min). This applies for two pepetides from one protein. any suggestions what the reason might be?
Thanks
best,
Bassel
Dear Bassel,
The whole concept of using heavy and light labeled peptide is to take advantage of the fact that both should, theoretically, elute at the same time, from your LC column. Were you able to obtain intact mass measurement of the peptides to know that they are indeed containing the correct molecular weights? If so, you should also try to obtain MS/MS spectra from each to see if the fragment ions are shifting by the isotopic labels.
It is possible that these peptides do not have the same purity. I would use a simple platform eg., MALDI-TOF to characterize them before spending any more time with the quantification experiments.
Best,
Hediye.
If you are trying to quantify a modified peptide (PTM) it might be that the signal you are seeing is coming from a positional isoform of the peptide. An isoform can have different elution time.
It could be one (or more) of the amino acids in the heavy peptide was the wrong stereoisomer, you might check with the company who made the heavy peptides to be sure this didn't happen. Another possibility is that the heavy chain peptides were synthesized as amides at the C terminus instead of carboxylates. The 1 amu difference is easy to overlook especially in heavy isotope labeled peptides; as suggested by others you need to confirm the molecular weights and fragmentation patterns of the synthetic peptides. Also possible is modification of the heavy chains with reagents used to deprotect the peptides and cleave them from the resins. Modifications such as conversion of arginine to homocitrulline or ornitihine, dehydration of serine and threonine, oxidation of Tryptophan and methionine etc. are possible.
Thank you all for your replies.
Hediye, I did perform MS/MS analysis for the peptides, the mass as well as fragments are correct however i did not check the purity yet, MALDI spectra provided by the company as well as LC analysis show that they are pure.
Ilian, the peptides are not modified
Bruce, i will ask the compnay about the stereoisomers. The other modifications can be excluded siince Mw is correct. It might be a problem of the synthesis.
Hi Bassel,
if the stereochemistry was messed up, you may try to denature the proteins prior analysis so that you do not have any effects of the secondary or tertiary structure. It will decrease the lifetime of the column, but you could run with urea solution. Alternatively, denature the proteins prior analysis. The elution times should overlap then.
In any way, if the company made a mistake, they should re-synthesize the right sample and send it to you for free!
Marcus
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