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Modifications in Trypsin Digestion Protocol for Increasing the Efficiency and Coverage

[ Vol. 22 , Issue. 4 ]

Author(s):

Kirtimaan Syal and Raghu Tadala   Pages 372 - 378 ( 7 )

Abstract:


Standard trypsin digestion protocol of proteins followed by MALDI-MS analysis has been realized as an important tool for the identification and characterization of proteins. In this article, we proposed the elimination of the step of ‘staining/de-staining of gel pieces’ in in-gel digestion protocol in order to improve the efficiency of trypsin digestion. Coomassie dye is known to interfere with digestion of proteins by trypsin and the procedure of staining-de-staining could result in loss of photoaffinity probe, post translational modifications and catalytic activities of enzymes. Further, we studied parameters like hydrophobicity and isoelectric point, and attempted to quantitatively relate it to the efficiency of trypsin digestion. We suggest that properties of proteins should be considered and trypsin digestion protocol should be appropriately modified as per sequence and other information.

Keywords:

Bottom-up approach, hydrophobicity, isoelectric point, post-translational modifications, staining/de-staining, trypsin digestion.

Affiliation:

Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.

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