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What is the importance of urea in mass spectrometry?
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Aart Van Beuzekom
What is the importance of urea in mass spectrometry?
I think that the OP was asking about relevance of using urea with respect to the FASP method. In the FASP method, the primary denaturant is SDS . Protein are denatured with a ~4% SDS solution (buffered to pH 7.5 - 8.0). Then 8 M urea solution is used to replace the SDS. Urea serves two purposes here, first it keeps the protein denatured and in solutions as SDS is replaced/washed off. Second, because urea is a chaotropic agent, it reduces the size of SDS micelles which otherwise would block the pores of the membrane filter.
I think that the OP was asking about relevance of using urea with respect to the FASP method. In the FASP method, the primary denaturant is SDS . Protein are denatured with a ~4% SDS solution (buffered to pH 7.5 - 8.0). Then 8 M urea solution is used to replace the SDS. Urea serves two purposes here, first it keeps the protein denatured and in solutions as SDS is replaced/washed off. Second, because urea is a chaotropic agent, it reduces the size of SDS micelles which otherwise would block the pores of the membrane filter.
One more thing I think should be added: it is important to understand that denaturants like urea, GuHCl, or SDS not only unfold/denature protein, but also keep them in solution thereby allowing their biochemical analysis. Otherwise heating to ~100 ºC or using Trichloroacetic acid can also denature protein, but they render protein insoluble.More
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8M urea is a denaturant, as @Chris mentioned. Denaturing proteins is important for molecular (aka classical) mass spectrometry. If you analyzed a non-denatured protein, you'd get information not only on the primary structure, but also on noncovalent interaction (2+°):
The classical MS approach, so-called “molecular MS,” analyzes, in the gas phase of the mass spectrometer, individual species initially present in solution after destruction of the noncovalent framework. On the other hand, “supramolecular MS” or “nondenaturing MS” aims at transferring intact noncovalent complexes that preexist in solution into the gas phase of the instrument.
Denaturing proteins is important for molecular/classical mass spec because it ensures your analysis only reflects the covalent bonds (primary structure) present in your target protein.
8M urea is a denaturant, as @Chris mentioned. Denaturing proteins is important for molecular (aka classical) mass spectrometry. If you analyzed a non-denatured protein, you'd get information not only on the primary structure, but also on noncovalent interaction (2+°):
The classical MS approach, so-called “molecular MS,” analyzes, in the gas phase of the mass spectrometer, individual species initially present in solution after destruction of the noncovalent framework. On the other hand, “supramolecular MS” or “nondenaturing MS” aims at transferring intact noncovalent complexes that preexist in solution into the gas phase of the instrument.
Denaturing proteins is important for molecular/classical mass spec because it ensures your analysis only reflects the covalent bonds (primary structure) present in your target protein.
I think that the OP was asking about relevance of using urea with respect to the FASP method. In the FASP method, the primary denaturant is SDS . Protein are denatured with a ~4% SDS solution (buffered to pH 7.5 - 8.0). Then 8 M urea solution is used to replace the SDS. Urea serves two purposes here, first it keeps the protein denatured and in solutions as SDS is replaced/washed off. Second, because urea is a chaotropic agent, it reduces the size of SDS micelles which otherwise would block the pores of the membrane filter.
Read the original article here: http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1322.html
I think that the OP was asking about relevance of using urea with respect to the FASP method. In the FASP method, the primary denaturant is SDS . Protein are denatured with a ~4% SDS solution (buffered to pH 7.5 - 8.0). Then 8 M urea solution is used to replace the SDS. Urea serves two purposes here, first it keeps the protein denatured and in solutions as SDS is replaced/washed off. Second, because urea is a chaotropic agent, it reduces the size of SDS micelles which otherwise would block the pores of the membrane filter.
Read the original article here: http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1322.html
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8M urea is a denaturant, as @Chris mentioned. Denaturing proteins is important for molecular (aka classical) mass spectrometry. If you analyzed a non-denatured protein, you'd get information not only on the primary structure, but also on noncovalent interaction (2+°):
Denaturing proteins is important for molecular/classical mass spec because it ensures your analysis only reflects the covalent bonds (primary structure) present in your target protein.
8M urea is a denaturant, as @Chris mentioned. Denaturing proteins is important for molecular (aka classical) mass spectrometry. If you analyzed a non-denatured protein, you'd get information not only on the primary structure, but also on noncovalent interaction (2+°):
Denaturing proteins is important for molecular/classical mass spec because it ensures your analysis only reflects the covalent bonds (primary structure) present in your target protein.
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