Does anyone know a protocol for membrane protein extraction that would be compatible with iTRAQ derivatization prior to gel-free mass spectrometry analysis with nanoLC-MS/MS?
Do you think that boiling with B-mercaptoethanol + SDS (Leammli sample buffer-like) of subcellular fraction enriched in cell membranes (centrifugate of a cell lysis), then precipitation with -20°C acetone (for sample buffer removing), and resolubilization could be suitable?
might be useful
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0020442
Thx !
these authors use Sarkosyl rather than SDS and DTT rather than B-mercaptoethanol, can we think these compounds would differently interact with iTRAQ labelling reagents or that acetone precipitation would efficiently remove both denaturing and reducing reagents ?
for itraq, a reverse phase before MS is mandatory for complex system, and better if online.
but the reverse phase column is damaged with high salt conc. and detergents like SDS.
I will say might be therefore these substitutions.
I will suggest running 1 cation exchange prior to reverse phase to remove theses damaging salts or else you might choke your reverse phase column.
We did iTRAQ analysis of myelin samples
http://www.ncbi.nlm.nih.gov/pubmed/22367995
See Mol Hum Reprod. 2010 Feb;16(2):68-79. doi: 10.1093/molehr/gap077 for a rveiew fdealing with this topic.
Maybe you could use TFE buffer to disolve and digest the samples. The 2,2,2 trifluoroethanol is specially used to redisolve membrane proteins. We usually use this buffer to digest+iTRAQ labelling and it's perfect.
http://www.chem.agilent.com/Library/posters/Public/5989-1781EN.pdf
I have used a protocol for microsomal fractions and did iTRAQ, you can see to it if useful....
http://www.ncbi.nlm.nih.gov/pubmed/22219345
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