Recovery of intact proteins from silver stained gels

Silver stained proteins of a wide molecular weight (MW) range (20-116 kDa) were successfully recovered by both electroblot and electroelution. The recovery was demonstrated for nanogram loads of proteins separated by SDS-PAGE and visualized by silver staining methods compatible and incompatible with mass spectrometry (MS). It was shown that the alcohol/acid and glutaraldehyde fixation steps present in a number of staining procedures did not prevent recovery of intact proteins from gels. It was found that the recovery of intact proteins from silver stained gels was substantially increased upon pre-equilibration in a buffer containing the reducing agent, dithiothreitol (DTT). The effect of destaining on the recovery of silver stained proteins was also investigated. Comparable recovery of intact proteins within a wide MW range from silver stained gels with and without destaining step was demonstrated. Recovery of model proteins from gels visualized using silver staining method compatible with MS showed 52 to 76% yield of that from the unstained gel, depending upon method of the transfer. Comparison of the recovery of intact proteins from gels visualized using other staining procedures was also made. The above findings have implications as to the supposed irreversible nature of protein "fixation" inside polyacrylamide matrix, and confirm lack of binding of proteins in the gel to metal silver deposited on its surface. This method has the potential to be suitable for direct characterization of proteins by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) without additional purification steps.     

Detection of noncovalent complex between alpha-amylase and its microbial inhibitor tendamistat by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is now routinely used for detection of noncovalent complexes. However, detection of noncovalent protein-protein complexes is not a widespread practice and still produces some challenges for mass spectrometrists. Here we demonstrate the detection of a noncovalent protein-protein complex between alpha-amylase and its microbial inhibitor tendamistat using ESI-MS. Crude porcine pancreatic alpha-amylase was purified using a glycogen precipitation method. Noncovalent complexes between porcine pancreatic alpha-amylase and its microbial inhibitor tendamistat are probed and detected using ESI-MS. The atmosphere-vacuum ESI conditions along with solution conditions and the ratio of inhibitor over enzyme strongly affect the detection of noncovalent complexes in the gas phase. ESI mass spectra of alpha-amylase at pH 7 exhibited charge states significantly lower than that reported previously, which is indicative of a native protein conformation necessary to produce a noncovalent complex. Detection of noncovalent complexes in the gas phase suggests that further use of conventional biochemical approaches to provide a qualitative, and in some cases even quantitative, characterization of equilibria of noncovalent complexes in solution is possible.     

Microwave-assisted protein staining: mass spectrometry compatible methods for rapid protein visualisation

The effects of microwave irradiation on the staining of electrophoresed and electroblotted proteins have been assessed using currently available detection methods. Although the absorption of microwave radiation was found to be uneven, band intensity following microwave-assisted protein staining (MAPS) was comparable and in some cases exceeded the intensity of the bands visualised by the original staining methods. It was found that microwave treatment drastically reduced the duration of the staining protocols for visualisation of the proteins separated by both one- and two-dimensional electrophoresis. Application of MAPS methods did not affect peptide mass fingerprinting analysis by mass spectrometry and subsequent identification of the protein by database searching. The peptide mass maps corresponding to the proteins visualised using both the conventional and MAPS methods did not show significant difference in signal/noise ratio. Moreover, it appeared that microwave treatment of the gels resulted in the increased recovery of the peptides following in-gel trypsin digestion. Briefly, microwave-assisted protein staining methods were rapid, compatible with mass spectrometry and were equally effective on thin (0.75-mm) and thick (1.5-mm) gels (such as those used in 2D electrophoresis).     

Potential drugs and nondrugs: prediction and identification of important structural features

Using decision trees, a model to discriminate between potential drugs and nondrugs has been developed. Compounds from the Available Chemical Directory and the World Drug Index databases were used as training set; the molecular structures were represented using extended atom types. The error rate on an independent validation data set is 17.4%. The number of false negatives can be reduced by penalizing the misclassification of drugs so that 92 out of 100 potential drugs are correctly recognized. At the same time, 34 out of 100 nondrugs are classified as potential drugs. The predictions of the model can be used to guide the purchase or selection of compounds for biological screening or the design of combinatorial libraries. The visualization of the generated models in the form of colored trees allowed us to identify a few, surprisingly simple features that explain the most significant differences between drugs and nondrugs in the training set: Just by testing the presence of hydroxyl, tertiary or secondary amino, carboxyl, phenol, or enol groups, already three quarters of all drugs could be correctly recognized. The nondrugs, on the other hand, are characterized by their aromatic nature with a low content of functional groups besides halogens. The general applicability of the model is shown by the predictions made for several Organon databases.     

Gas-phase binding of non-covalent protein complexes between bovine pancreatic trypsin inhibitor and its target enzymes studied by electrospray ionization tandem mass spectrometry

The potential of electrospray ionization (ESI) mass spectrometry (MS) to detect non-covalent protein complexes has been demonstrated repeatedly. However, questions about correlation of the solution and gas-phase structures of these complexes still produce vigorous scientific discussion. Here, we demonstrate the evaluation of the gas-phase binding of non-covalent protein complexes formed between bovine pancreatic trypsin inhibitor (BPTI) and its target enzymes over a wide range of dissociation constants. Non-covalent protein complexes were detected by ESI-MS. The abundance of the complex ions in the mass spectra is less than expected from the values of the dissociation constants of the complexes in solution. Collisionally activated dissociation (CAD) tandem mass spectrometry (MS/MS) and a collision model for ion activation were used to evaluate the binding of non-covalent complexes in the gas phase. The internal energy required to induce dissociation was calculated for three collision gases (Ne, Ar, Kr) over a wide range of collision gas pressures and energies using an electrospray ionization source. The order of binding energies of the gas-phase ions for non-covalent protein complexes formed by the ESI source and assessed using CAD-MS/MS appears to differ from that of the solution complexes. The implication is that solution structure of these complexes was not preserved in the gas phase.     

Integrated receiver architectures for board-to-board free-space optical interconnects

In many computer and server communications copper cables and wires are currently being used for data transmission and interconnects. However, due to significant shortcomings, such as long transmission time, high noise level, unstable electrical properties, and high power consumption for cooling, researchers are increasingly turning their research interests toward alternatives, such as fiber optic interconnects and free-space optical communication technologies. In this paper, we present design considerations for an integrated receiver for high-speed free-space line-of-sight optical interconnects for distortion-free data transmission in an environment with mechanical vibrations and air turbulences. The receiver consists of an array of high-speed photodiodes for data communication and an array of quadrant photodiodes for real-time beam tracking in order to compensate for the beam misalignment caused by vibrations in servers. Different configurations for spatially positioning the quadrant and data photodiodes are discussed for 4×4 and 9×9 multielement optical detector arrays. We also introduce a new beam tracking device, termed the strip quadrant photodiodes, in order to accurately track highly focused optical beams with very small beam diameter.     

Identification of the estrogen receptor Cd-binding sites by chemical modification

The widely reported interactions of the estrogen receptor (ER) with endocrine disrupting chemicals (EDCs) present in the environment gave raise to public concern and led to a number of screening and testing initiatives on the international level. Recent studies indicated that certain heavy metals, including cadmium, can mimic the effects of the endogenous estrogen receptor agonist 17beta-estradiol, and lead to estrogen receptor activation. Previous studies of the chimeric proteins, which incorporate the ligand-binding domain of the human ER, identified Cys 381, Cys 447, Glu 523, His 524 and Asp 538 as possible sites of interactions with cadmium. In the present study we utilized the rainbow trout ER ligand-binding domain fused to glutathione-S-transferase, and used Cd-shielding against various types of chemical modification of the fusion protein to study non-covalent interactions between the ER and Cd. The distribution of exposed and shielded residues allowed to identify amino acid residues involved in the interaction. Our data indicated preferential protection of Cys groups by cadmium, suggesting their involvement in the interaction. This supports data found in the literature on the strong binding affinity of the thiol group towards metals. However, not all Cys in the fusion protein sequence were protected against chemical modification, illustrating the importance of their chemical environment. In general, the location of rtER-LBD Cys residues implicated in Cd interactions did not confirm assignments made by alanine-scanning mutagenesis for the hER, probably due to differences in experimental setup and fusion proteins used. The involvement of other functional groups such as carboxylic acids in the Cd interactions, though not confirmed, can not be completely ruled out due to the general limitations of the chemical modification approach discussed in detail. Suggestions for an improved experimental setup were made.     

Line-addressable digital-deflection programmable micromirror array

We report a novel digital-deflection programmable micromirror array driven by micromechanical digital-to-analog converters that eliminates the need for electrical digital-to-analog converters for analog displacement control, thus simplifying the driving circuitry and reducing the overall system cost. Furthermore, owing to the bistable and hysteretic characteristics of parallel-plate electrostatic actuators, an array of micromirrors can be controlled by means of row- and column-addressing lines, which drastically reduce the number of routing wires and allow array sizes to increase while they maintain high array quality.