Question:
I’m interested in isolating and identifying cell surface receptor proteins. They typically have high Mw, are glycosylated, and also transmembrane domains. I’m considering 2D electrophoresis, but do you have any recommendations?
Answer:
2D PAGE is the classical approach to separate and visualize many proteins from complex proteomics samples. However, 2D PAGE has some known disadvantages. As an example, hydrophobic membrane proteins precipitate during IEF and are almost never observed. So most likely, you will only observe proteins within the pI range of the gel, typically pH 4-7, or 3-10, and the Mw range of proteins in the gel is approximately 10-130 kDa.
For large and hydrophobic proteins it is therefore better to use 1D SDS PAGE. Mainly because the proteins can be dissolved in the 1D SDS PAGE buffer containing 0.1% SDS. In addition, the gels have no pI limits, and the Mw range can go up as high as 1.000 kDa.
Another possibility is to use in-solution digestion of the protein mixture. You would then make protein identification by LC-MS/MS.
Some relevant references:
Albert Sickmann et al. PNAS 2003 vol. 100, 23, 13207-13212. The proteome of Saccharomyces cerevisiae mitochondria. www.pnas.org/content/100/23/13207
Jens S. Andersen et al. Current Biology, vol.12, 1, 2002, 1-11 Directed Proteomic Analysis of the Human Nucleolus.
Wiśniewski JR et al. Nat Methods. 2009 May;6(5):359-62. Epub 2009 Apr 19. Universal sample preparation method for proteome analysis. https://www.ncbi.nlm.nih.gov/pubmed/19377485