Biomedical Engineering Reference
In-Depth Information
are comprised of detergent-resistant membrane microdomains,
presents an even greater challenge. Although several methods
have been explored to improve the in-solution digestion of mem-
robust analysis was performed by 1D SDS-polyacrylamide gel
based methods can facilitate membrane proteomics as the sam-
ples can be solubilised in strong denaturing conditions and the
proteins resolved and isolated in the gel matrix. Chaotropes and
detergents used for sample solubilisation can be removed by
washes and in-gel digestion of the gel matrix-suspended pro-
teins can be efficiently performed in a concentrated volume with-
out precipitation of the proteins occurring. However, the sep-
aration of proteins on 1D SDS-PAGE resolving gels can com-
plicate quantitation using post-digestion chemical labelling, such
as isobaric tagging, due to variations in sample running, mul-
tiple gel band excisions and in-gel digestions, and final peptide
extractions.
To solve this problem, we developed a method to isolate the
protein population from membrane and membrane raft samples
Cell culture and
differential treatments
Sucrose gradient isolation of
membranes/membrane rafts
1234
Isolate protein populations as
unresolved bands
1. Excise and digest in gel
2. Label and combine samples
1. SCX fractionation
2. LC/MS/MS
1. Combine and search data
2. Identify and quantify proteins
Fig. 14.1 Schematic of the protocol workflow.
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