Glycerol affects the quantification of aspartate and glutamate in acid-hydrolyzed proteins |
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Authors: | J -F Juranville B Pöschl G Oesterhelt H -J Schönfeld M Fountoulakis |
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Institution: | (1) F. Hoffmann-La Roche Ltd., Preclinical Central Nervous System Research-Gene Technology, Building 93-444, CH-4070 Basel, Switzerland;(2) Infectious Diseases, F. Hoffmann-La Roche Ltd., Pharma Division, Basel, Switzerland |
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Abstract: | Summary Glycerol is widely used in protein isolation pathways to improve folding and solubility of the proteins of interest. Amino acid composition analysis of protein samples hydrolyzed in the presence of glycerol resulted in underestimation of aspartate and glutamate, when compared to hydrolysis in the absence of glycerol. Quantification of free asparagine, aspartic acid, glutamine and glutamic acid hydrolyzed with hydrochloric acid or methanesulfonic acid in the presence of glycerol resulted in poor recoveries of aspartate and glutamate (between 6 and 66%). Gas chromatography-mass spectrometry analysis of the hydrolyzates revealed, as expected, the presence of esterification products. The esters were formed between the primary and secondary hydroxyl groups of the glycerol and both carboxyl groups of the amino acids. Protein samples intended for compositional analysis should be free of glycerol.Abbreviations EI
electron impact
- GC-MS
gas chromatography-mass spectrometry
- MS
mass spectrum
- MSA
methanesulfonic acid
- BSTFA
bis(trimethylsilyl)trifluoroacetamide
- TMS
trimethylsilyl |
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Keywords: | Amino acid analysis Hydrolysis Glycerol Aspartate Glutamate Protein quantification |
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