title: Implications of partial tryptic digestion in organic-aqueous solvent systems for bottom-up proteome analysis.
abstract: 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.
authors: Department of Chemistry, Dalhousie University, 6274 Coburg Road, Halifax, NS, Canada B3H 4J3.
authors: Mark J
authors: MJ
authors: Wall
authors: Andrew M J
authors: AM
authors: Crowell
authors: Gordon A
authors: GA
authors: Simms
authors: Graham H
authors: GH
authors: Carey
authors: Fang
authors: F
authors: Liu
authors: Alan A
authors: AA
authors: Doucette
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D011506:Proteins / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D020539:Sequence Analysis, Protein / Q000379:methods; D012995:Solubility; D012997:Solvents / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013696:Temperature; D014357:Trypsin / Q000378:metabolism*; D014867:Water / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D011506:Proteins; D020543:Proteome; D012997:Solvents; D014867:Water; D014357:Trypsin; C032159:acetonitrile