Trypsin, protein digestion using

Modification of protein digestions using physical methods has also contributed to improved digestion efficiency and proteomic coverage. Covalent and dytramic immobilization of trypsin on micro- and nanoparhcles, the use of pressure cycling... 

As described above all samples were separated online using LCT ESI-TOF-MS then normalized for relative quantitation using a bovine insulin internal standard. Fractions were then collected for MAFDI-TOF-MS PMF, digested with modified porcine trypsin, and analyzed using the TofSpec2E. Following this analysis, three major classes of differentially expressed including proteins were revealed in these... 

Trypsin is the most frequently used serine protease. Generally, porcine or bovine pancrease is used as the source of pure trypsin. Trypsin usually digests proteins at their lysine and arginine residues. The superiority of trypsin is that it displays good activity both in solution and in-gel digestion protocols. 

Similar to the potential utility of patterns generated from mass spectra analyses, patterns of protein expression observed by 2D-PAGE may also be used for diagnostic purposes [6, 46, 47]. 2D-PAGE resolves complex protein mixtures into numerous unique spots, forming a distinctive pattern. The pattern formed by the protein spots from normal and diseased tissue can then be visually compared. Proteins of interest can be excised from the gel, digested using trypsin, and analyzed by MS to reveal their identity. 

Routinely, common chemical and enzymatic techniques are used to obtain protein fragments. Unfortunately, when enzymatic digestion techniques and nanograms quantities of proteins are used, the method become ineffective due to dilution and reduced enzymatic activity. An alternative approach to overcome this problem is the use of proteolytic enzymes immobilized to a solid support and a small-bore reactor column. Using trypsin immobilized to agarose, tryptic digests of less than 100 ng of protein can be reproducible obtained (49). 

The membrane proteins isolated using the colloidal silica method described above are especially difficult to solubilize, and difficult to separate by 2D gel electrophoresis. Consequently the membranes were solubilized in Laemmli buffer with sonication in a 60 °C water bath, and separated by 1D S DS gel electrophoresis. This ID gel was cut into 1 mm bands, each of which was subjected to trypsin digestion. The peptides were recovered, separated and analyzed by LC/ESl MS/ MS. This strategy is illustrated in Eigure 13.5. More than 3200 peptides were... 

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