Preparation and Proteomic Analysis of Chloroplast Ribosomes

Abstract

Proteomics of chloroplast ribosomes in spinach and Chlamydomonas revealed unique protein composition and structures of plastid ribosomes. These studies have suggested the presence of some ribosomal proteins unique to plastid ribosomes which may be involved in plastid-unique translation regulation. Considering the strong background of genetic analysis and molecular biology in Arabidopsis, the in-depth proteomic characterization of Arabidopsis plastid ribosomes would facilitate further understanding of plastid translation in higher plants. Here, I describe simple and rapid methods for the preparation of plastid ribosomes from Chlamydomonas and Arabidopsis using sucrose gradients. I also describe purity criteria and methods for yield estimation of the purified plastid ribosomes and subunits, methods for the preparation of plastid ribosomal proteins, as well as the identification of some Arabidopsis plastid ribosomal proteins by matrix-assisted laser desorption/ionization mass spectrometry.

The Arabidopsis Cytosolic Ribosomal Proteome: From form to Function

“…Methods are available to resolve 80S and 70S chloroplast ribosomes (Yamaguchi, 2011). However, given that the 70S and 80S ribosomes of C. reinhardtii sedimented closely on sucrose gradients (Yamaguchi et al, 2003) and that mitochondrial ribosomes from higher plants have been observed to sediment anywhere between 70S (Vasconcelos and Bogorad, 1971; Pinel et al, 1986) and 78S (Leaver and Harmey, 1973, 1976; Pring, 1974), the above observations highlight the importance of early fractionation steps, orthogonal to sucrose gradient purification, in obtaining pure cytosolic ribosomes required for confident discrimination of cytosolic and organellar ribosomal proteomes.…”

RT-PCR- and MALDI-TOF Mass Spectrometry-Based Identification and Discrimination of Isoforms Homologous to Pufferfish Saxitoxin- and Tetrodotoxin-Binding Protein in the Plasma of Non-Toxic Cultured Pufferfish (Takifugu rubripes)

“…14) Reductive alkylation and in-gel digestion were carried out as described by Shevchenko et al 15) Peptide extraction and MALDI mass spectrometry were performed as previously described. 16) MS and MS/MS spectra were obtained using a MALDI-QIT-TOF mass spectrometer (AXIMA Resonance, Shimadzu, Kyoto, Japan) in positive mode. Protein electroblotting onto a polyvinylidene difluoride (PVDF) membrane (Sequi-Blot, Bio-Rad, Hercules, CA, USA) was performed using a tank-blotter (Criterion Trans-Blot Cell, plate electrode type, Bio-Rad, Hercules, CA, USA) in ice-cold blotting buffer (25 mM Tris, 192 mM glycine, pH 8.3) at 100 V for 30 min.…”

A small-scale proteomic approach reveals a survival strategy, including a reduction in alkaloid biosynthesis, in Hyoscyamus albus roots subjected to iron deficiency

“…In-gel tryptic digestion and peptide extraction were carried out as previously described by Yamaguchi (2011). The dried samples were dissolved in 2 μL of DHBA solution (5 mg mL −1 of 2, 5-dihydroxybenzoic acid, in 33% v/v acetonitrile and 0.1% v/v trifluoroacetic acid), and 1 μL samples of the solutions were spotted onto a stainless 384-well MALDI target plate (Shimadzu GLC, Tokyo, Japan).…”