Ca(2+) -complex stability of GAPAGPLIVPY peptide in gas and aqueous phase, investigated by affinity capillary electrophoresis and molecular dynamics simulations and compared to mass spectrometric results.

Strong, sequence-specific gas-phase bindings between proline-rich peptides and alkaline earth metal ions in nanoESI-MS experiments were reported by Lehmann et al. (Rapid Commun. Mass Spectrom. 2006, 20, 2404-2410), however its relevance for physiological-like aqueous phase is uncertain. Therefore, the complexes should also be studied in aqueous solution and the relevance of the MS method for binding studies be evaluated. A mobility shift ACE method was used for determining the binding between the small peptide GAPAGPLIVPY and various metal ions in aqueous solution. The findings were compared to the MS results and further explained using computational methods. While the MS data showed a strong alkaline earth ion binding, the ACE results showed nonsignificant binding. The proposed vacuum state complex also decomposed during a molecular dynamic simulation in aqueous solution. This study shows that the formed stable peptide-metal ion adducts in the gas phase by ESI-MS does not imply the existence of analogous adducts in the aqueous phase. Comparing peptide-metal ion interaction under the gaseous MS and aqueous ACE conditions showed huge difference in binding behavior.

Optimization of affinity capillary electrophoresis for routine investigations of protein-metal ion interactions.

To facilitate the implementation of affinity capillary electrophoresis into routine binding screening studies of proteins with metal ions, method acceleration, transfer and precision improvement were investigated. Affinity capillary electrophoresis was accelerated by using shorter capillaries, employing lower sample concentrations and smaller injection volumes. Intra- and inter-instrument method transfers were investigated considering the temperature setting of the capillary cooling system. For intra-instrument method transfer, similar results were obtained when transferring a method from a long (62 cm) to a short (31 cm) capillary. The analysis time was reduced from 9 to 4 min. In case of inter-instrument method transfer, interaction results showed small variation on the capillary electrophoresis instrument with inefficient capillary cooling system. Binding measurement precision was enhanced by slightly pushing the sample above the beginning of the capillary. Changing the buffer vials after each 30 runs and employing extra flushing after each 60 subsequent runs further enhanced the precision. The use of 0.1 molar ethylenediaminetetraacetic acid in the rinsing solution successfully desorbs the remaining metal ions from the capillary wall. Excellent precision for apparent mobility ratio measurements was achieved for different protein-metal ion interactions (relative standard deviation of 0.16-0.89%, 15 series, 12 runs for each).

A comprehensive platform to investigate protein-metal ion interactions by affinity capillary electrophoresis.

In this work, the behavior of several metal ions with different globular proteins was investigated by affinity capillary electrophoresis. Screening was conducted by applying a proper rinsing protocol developed by our group. The use of 0.1M EDTA in the rinsing solution successfully desorbs metal ions from the capillary wall. The mobility ratio was used to evaluate the precision of the method. Excellent precision for repeated runs was achieved for different protein metal ion interactions (RSD% of 0.05-1.0%). Run times were less than 6 min for all of the investigated interactions. The method has been successfully applied for the interaction study of Li(+), Na(+), Mg(2+), Ca(2+), Ba(2+), Al(3+), Ga(3+), La(3+), Pd(2+), Ir(3+), Ru(3+), Rh(3+), Pt(2+), Pt(4+), Os(3+), Au(3+), Au(+), Ag(+), Cu(1+), Cu(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), Cr(3+), V(3+), MoO4(2-) and SeO3(2-) with bovine serum albumin, ovalbumin, ß-lactoglobulin and myoglobin. Different interaction values were obtained for most of the tested metal ions even for that in the same metal group. Results were discussed and compared in view of metal and semimetal group's interaction behavior with the tested proteins. The calculated normalized difference of mobility ratios for each protein-metal ion interaction and its sign (positive and negative) has been successfully used to detect the interaction and estimate further coordination of the bound metal ion, respectively. The comprehensive platform summarizes all the obtained interaction results, and is valuable for any future protein-metal ion investigation.

Precise, fast, and flexible determination of protein interactions by affinity capillary electrophoresis: part 3: anions.

The binding of physiologically anionic species or negatively charged drug molecules to proteins is of great importance in biochemistry and medicine. Since affinity capillary electrophoresis (ACE) has already proven to be a suitable analytical tool to study the influence of ions on proteins, this technique was applied here for comprehensively studying the influence of various anions on proteins of BSA, ß-lactoglobulin, ovalbumin, myoglobin, and lysozyme. The analysis was performed using different selected anions of succinate, glutamate, phosphate, acetate, nitrate, iodide, thiocyanate, and pharmaceuticals (salicylic acid, aspirin, and ibuprofen) that exist in the anionic form at physiological pH 7.4. Due to the excellent repeatability and precision of the ACE measurements, not necessarily strong but significant influences of the anions on the proteins were found in many cases. Different influences in the observed bindings indicated change of charge, mass, or conformational changes of the proteins due to the binding with the studied anions. Combining the mobility-shift and pre-equilibrium ACE modes, rapidity and reversibility of the protein-anion bindings were discussed. Further, circular dichroism has been used as an orthogonal approach to characterize the interactions between the studied proteins and anions to confirm the ACE results. Since phosphate and various anions from amino acids and small organic acids such as succinate or acetate are present in very high concentrations in the cellular environment, even weak influences are certainly relevant as well.

Precise, fast and flexible determination of protein interactions by affinity capillary electrophoresis. Part 2: cations.

The influence of various cations as metal ions (barium, calcium, copper, magnesia, manganese, and nickel), pharmaceuticals (ephedrine, ethambutol, pilocarpine, and pirenzepine), arginine, and guanidine has been tested on BSA, ß-lactoglobulin, and ovalbumin. Influences on proteins regarding changes in size, charge, or mass were of particular interest. ACE proved to be a suitable method to investigate these effects. ACE is able to observe slight but significant changes on proteins due to the excellent precision of the measurements. Therefore, some unexpected behaviors of protein-ligand interactions were found. The protein charge becomes more negative under metal ion influence and some pharmaceutical cations. Probably metal ions bound to the proteins form additional complexes with anions from the surrounding solution. Furthermore, already bound cations could be displaced at the protein surface. Both effects would change the overall charge of the ligand-protein complexes. In all studied cases, multiple-binding stoichiometries have been observed.

Precise, fast, and flexible determination of protein interactions by affinity capillary electrophoresis: part 1: performance.

A generic screening approach was performed to investigate the binding of various potential ligands to proteins in order to investigate how proteins interact with ions and the complete surrounding solution. This also allows to study binding behavior and its regulation and protein functionality in general in a native environment. The ACE technique affords an excellent precision by applying appropriate rinsing procedures using a pressure of 2.5 bar and a continuous flow concept. Confidence intervals were estimated to proof significance of the interactions. This enables to investigate smaller yet important interactions, which were not possible with less precise techniques before. The influence of various ions on ovalbumin, ß-lactoglobulin, and BSA were screened by comparing the mobility ratios of the free protein and the influenced one. The analysis was performed using the metal ions Ba(2) +, Ca(2) +, Cu(2) +, Mg(2) +, Mn(2) +, Ni(2) +, the pharmaceutical cations ephedrine hydrochloride, ethambutol dihydrochloride, pilocarpine hydrochloride and pirenzepine dihydrochloride, and various anions, in particular phosphate, acetate, succinate, glutamate, and salicylate. For the anion influence study, myoglobin was also included to the screened proteins. The influence of these ions on the proteins was well diversified. The interactions could be distinguished with a fast and precise screening method since 90% of all mobility ratios had a RSD% below 2% and 79% had a RSD% lower than 1%. Hence, for more than 75% of the protein-ligand pairs significant interactions are observed with a very small confidence interval due to the very excellent precision of these used method.

The challenge to quantify proteins with charge trains due to isoforms or conformers.

Single proteins separated by 2-DE often show multiple spots spreading along the first dimension. In many cases, such charge trains are explained by isoform differences or by putative post-translational modifications including phosphorylation, glycosylation and others. We now report that individual spots of such charge trains on 2-D gels in fact often represent the same protein, but, apparently due to conformational changes, segregate to different isoelectric points. If MS analysis reveals protein identity, we therefore suggest integrating all individual spots within a charge train for quantification. Especially in quality control of pharmaceutical proteins, the integration of the spot groups of all active contents is preferable in order to obtain reproducible and reasonable quantitative results. However, most commercial software packages for gel analysis integrate the signals spot-wise. We provide an improved quantification tool for proteins with charge train groups. This calculation can be implemented using the MATLAB software and the self-developed "Correct Integration Software System" or the commercial software package Delta2D.

Quantitative gel electrophoresis: new records in precision by elaborated staining and detection protocols.

Gel electrophoresis (GE) is a very common analytical technique for proteome research and protein analysis. Despite being developed decades ago, there is still a considerable need to improve its precision. Using the fluorescence of Colloidal Coomassie Blue -stained proteins in near-infrared (NIR), the major error source caused by the unpredictable background staining is strongly reduced. This result was generalized for various types of detectors. Since GE is a multi-step procedure, standardization of every single step is required. After detailed analysis of all steps, the staining and destaining were identified as the major source of the remaining variation. By employing standardized protocols, pooled percent relative standard deviations of 1.2-3.1% for band intensities were achieved for one-dimensional separations in repetitive experiments. The analysis of variance suggests that the same batch of staining solution should be used for gels of one experimental series to minimize day-to-day variation and to obtain high precision.