Prince:Filter-aided Sample Preparation: Difference between revisions
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Filter-aided sample preparation (FASP) prepares protein for mass spectrometry. After lysis, proteins are placed on a filter and washed repeated to remove detergent. Detergents creates noise in the mass spectrometer and contaminate the chromatography. After a number of washing steps the proteins are ready for protease digestion. Proteases are enzymes that cut proteins into peptides. We normally use trypsin, which cuts after Lysine and Arginine amino acids. Trypsin digestion normally takes 18 hours. | Filter-aided sample preparation (FASP) prepares protein for mass spectrometry. After lysis, proteins are placed on a filter and washed repeated to remove detergent. Detergents creates noise in the mass spectrometer and contaminate the chromatography. After a number of washing steps the proteins are ready for protease digestion. Proteases are enzymes that cut proteins into peptides. We normally use trypsin, which cuts after Lysine and Arginine amino acids. Trypsin digestion normally takes 18 hours. | ||
''Figure: Filter aided sample preparation (FASP). 1. Sodium dodecyl sulfate (SDS) denatures and dissolves proteins. 2. Proteins are placed on a filter and washed repeated with 8 M urea to remove SDS. After urea washings, protein is washed with 50 mM ammonium bicarbonate to remove urea and provide a suitable environment for trypsin digestion. 3. Trypsin digests protein into peptides. | ''Figure: Filter aided sample preparation (FASP). 1. Sodium dodecyl sulfate (SDS) denatures and dissolves proteins. 2. Proteins are placed on a filter and washed repeated with 8 M urea to remove SDS. After urea washings, protein is washed with 50 mM ammonium bicarbonate to remove urea and provide a suitable environment for trypsin digestion. 3. Trypsin digests protein into peptides.'' | ||
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[[Prince:Yeast FASP|Yeast FASP]] | |||
=Literature= | =Literature= | ||
Revision as of 17:24, 3 February 2012
Brief Explanation
Filter-aided sample preparation (FASP) prepares protein for mass spectrometry. After lysis, proteins are placed on a filter and washed repeated to remove detergent. Detergents creates noise in the mass spectrometer and contaminate the chromatography. After a number of washing steps the proteins are ready for protease digestion. Proteases are enzymes that cut proteins into peptides. We normally use trypsin, which cuts after Lysine and Arginine amino acids. Trypsin digestion normally takes 18 hours.
Figure: Filter aided sample preparation (FASP). 1. Sodium dodecyl sulfate (SDS) denatures and dissolves proteins. 2. Proteins are placed on a filter and washed repeated with 8 M urea to remove SDS. After urea washings, protein is washed with 50 mM ammonium bicarbonate to remove urea and provide a suitable environment for trypsin digestion. 3. Trypsin digests protein into peptides.
Literature
1. Wisniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M., Universal sample preparation method for proteome analysis. Nat. Methods 2009, 6 (5), 359-U60. http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1322.html
