Victor J. Nesatyy

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). Copyright © 2002 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.

The potential of electrospray ionization (ESI) mass spectrometry (MS) to detect non‐covalent protein complexes has been demonstrated repeteadly. 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. Copyright © 2001 John Wiley & Sons, Ltd.