title: Mass Spectrometry of Intact Proteins Reveals +98 u Chemical Artifacts Following Precipitation in Acetone.
abstract: Protein precipitation in acetone is frequently employed ahead of mass spectrometry for sample preconcentration and purification. Unfortunately, acetone is not chemically inert; mass artifacts have previously been observed on glycine-containing peptides when exposed to acetone under acidic conditions. We herein report a distinct chemical modification occurring at the level of intact proteins when incubated in acetone. This artifact manifests as one or more satellite peaks in the MS spectrum of intact protein, spaced 98 u above the mass of the unmodified protein. Other artifacts (+84, +112 u) also appear upon incubation of proteins or peptides in acetone. The reaction is pH-sensitive, being suppressed when proteins are exposed to acetone under acidic conditions. The +98 u artifact is speculated to originate through an intermediate product of aldol condensation of acetone to form diacetone alcohol and mesityl oxide. A +98 u product could originate from nucleophilic attack on mesityl oxide or through condensation with diacetone alcohol. Given the extent of modification possible upon exposure of proteins to acetone, particularly following overnight solvent exposure or incubation at room temperature, an awareness of the variables influencing this novel modification is valued by proteomics researchers who employ acetone precipitation for protein purification.
authors.affiliation: Department of Chemistry, Dalhousie University , PO Box 15000, 6274 Coburg Road, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Melda Z
authors.initials: MZ
authors.lastname: Güray
authors.affiliation: Department of Chemistry, Dalhousie University , PO Box 15000, 6274 Coburg Road, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Shi
authors.initials: S
authors.lastname: Zheng
authors.affiliation: Department of Chemistry, Dalhousie University , PO Box 15000, 6274 Coburg Road, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Alan A
authors.identifier: 0000-0002-9467-1002
authors.initials: AA
authors.lastname: Doucette
mesh_terms: D000096:Acetone / Q000737:chemistry*; D000818:Animals; D016477:Artifacts; D011232:Chemical Precipitation; D045304:Cytochromes c / Q000032:analysis* / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000737:chemistry; D006454:Hemoglobins / Q000032:analysis / Q000737:chemistry; D006588:Hexanones / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000592:standards*; D009211:Myoglobin / Q000032:analysis / Q000737:chemistry; D000439:Pentanols / Q000737:chemistry; D010422:Pentanones / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000592:standards; D025801:Ubiquitin / Q000032:analysis / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D006454:Hemoglobins; D006588:Hexanones; D009211:Myoglobin; D000439:Pentanols; D010422:Pentanones; D010455:Peptides; D025801:Ubiquitin; D000096:Acetone; C008374:mesityl oxide; D045304:Cytochromes c; C080344:diacetone alcohol
keywords: 98 u; acetone; aldol; diacetone alcohol; mass artifact; mesityl oxide; protein modification