Construction of a directory of tobacco plasma membrane proteins by combined two‐dimensional gel electrophoresis and protein sequencing

Abstract

The polypeptide pattern of the plasma membrane from tobacco was studied by two-dimensional gel electrophoresis. When using classical carrier ampholyte isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) approximately 400 polypeptide spots were detected after silver staining and computer analysis using the QUEST software. This resolution was sufficient to assess physiological effects such as changes in a phytohormone concentration. By using pH 4-8 immobilized pH gradient (IPG)-IEF and 10%T SDS-PAGE gels, approximately 600 polypeptides, corresponding to ca. 80% of the total population expected, were resolved. This cross-section of the plasma membrane polypeptide population was mainly constituted by low or intermediate molecular mass (25 to 45 kDa) and acidic (5.2 < pI < 6.1) polypeptides. After sample application by in-gel rehydration, large amounts of plasma membrane protein (between 5 mg and 10 mg protein) were analyzed using IPG-IEF, and N-terminal protein sequencing was performed for polypeptides collected from one gel. Internal protein sequences were also obtained. Nearly all protein sequences corresponded to unidentified proteins but several of them matched translated sequences from unidentified plant expressed sequence tags (ESTs). It is concluded that the combined use of IPG-IEF gels and in-gel rehydration allows, in the case of plant membrane protein, both analytical and micropreparative separations with an efficiency comparable to that demonstrated for soluble proteins. Finally, it is suggested that a systematic investigation of plant plasma membrane polypeptides is feasible and would constitute a source of new and plant-specific genes.

Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative Proteomics

“…The IPG strips were rehydrated overnight with 150 mg of proteins solubilized in 9 M urea, 4% 3-([3-cholamidopropyl]dimethylammonio)-1-propane-sulfonate, 2 mM tributylphosphine, 2% pharmalyte, pH 4.0 to 7.0, 0.5% Triton X-100, and a few crystals of bromophenol blue (to evaluate the uniformity of the rehydration) in a reswelling tray at room temperature. Isoelectric focusing was conducted at 188C in a Multiphor II (Amersham Biosciences) following the running conditions as described (Rouquie et al, 1997) for up to 72 kV/h. The focused strips were denaturated and reduced in an equilibration buffer (6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 6.8, and 2% SDS) with addition of 5 mM tributylphosphine for 20 min and subsequently alkylated in equilibration buffer with 15 mM iodoacetamide for 20 min (Rabilloud et al, 1997;Herbert et al, 1998).…”

Challenges and Prospects of Plant Proteomics

“…This resulted in a number of fairly methodological studies, the construction of 2-DE reference maps (Rouquie et al, 1997;, and some of the data appeared on a Web site (http://sphinx.rug.ac.be:8080/ppmdb/). A number of plasma membrane-specific proteins were identified and most of these papers highlighted the failure to use 2-DE gels for reproducible and complete mapping of membrane proteins.…”

Proteomics of the Chloroplast: Systematic Identification and Targeting Analysis of Lumenal and Peripheral Thylakoid Proteins

“…For the analytical and preparative gels, individual 13-cm IPG strips (pH 4.0 to 7.0 or 6.0 to 11.0) were rehydrated overnight with 250 L of protein sample in solution A (for lumen) or B (for peripheral) in a reswelling tray at room temperature. The isoelectric focusing was conducted at 18ЊC by using a Pharmacia Multiphor II with a DryStrip kit and a Pharmacia 3500XL power supply, following the running conditions in Rouquié et al (1997). Isoelectric focusing strips were focused for ‫08ف‬ kVhr.…”