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Background and purpose: Phenyl-chloroethyl ureas (CEUs) are a class of anticancer drugs that mainly react with proteins. Two molecules of this family, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU) induced G1/S and G2/M cell cycle blocks, respectively. We hypothesised that these observations were linked to a differential protein alkylation pattern. Experimental approach: Proteins from B16 cells incubated with [14C-urea]-CCEU and [125I]-ICEU were compared by 2D-analyses followed by MALDI-TOF identification of modified proteins and characterisation of the CCEU binding. Protein expression was investigated by Western blot analyses and cell cycle data were obtained by flow cytometry. Key results: Several proteins (PDIA1, PDIA3, PDIA6, TRX, VDAC2) were alkylated by both ICEU and CCEU but β-tubulin and prohibitin (PHB) were specifically alkylated by either ICEU or CCEU respectively. Specific alkylation of these two proteins might explain the observed difference in B16 cell cycle arrest in G2 and G1 phases respectively. Mass spectrometry studies on the alkylated prohibitin localised the modified peptide and identified Asp-40 as the target for CCEU. This alkylation induced an increased cellular content of PHB that should contribute to the accumulation of cells in G1 phase. Conclusions and implications: This study reinforces our findings that CEUs alkylate proteins through an ester linkage with an acidic amino acid and shows that PHB alkylation contributes to G1/S arrest in CCEU treated B16 cells. Modification of PHB status and/or activity is an open route for new cancer therapeutics. British Journal of Pharmacology (2007) 152, 449–455; doi:10.1038/sj.bjp.0707415; published online 20 August 2007

An ultrasonic bath, an ultrasonic probe and a sonoreactor were used to speed up the kinetics of the reactions involved in each step of the sample handling for in-gel protein identification by peptide mass fingerprint, PMF, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The following steps were successfully accelerated using ultrasonic energy: gel washing, protein reduction, and protein alkylation. As a result, a reduction comprising 80% to 90% of the total time involved in the classic approach was achieved. In addition the sample handling was also drastically simplified. The number of peptides identified and the protein sequence coverage obtained for the new procedure were comparable to those obtained with the traditional sample treatment for the following protein standards: glycogen phosphorylase b, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor and α-lactalbumin. Finally, as a proof of the procedure, specific proteins were identified from complex protein mixtures obtained from three different sulphate-reducing bacteria: Desulfovibrio desulfuricans G20, Desulfuvibrio gigas NCIB 9332, and Desulfuvibrio desulfuricans ATCC 27774. Copyright © 2007 John Wiley & Sons, Ltd.

O(6)-alkylguanine-DNA alkyltransferase (AGT) repairs pro-mutagenic O(6)-alkylguanine and O(4)-alkylthymine lesions in DNA. The alkylated form of the protein is not reactivated; instead, it is rapidly ubiquitinated and degraded. Here, we show that alkylation destabilizes the native fold of the protein by 0.5-1.2 kcal/mole and the DNA-binding function by 0.8-1.4 kcal/mole. On this basis, we propose that destabilization of the native conformational ensemble acts as a signal for ubiquitination.

All existing protocols for protein separation by two-dimensional (2-D) gel electrophoresis require the full reduction, denaturation, and alkylation as a precondition for an efficient and meaningful separation of such proteins. Existing literature provides a strong evidence to suggest that full reduction and denaturation can be achieved in a relatively short time; the same thing, however, can not be said for the alkylation process, which the present study shows that more than 6 h are required for a complete alkylation. We have used matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) to monitor protein alkylation by iodoacetamide over the period 0-24 h at pH 9. The present, fast and specific MS method provided clear indication on the extent and speed of alkylation which reached approximately 70% in the first 2 min, yet the remaining 30% resisted complete alkylation up to 6 h. The use of sodium dodecyl sulfate (SDS) during the alkylation step resulted in a strong quenching of this reaction, whereas 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) exerted a much reduced inhibition. The implications of the present measurements on 2-D gel analysis in particular and proteomics in general are discussed.

The present review highlights some important alkylation pathways of proteins, as measured by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometric analysis, engendered by acrylamide and a number of its derivatives, including N-substituted acrylamides, cross-linkers and Immobilines (the acrylamido weak acids and bases used to create immobilized pH gradients). The present data are of relevance in two-dimensional maps and proteome analysis. It is shown that acrylamide can alkylate the -SH group of proteins even when engaged in disulfide bridges. An order of reactivity is obtained for a series of cross-linkers, which are shown to have an extremely reacting double bond, with the second one almost unreactive, originating "pendant, unreacted ends", which can subtract proteins migrating in a gel by covalently affixing them to it. An analogous reactivity scale is constructed also for the Immobiline chemicals, whose reactivity is shown to be linearly dependent on the pK values, the least reacting species being the acidic compounds. When analyzing real-life samples by two-dimensional (2-D) maps, like milk powders, a number of modifications can be detected by MALDI-TOF mass spectra of eluted spots, including variable phosphorylation sites (up to nine) and lactosyl moieties. If, for eluting such spots, formic acid is used, MALDI-TOF mass spectrometry (MS) reveals an incredible number of formylation sites, on Ser and Thr residues.

A number of cross-linkers that are commonly used in polyacrylamide gels have been incubated with bovine beta-lactoglobulin B and the resulting reaction mixtures were examined by matrix assisted laser desorption/ionization-mass spectrometry. At concentrations of 0.1, 1, and 20 mM of each cross-linker incubated for 1 h with 50 pmol/microL of the protein, a reactivity scale can be expressed as polyethylene glycol diacrylate > N,N'-bisacrylylcystamine > bisacrylyl piperazine > N,N'-methylenebisacrylamide >> N,N'-diallyltartardiamide (PEGDA>BAC>BAP>Bis>>DATD). Relatively short incubation times indicated one of the five Cys residues as the target of reaction, which was confirmed by post-source decay measurements. Longer incubation times (24 h) with bisacrylamide extended the reaction to all five Cys residues and a number of Lys residues. A second consequence of longer reaction time is the involvement of both terminals of the cross-linker in the observed reaction. This experimental evidence is the first to demonstrate a different reactivity of both ends of one of the most commonly used cross-linkers. Investigation of solutions containing a cross-linker and acrylamide monomers provided useful information on the competition between the two identities for reaction with the protein. Possible implications of these experimental observations for isoelectric focusing separations in polyacrylamide gels are discussed.

A number of Immobilines, with pK 1.0-10.3, were incubated with two proteins, bovine alpha-lactalbumin (pI 4.80) and chicken egg lysozyme (pI 9.32), at pH approximately 9-10 and the resulting solutions were examined by matrix assisted laser desorption/ionization mass spectrometry. The reflectron mode of the same technique was also used to analyze a number of tryptic digests of some solutions. The extent and the number of detected alkylation sites associated with the acidic protein were found to be linearly proportional to the pK values of the investigated Immobilines, an effect which was less evident for the basic protein. The high resolution measurements of some tryptic digests indicate the cysteine residues as the likely sites of alkylation. The implications of the present data for isoelectric focusing separations on immobilized pH gradients and for two-dimensional maps are discussed.

In addition to taking into account the more than 200 endogenously produced posttranslational modifications, a detailed analysis of proteins separated by two-dimensional electrophoresis must consider other modifications that a protein can experience during the various steps of its separation. Keywords: proteomics; protein alkylation; iodoacetamide; acrylamide; 4-vinylpyridine; electrophoresis