In-solution Enzymatic Digestion of Proteins Using a Mass Spectrometry Compatible Denaturant

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Ying Qing Yu, Martin Gilar, Peter J. Lee, John C. Gebler [Waters]
ISPPP 2002 Heidelberg Germany, Nov. 10-13
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Proteins, Protein Digest, Trypsin, BAEE, Tryptic Digest, Myoglobin, Bovine Ubiquitin, Amino Acids, Ovalbumin, Bacteriorhodopsin, Bovine Serum Albumin, BSA, BA
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Rapid advancements in the mass spectrometry field have enabled more sensitive and accurate identification of proteins. The common strategy is to digest proteins with enzymes and analyze resulting peptides via mass spectrometric methods. Hydrophobic proteins are troublesome to identify due to their low solubility; surfactants are commonly used to disrupt the aggregation of hydrophobic biomolecules prior to enzymatic digestions. However, ionic surfactants such as SDS are disruptive to enzyme activity (e.g., trypsin) and interfere with mass spectrometry analysis. We employed a novel denaturant, RapiGestTM SF, as an aid for enzymatic digestion of proteins. Trypsin activity inhibition was investigated with 0.1% - 2.5% (w/v) RapiGestTM SF and compared with SDS. Experimental results showed that in contrast to SDS, RapiGestTM SF does not inhibit trypsin activity. Enzymes such as Asp-N, Glu-C and Lys-C also maintain their activity in the presence of RapiGestTM SF. Drastic improvements of protein digestion in terms of speed and peptide recovery were observed. RapiGestTM SF reduces the time needed for a complete enzymatic digestion of proteins. For example, a complete myoglobin tryptic digestion was achieved at 5 minutes in the presence of 0.1% RapiGestTM SF, while the control sample (without a denaturant) requires overnight digestion. Upon digestion of membrane proteins, e.g., bacteriorhodopsin, more peptide coverages and reduced digestion time are achieved with the use of RapiGestTM SF. RapiGestTM SF is compatible with mass spectrometry. It rapidly hydrolyzes under low pH conditions and the degradation products are easily removed prior to LC/MS analysis. It is optional to remove these degradation products prior to MALDI-MS analysis since no ion suppression was observed.

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