Aqueous solutions of dichloro(ethylenediamine)palladium(II) were investigated using electrospray mass spectrometry (ESMS). The most abundant peak (m/z 436.8) was attributed to the dimeric Pd(en)Cl 2 ·Pd(en)Cl ϩ ion. We conjecture that the structures of the observed ions arise from the clustering of the hydrolysis products of the parent compound. This hypothesis was tested experimentally by carrying out a series of collision-induced dissociation (CID) experiments and deuterium exchange reactions. It was also assessed by performing density functional theory (DFT) calculations, from which optimized structures and reaction energetics were obtained. These results were compared with our earlier ESMS study of an aqueous Pd(en)Br 2 solution. Calculations were also carried out on the Pd(en)Br 2 system to facilitate the comparisons. Conclusions are drawn regarding the species present in the two aqueous solutions. (J Am Soc Mass Spectrom 2007, 18, 769 -777) © 2007 American Society for Mass Spectrometry T he last decade has seen a resurgence of interest in the coordination chemistry of transition metals. This is due in large part to their therapeutic value as antitumor agents. Many of these agents are platinum(II) complexes, the four best known being cisplatin, carboplatin, oxaliplatin, and nedaplatin [1][2][3]. Palladium(II) complexes have also attracted attention for similar reasons [4][5][6][7].In vivo, hydrolysis of these compounds occurs, and it is these hydrolytic products that yield their medicinal activity [8,9]. The hydrolytic products are also strongly linked to issues of renal toxicity [10]. The mechanisms of action for these coordination complexes are closely tied to their hydrolysis products, and these products and mechanisms are poorly understood, partly due to the possibility of multiple equilibria with similar energetics [11,12]. It is for these reasons that coordination complexes of Ni(II), Pd(II), and Pt(II) in aqueous solutions have been the focus of a number of investigations [13][14][15][16][17].Electrospray mass spectrometry (ESMS) is particularly well suited for the study of metal-containing aqueous solutions, and a variety of such studies have been conducted [18,19]. We recently reported the results of one such study into aqueous solutions of dibromo(ethylenediamine)palladium(II) [20]. When the solution was electrosprayed into a quadrupole ion-trap mass spectrometer, a number of previously unreported species were detected, the principle one being a dimeric ion formed from the Pd(en)Br 2 parent compound and the Pd(en)Br ϩ ion. The dimeric ion was subjected to collision-induced dissociation (CID), and these MS n experiments revealed a series of ions formed through the sequential loss of HBr molecules. Complexes observed from the electrospray process are not necessarily a reflection of those complexes in solution, but they may instead simply represent species in solution from which the complexes are produced in the electrospray desolvation process. This has been observed previously in solutions containi...
Transcription factors (TFs) are essential for the expression of all proteins, including those involved in human health and disease. However, TFs are resistant to proteomic characterization because they are frequently masked by more abundant proteins due to the limited dynamic range of capillary liquid chromatography-tandem mass spectrometry and protein database searching. Purification methods, particularly strategies that exploit the high affinity of TFs for DNA response elements on gene promoters, can enrich TFs prior to proteomic analysis to improve dynamic range and penetrance of the TF proteome. For example, trapping of TF complexes specific for particular response elements has been achieved by recovering the element DNA-protein complex on solid supports. Additional methods for improving dynamic range include two- and three-dimensional gel electrophoresis incorporating electrophoretic mobility shift assays and Southwestern blotting for detection. Here we review methods for TF purification and characterization. We fully expect that future investigations will apply these and other methods to illuminate this important but challenging proteome.
Southwestern blotting is a technique used to study DNA-protein interactions. This method detects specific DNA-binding proteins by incubating radiolabeled DNA with a gel blot, washing, and visualizing through autoradiography. A blot resulting from 1-dimensional SDS-PAGE reveals the molecular weight of the binding proteins. To increase separation and determine isoelectric point a 2-dimensional gel can be blotted. Additional dimensions of electrophoresis, such as a gel shift (EMSA), can precede isoelectric focusing and SDS-PAGE to further improve separation. Combined with other techniques, such as mass spectrometry, the DNA-binding protein can be identified.
A method to purify enzymes utilizing their specific biological affinity and catalytic specificity is described. For this chromatographic technique, an enzyme binds immobilized substrate coupled to a column in the absence of a cofactor required for catalysis but permissive for substrate binding. After washing, the missing cofactor is added to the column mobile phase, and the enzyme converts substrate into product and elutes from the column. A single-step purification of EcoRI endonuclease using a sequence-specific DNA column (containing the GAATTC motif coupled to cyanogen bromide-activated Sepharose 4B) binds EcoRI in the absence of Mg2+ and elutes when Mg2+ is applied in a highly purified state. Although the method described is specific for EcoRI, it can be readily modified for the purification of DNA polymerases and other enzymes. Furthermore, many of the same materials are also used for transcription factor purification. This protocol can be completed within 4-6 d.
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