Quantification of potassium levels in cells treated with Bordetella adenylate cyclase toxin.

The aim of this study was to compare two methods for quantification of changes in intracellular potassium concentration (decrease from ∼140 to ∼20mM) due to the action of a pore-forming toxin, the adenylate cyclase toxin (CyaA) from the pathogenic bacterium Bordetella pertussis. CyaA was incubated with stably transfected K1 Chinese hamster ovary cells expressing the toxin receptor CD11b/CD18 and the decrease in potassium concentration in the cells was followed by inductively coupled plasma mass spectrometry (ICP-MS). It is shown that this method is superior in terms of sensitivity, accuracy, and temporal resolution over the method employing the potassium-binding benzofuran isophthalate-acetoxymethyl ester fluorescent indicator. The ICP-MS procedure was found to be a reliable and straightforward analytical approach enabling kinetic studies of CyaA action at physiologically relevant toxin concentrations (<1000ng/ml) in biological microsamples.

Mercury volatile species generation from HCl and TRIS buffer media: Quantification of generation efficiency and characterization of severe changes in speciation information due to de-alkylation.

Severe changes in speciation information were observed during volatile species generation (VSG) due to de-alkylation of generated Hg species as proven by cryogenic trapping with inductively coupled plasma mass spectrometric detection (CT-ICP-MS). Methyl mercury hydride is de-methylated to Hg0 by 45% and 6%, respectively, in HCl and TRIS buffer media. Ethylmercury hydride is de-ethylated to Hg0 by 71% and 28%, respectively in HCl and TRIS buffer media. Only Hg0 as a volatile product was observed when generating from phenylmercury regardless of the reaction medium employed. These findings limit significantly the application of VSG to Hg speciation analysis, especially the possibility of generation of alkyl-substituted mercury hydrides for cryogenic trapping/separation or gas chromatography. On the contrary, post-column VSG of Hg species prior to spectrometric detection can be employed to enhance sensitivity without any negative effects on accuracy and precision of the results.

Influence of different proteomic protocols on degree of high-coverage identification of nonspecific lipid transfer protein 1 modified during malting.

Both top-down (combining protein separation with MS analysis of intact proteins) and bottom-up (MS analysis of digested proteins) proteomic approaches were used for detailed characterization of nonspecific lipid transfer protein from barley malt. The aim was obtaining high-coverage of the primary structure of the proteins and the determination of PTMs such as lipid adduction and glycation. Here we present an influence of 15 proteomic protocols (differing in applied separation technique, enzyme and digestion procedure) on the extent of the coverage of the protein primary structure. The most successful protocols were in-gel digestion with trypsin of alkylated protein and in-solution digestions with trypsin or trypsin/chymotrypsin mixture of the nonalkylated protein. Totally, full sequence coverage based on the PMF and 85% sequence coverage based on the peptide fragmentation including PTMs was obtained.

Transmembrane segments of complement receptor 3 do not participate in cytotoxic activities but determine receptor structure required for action of Bordetella adenylate cyclase toxin.

Adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of the whooping cough agent Bordetella pertussis penetrates phagocytes expressing the integrin complement receptor 3 (CR3, CD11b/CD18, α(M)ß(2) or Mac-1). CyaA translocates its adenylate cyclase (AC) enzyme domain into cell cytosol and catalyzes unregulated conversion of ATP to cAMP, thereby subverting cellular signaling. In parallel, CyaA forms small cation-selective membrane pores that permeabilize cells for potassium efflux, contributing to cytotoxicity of CyaA and eventually provoking colloid-osmotic cell lysis. To investigate whether the single-pass α-helical transmembrane segments of CR3 subunits CD11b and CD18 do directly participate in AC domain translocation and/or pore formation by the toxin, we expressed in CHO cells variants of CR3 that contained artificial transmembrane segments, or lacked the transmembrane segment(s) at all. The results demonstrate that the transmembrane segments of CR3 are not directly involved in the cytotoxic activities of CyaA but serve for maintaining CR3 in a conformation that is required for efficient toxin binding and action.

2D-HPLC and MALDI-TOF/TOF analysis of barley proteins glycated during brewing.

The barley proteins have been the subject of interests of many research groups dealing with barley grains, malt and beer. The proteins which remain intact after harsh malting conditions influence the quality and flavor of beer. The characteristic feature of the proteins present in malt and beer is their extensive modification with carbohydrates, mainly glucose that comes from the starch degradation during technological processes. The degree of the protein glycation has an effect on the quality of malt and beer and on the properties of the beer foam. A combination of two-dimensional high performance liquid chromatography (2D-HPLC) and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF MS) was used for the analysis of the protein extracts that were reduced, alkylated, and degraded enzymatically without prior protein separation. This so-called "shot-gun" approach enabled us to determine glycation sites in one third of the proteins identified in the study and to propose potential glycation markers for fast and efficient monitoring during malting.

Electrospray ionization mass spectrometry as a method for studying the high-pressure denaturation of proteins.

High-pressure denaturation of proteins can provide important information concerning their folding and function. These studies require expensive and complicated equipment. In this paper, we present a new convenient method for studying high-pressure denaturation of proteins combining DHX (deuterium-hydrogen exchange) and electrospray ionization MS. Application of various values of pressure causes different degrees of protein unfolding resulting in molecules with a different number of protons available for exchange with deuterons. After decompression a protein refolds and a certain number of deuterons are trapped within the hydrophobic core of a refolded protein. Redissolving the deuterated protein in an aqueous buffer initiates the DHX of amides located on the protein surface only, which can be monitored under atmospheric pressure by MS. Depending on the degree of deuteration after high-pressure treatment, the DHX kinetics are different and indicate how many deuterons were trapped in the protein after refolding. The dependence of this number on pressure gives information on the denaturation state of a protein. The distribution of deuterium along the sequence of a high-pressure-denatured protein was studied the ECD (electron-capture-induced dissociation) on a Fourier-transform mass spectrometer, enabling the monitoring of high-pressure denaturation with single amino acid resolution.