Hi (Salam),
I'm studying the expression of some proteins in cell lines using LC-MS. In order to do so, I'll go with the "bottom-up" approach. After selecting the suitable signature peptides, I wanna do stable isotope labelling. I found various isotope options (13C, 15N, 18O, 2H, deuterium) which made me uncertain of the next step so here are my questions:
1. How to know which isotope is the best for my peptides?
2. Is there an isotope that's better than the others when using mass spect.?
3. Is it better to label my signature peptide with more than one isotope or one will do the job?
4. When I looked up Deuterium, which I've used before, I found options like "3,3-d2" or "2,3,3-d7". Can someone kindly explain to me what these numbers mean and how it shall influence my choice?
I highly appreciate your answers :)
Hi,
Interesting!
I think it is quite common to use 13C and/or 15N-labeled amino acids these days, for the type of experiment you're envisioning .
If you're planning to use trypsin as your protease of choice, you might want to consider using 13C and 15N-labeled lysine and arginine. In that way, you'll always have at least one labeled amino acid in your peptides (apart from C-terminal peptides, perhaps). Moreover, the mass difference introduced by the stable isotope labels in these amino acids are also relatively large, allowing for easy distinction between labeled and non-labeled peptides.
The numbers in the coding usually indicate where the stable isotope labels reside in the amino acid.
Hope this helps!
Hi Mariam (Aleykum Selam),
Depending on your cell lines, possibly the method that you would need is "SILAC" (Stable isotope labeling using amino acids in cell culture) which is using the labels and amino acids mentioned by EeF. You can find a lot of good publications from Pubmed, describing this method and also companies selling all required media and labels for this method.
Good Luck!
Murat
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