amino acid choosing for SILAC

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sziaee
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amino acid choosing for SILAC

Postby sziaee » Sat May 12, 2012 8:23 pm

I want to start an expremint with SILAC technique in phospho proteomics, but im wondering how can i choose the right amino acid for SILAC. what is differences between 13C6 15N4 L-Arginine-HCl and 13C6 L-Arginine-HCl in final resaults and why some of expriments had used arginin and lysin together.

am i used only one havy amino acid for my expreminet ? for axample 13C6 L-Arginine-HCl ?

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Doug
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Postby Doug » Sat May 12, 2012 10:42 pm

First I should say that I have very little experience with SILAC but I think I can explain a little bit..

I believe the only difference is that 13C6 15N4 L-Arginine-HCl adds 10 Daltons in mass and 13C6 L-Arginine-HCl adds 6 Daltons. I think you could use both of these as well as normal media to do 3-plex SILAC experiments. If you only want to do 2-plex you could just use 13C6 L-Arginine-HCl.

The reason people label both R and K is so that if they digest with trypsin every peptide is labeled. However, as mentioned by several papers, including the one below, Arginine can be converted to Proline causing errors in quantitation. I believe this is especially a problem in Yeast and requires using specific yeast strains in which key enzymes in certain pathways have been knocked out.

You might want to consider simply labeling lysines (not arginines) and digesting with LysC. This should be fine for resonant excitation CAD and should work great for HCD/beam-type CAD. Plus, you can digest in higher urea concentrations. Good luck.


Bendall SC, Hughes C, Stewart MH, Doble B, Bhatia M, Lajoie GA. Prevention of
amino acid conversion in SILAC experiments with embryonic stem cells. Mol Cell
Proteomics. 2008 Sep;7(9):1587-97. Epub 2008 May 16. PubMed PMID: 18487603;
PubMed Central PMCID: PMC2556023.

sziaee
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Postby sziaee » Mon May 14, 2012 2:51 am

thanks Doug.

gadsouza
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Postby gadsouza » Tue May 15, 2012 3:30 am

I would say that even if you are '2-plexing', I would use both Lys6 and Arg10, instead of Lys6 and Arg6. If you are using a good identification engine that take into consideration SILAC pair features, having different delta masses is a plus for improved scoring (i.e., a 6 Da pair must be a Lys containing peptide, etc, not an Arg if you use Arg10).

Arg conversion can be easily avoided by adding extra Proline to the media. At least works fine for Jurkat over here.

Indeed, if money is a concern heavy Lys and LysC or endo-Lys as enzymes of choice are an alternative. Advantage would be a less complex peptide mixture (compared to tryptic peptides), and disadvantage would be missing some protein IDs or sequence coverage depending on individual protein sequence features.


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