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Andrew J. Alpert
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Speaker: Andrew J. Alpert, PolyLC, Inc., Columbia, MD 21045
Topic: HPLC of Really Difficult Protein Mixtures: Prions to Proteomics
Place: Building 549, Auditorium, NCI at Frederick, Frederick, MD
Time: Tuesday, September 9, 2003, at 2:00 PM
Abstract: Recent growth in fields such as proteomics is creating a demand for methods that are compatible with modern analytical instruments. However, standard biochemistry methods were often developed with the objective of maintaining enzymatic activity. The use of detergents, urea, and nonvolatile salts produces samples unfriendly to chromatography and incompatible with mass spectroscopy. Also, 2-D electrophoresis does not work well for many proteins that are quite large, quite basic, or from membranes, and has too limited a dynamic range for many proteomics applications. The following alternatives to standard methods will be described: [1] solubilization of prions, membrane proteins, and whole cell pellets with organic solvents; [2] use of organic solvents in chromatography of intact proteins; [3] trypsinization in totally volatile solvents; [4] strategies for fractionation of samples containing tens of thousands of proteins or peptides (including, hydrophilic interaction and mixed-bed ion-exchange for intact proteins, and multidimensional liquid chromatography for complex tryptic digests); [5] selective isolation of specific classes of peptides such as glycopeptides and phosphopeptides; and [6] quick, cheap ways to get rid of detergents, lipids, salts and urea from samples prior to analysis by mass spectrometry or capillary electrophoresis.
The slides to this seminar are in a 1.2 Megabyte PDF file, which can be opened and read by using the free Adobe Acrobat Reader®.
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