Implications of partial tryptic digestion in organic-aqueous solvent systems for bottom-up proteome analysis.

Autor: Wall MJ; Department of Chemistry, Dalhousie University, 6274 Coburg Road, Halifax, NS, Canada B3H 4J3., Crowell AM, Simms GA, Carey GH, Liu F, Doucette AA
Jazyk: angličtina
Zdroj: Analytica chimica acta [Anal Chim Acta] 2011 Oct 10; Vol. 703 (2), pp. 194-203. Date of Electronic Publication: 2011 Aug 05.
DOI: 10.1016/j.aca.2011.07.025
Abstrakt: For bottom-up MS, the digestion step is critical and is typically performed with trypsin. Solvent-assisted digestion in 80% acetonitrile has previously been shown to improve protein sequence coverage at shorter digestion times. This has been attributed to enhanced enzyme digestion efficiency in this solvent. However, our results demonstrate that tryptic digestion in 80% acetonitrile is less efficient than that of conventional (aqueous) digestion. This is a consequence of decreased enzyme activity beyond ~40% acetonitrile, increased enzyme autolysis and lower protein solubility in 80% acetonitrile. We observe multiple missed cleavages and reduced concentration of fully cleaved digestion products. Nonetheless we confirm, through room temperature solvent-assisted digestion, a consistent improvement in protein sequence coverage when analyzed by mass spectrometry. These results are explained through the increased number of unique digestion products available for detection. Thus, while solvent-assisted digestion has clear merits for proteome analysis, one should be aware of the inefficiency of protein digestion though this protocol, particularly with absolute protein quantitation experiments.
(Copyright © 2011 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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