1. DHB is believed to increase the roughness of the tissue surface and create pores. The pores may be the result of tissue collapse upon exposure to acidic pHs. It is believed that the pores allow the extraction solvent to penetrate deeper layers of tissue and thus allows peptides to diffuse through the pores.
2. It is better to use a matrix soluble in water because it favors peptide extraction while limiting the number of lipids that can be extracted. This makes the MS analysis cleaner and it allows cell membranes to be preserved. Hydrophobic solvents would dissolve lipids, distrupt cell membranes, and make MS analysis much more difficult.
1. DHB is believed to increase the roughness of the tissue surface and create pores. The pores may be the result of tissue collapse upon exposure to acidic pHs. It is believed that the pores allow the extraction solvent to penetrate deeper layers of tissue and thus allows peptides to diffuse through the pores.
2. It is better to use a matrix soluble in water because it favors peptide extraction while limiting the number of lipids that can be extracted. This makes the MS analysis cleaner and it allows cell membranes to be preserved. Hydrophobic solvents would dissolve lipids, distrupt cell membranes, and make MS analysis much more difficult.
I'm curious about question one. If the purpose of the DHB was to isolate the peptides and create pores in the tissues so more peptides could be extracted, why couldn't the tissue just be digested with enzymes to break it apart? Are the isolated peptides they are looking for already fragmented into smaller sections?
If they are, it seems like digesting the tissue would result in increased peptide isolation.
The point of the paper was to extract the signaling peptides while leaving the protein intact. Peptides formed from the degradation of larger proteins negatively affect the analysis of the smaller signaling peptides. So digesting the tissue would obviously make the sample more complex to analyze because it would be difficult to determine the difference between SPs and protein fragments.
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Comments
I have the questions below for Melissa and the rest of the class.
1. What is current hypothesis on the role of DHB in peptide extraction?
2. Why is it better to use a matrix that is highly soluble in water as opposed to a matrix that is hydrophobic?
Posted by: Edgar Arriaga | March 25, 2008 12:54 PM
1. DHB is believed to increase the roughness of the tissue surface and create pores. The pores may be the result of tissue collapse upon exposure to acidic pHs. It is believed that the pores allow the extraction solvent to penetrate deeper layers of tissue and thus allows peptides to diffuse through the pores.
2. It is better to use a matrix soluble in water because it favors peptide extraction while limiting the number of lipids that can be extracted. This makes the MS analysis cleaner and it allows cell membranes to be preserved. Hydrophobic solvents would dissolve lipids, distrupt cell membranes, and make MS analysis much more difficult.
Posted by: Melissa Maurer-Jones | March 26, 2008 12:52 PM
1. DHB is believed to increase the roughness of the tissue surface and create pores. The pores may be the result of tissue collapse upon exposure to acidic pHs. It is believed that the pores allow the extraction solvent to penetrate deeper layers of tissue and thus allows peptides to diffuse through the pores.
2. It is better to use a matrix soluble in water because it favors peptide extraction while limiting the number of lipids that can be extracted. This makes the MS analysis cleaner and it allows cell membranes to be preserved. Hydrophobic solvents would dissolve lipids, distrupt cell membranes, and make MS analysis much more difficult.
Posted by: Melissa Maurer-Jones | March 26, 2008 12:53 PM
I'm curious about question one. If the purpose of the DHB was to isolate the peptides and create pores in the tissues so more peptides could be extracted, why couldn't the tissue just be digested with enzymes to break it apart? Are the isolated peptides they are looking for already fragmented into smaller sections?
If they are, it seems like digesting the tissue would result in increased peptide isolation.
Posted by: Josh Ochocki | March 30, 2008 06:49 PM
The point of the paper was to extract the signaling peptides while leaving the protein intact. Peptides formed from the degradation of larger proteins negatively affect the analysis of the smaller signaling peptides. So digesting the tissue would obviously make the sample more complex to analyze because it would be difficult to determine the difference between SPs and protein fragments.
Posted by: Melissa Maurer-Jones | March 30, 2008 07:06 PM