Dear
leopardyu,
Thanks for the questions. The basic principle underlying the STrap methodology is creation of the fine particles from the SDS-solubilized proteins and trapping the particles in the depth filter media. The quartz depth filters were chosen because of their pure composition (SiO2), low metal content, low peptide background binding under the digest conditions, as well as commercial availability (you may also try to use glass depth filters, for example). While the particles are indeed retained throughout the depth filter dimensions, SDS and other contaminants are removed in the flow through, and then a protease is introduced which processes the fine protein particulate into the peptides. This is, in my opinion, as close as one can get to the high-throughput robust unbiased comprehensive etc. proteome profiling.
1. How the "particulate matter” is generated?
It seems when you add the SDS-containing lysate into methanol (90% in 100 mM Tris/HCl, pH 7.1), then you’ll get the protein particulate suspension, is that right? But why is that? Is it because the proteins are not soluble in methanol?
When you add the SDS-protein solution into the methanolic buffer the SDS-micelles are broken, the proteins are exposed to the alcohol medium which has a low dielectric constant and thus the proteins start precipitating out.
2. What if you add the SDS-containing lysate into the tip and wash with 8M urea directly? Are the SDS detergents going to be depleted in this case?
If you do such a thing you will loose your proteins in the flow through because urea retains the proteins in solution and thus the proteins are going to simply pass through the filter pores.
3. What’s the purpose of acidifying protein samples with phosphoric acid? Otherwise you will not get particulate matter?
The acidification helps to obtain the fine protein particles which fit the quartz depth filter particle retention characteristics. This was found by trial and error.
4. If methanol can solubilize SDS (up to 2%), can we replace 8M urea in the traditional FASP method for SDS depletion? If so, we don’t have to deal with high concentration urea buffer then.
Providing your centrifugal filter unit withstands the action of methanol, the particulate matter created in such an exercise is going to block the filter membrane and, basically, that is it
Just to add, when working with the STrap tip please do not overload it - it will result in the increased backpressure and reduced performance.
Best,
Alexandre
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