Proteins from silver-stained gels can be digested enzymatically and the resulting peptides analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nanoelectrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.

Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels

Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.

Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry

A cellular gene encodes scrapie PrP 27-30 protein

Deoxyoligonucleotides can now be synthesized rapidly and in high yield because of recent advances in nucleic acid chemistry. Key innovations include solid-phase synthesis on silica-based supports and the development of stable deoxynucleoside phosphoramidites as synthons. When incorporated into manual, semiautomatic, or automatic instruments, these new procedures can be used to prepare probes, mixed probes, deoxyoligonucleotides for priming DNA synthesis, analogues of deoxyoligonucleotides, and DNA segments containing more than 100 deoxynucleotides.

https://science.sciencemag.org/content/sci/230/4723/281.full.pdf

Gene synthesis machines: DNA chemistry and its uses

The two-dimensional electrophoresis (2-DE) technique developed by Klose in 1975 (Humangenetik 1975, 26, 211-234), independently of the technique developed by O'Farrell (J. Biol. Chem. 1975, 250, 4007-4021), has been revised in our laboratory and an updated protocol is presented. This protocol is the result of our experience in using this method since its introduction. Many modifications and suggestions found in the literature were also tested and then integrated into our original method if advantageous. Gel and buffer composition, size of gels, use of stacking gels or not, necessity of isoelectric focusing (IEF) gel incubation, freezing of IEF gels or immediate use, carrier ampholytes versus Immobilines, regulation of electric current, conditions for staining and drying the gels - these and other problems were the subject of our concern. Among the technical details and special equipment which constitute our 2-DE method presented here, a few features are of particular significance: (i) sample loading onto the acid side of the IEF gel with the result that both acidic and basic proteins are well resolved in the same gel; (ii) use of large (46 x 30 cm) gels to achieve high resolution, but without the need of unusually large, flat gel equipment; (iii) preparation of ready-made gel solutions which can be stored frozen, a prerequisite, among others, for high reproducibility. Using the 2-DE method described we demonstrate that protein patterns revealing more than 10 000 polypeptide spots can be obtained from mouse tissues. This is by far the highest resolution so far reported in the literature for 2-DE of complex protein mixtures. The 2-DE patterns were of high quality with regard to spot shape and background. The reproducibility of the protein patterns is demonstrated and shown to be thoroughly satisfactory. An example is given to show how effectively 2-DE of high resolution and reproducibility can be used to study the genetic variability of proteins in an interspecific mouse backcross (Mus musculus x Mus spretus) established by the European Backcross Collaborative Group for mapping the mouse genome. We outline our opinion that the structural analysis of the human genome, currently pursued most intensively on a worldwide scale, should be accompanied by a functional analysis of the genome that starts from the proteins of the organism.

Two‐dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome

A series of automated instruments that use state-of-the-art chemical methods has been developed for high-sensitivity protein sequencing, DNA synthesis and peptide synthesis. These intruments have been integrated into a centralized microchemical facility in order to promote their use for the study of a variety of biologically interesting problems. This facility has as one of its major functions the development of new chemistries and instrumentation for the structural analysis and synthesis of genes and proteins.

J. Firca

Papers

A microchemical facility for the analysis and synthesis of genes and proteins