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.

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“…This is a common occurrence due to alkylation by excess iodoacetamide during sample preparation. 33,34 Another artifact observed was the lauryl sulfuric acid adduct (266 Da), which likely results from incomplete detergent (SDS) removal; 35 the heaviest proteoform (E323) in this family had three such adducts. While sample preparation artifacts are not biologically relevant, it is still important to correctly assign them so as not to misidentify them as some other PTM.…”

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“…As an alternative method, acetone precipitation has the distinct advantage of leaving many proteins folded. This method, however, has been shown to modify proteins with +98 Da adducts 49 , requires longer incubation at −20 °C (at least 1 h), requires that all steps be performed at or below 0 °C to maximize resolubilization, and can be compromised by detergents.…”

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“…However, this solvent is not inert and can cause undesirable modifications to lysine (K), arginine (R), and histidine (H) residues. Guray et al [55] showed that the amount of artifacts in a sample rapidly increases with increasing pH. The authors explained this by the interaction of mesityl oxide or diacetone alcohol formed in the basic medium with the proteins of the sample.…”