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.

Human serum response factor (SRF) bearing a histidine tag was expressed using vaccinia virus. The recombinant protein was purified and shown to be phosphorylated mainly in its N‐terminal part. The corresponding phosphorylation sites were mapped by microsequencing and also appear to be phosphorylated in endogenous serum response factor. Four phosphorylation sites are located on serines within amino acids 77–85, while another phosphorylation site has been identified at Ser103. Mutations that considerably reduced or abolished phosphorylation at amino acids 77–85 caused a decrease in binding to the c‐fos serum response element accompanied by markedly reduced association and dissociation rates. In contrast, replacing Ser103 by alanine decreased DNA binding activity without drastically affecting the on/off rates. The combination of abolishing phosphorylation at amino acids 77–85 and 103 displayed greatly reduced on/off rates of DNA binding, but the reduction of DNA binding activity was partially alleviated. None of these mutations affect either the ability to interact with p62TCF or stimulation of transcription in vitro. These findings imply possible roles for SRF phosphorylation in the regulation of c‐fos transcription.

To achieve high throughput, protein microcharacterization sample preparation must be automated. We describe a cartesian robot capable of processing 32 protein samples in parallel. The system is based on specially designed flow-through reactors for contamination-free reagent delivery and removal. Washing of excised gel pieces, reduction and alkylation, proteolytic cleavage and peptide extraction are performed in these reactors. Compatibility of the system with HPLC peptide separation and Edman degradation as well as with laser desorption mass spectrometry of the unseparated mixture is demonstrated. This is the first report describing automated preparation and processing of multiple protein samples.

We here describe the use of added reversed-phase chromatographic beads to concentrate peptides from highly diluted solutions. In the procedure developed, peptide-bead suspensions are dried under vacuum to complete dryness; peptides are subsequently eluted in a small volume of matrix-assisted laser desorption/ionization (MALDI)-matrix containing organic/aqueous solvent and transferred to a MALDI-target for mass analysis. We show that by using this bead-peptide concentration procedure, low femtomole amounts of peptides are efficiently concentrated, up to 1000 times, to volumes smaller than 0.7 microL. We have used this concentration procedure in combination with MALDI-post-source decay analysis to identify subpicomole amounts of proteins present in polyacrylamide gels. Furthermore, we show that the bead-peptide concentration method can be elegantly used to clean up samples contaminated with high concentrations of substances normally deleterious to MALDI-mass spectrometry (MS) experiments. We have found additionally that the bead-peptide concentration procedure can be successfully used to store low femtomole amounts of peptide for prolonged periods of time without severe losses of peptide material. This bead-peptide concentration procedure therefore seems to be a simple and convenient step in the MALDI-MS sample preparation process.