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.

O-methylation of the side chains of glutamic acid (E) and aspartic acid (D) residues is generally observed modification when an acidified methanol/water (MeOH/dHO) mixture is used as a solvent system during sample preparation for proteomic research. This chemical modification may result misidentification with endogenous protein methylation; therefore, a special care should be taken during sample handling prior to mass spectrometric analysis. In the current study, we systematically examined the extent of E/D methylation and C-terminus carboxyl group of synthetic model peptides in terms of different incubation temperatures, storage times, and added acid types as well as its percentages. To monitor these effects, C-terminus amidated and free acid forms of synthetic model peptides comprised of E or D residue(s) have been analyzed by electrospray ionization-mass spectrometry (ESI-MS). Additionally, LC-MS/MS experiments were performed to confirm the formation of methylated peptide product. The results showed that the rate of methylation was increased as the temperature increases along with prolong incubation times. Moreover, the extent of methylation was remarkably high when formic acid (FA) used as a protonation agent instead of acetic acid (AA). In addition, it was found that the degree of methylation was significantly decreased by lowering acid percentages in ESI solution. More than one acidic residue containing model peptides have been also used to explore the extent of multiple methylation reaction. Lastly, the ethanol (EtOH) and isopropanol (iPrOH) have been substituted separately with MeOH in sample preparation step to investigate the extent of esterification reaction under the same experimental conditions. However, in the positive perspective of view, this method can be used as a simple, rapid and cheap method for methylation of acidic residues under normal laboratory conditions.

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Proteomik yaklaşımları 2000 li yılların başlarına kadar mikroorganizmalar ve hayvansal kaynaklı örneklerde ağırlıklı olarak kullanıldı. Bu dönemde bitki proteomik çalışmaları yok denecek kadar azdır. Bitkisel dokulardaki sert hücre çeperleri, karmaşık ve çok çeşitli sekonder metabolitlerin varlığı, fazla miktardaki pigmentler, proteazlar, polifenoller, polisakkaritler, nişasta ve lipitler total protein örneklerinin hazırlanması ve proteinlerin ayrımı sırasında pek çok soruna neden olmuştur. Ancak her bir sorunun üstesinden gelmek üzere sürdürülen çabalar sayesinde bitki dünyasında da proteomik yaklaşım kullanımı yaygınlaşmıştır. Bu derlemede, örnek hazırlığından protein tanımlamaya kadar tüm basamaklar yöntemsel gelişmeleri de kapsayacak şekilde ayrıntılı olarak ele alınmış ve konuyla ilgili araştırıcıların maksimum yararlanabileceği bir kaynak oluşturulmaya çalışılmıştır.