Proteomic LC–MS analysis of Arabidopsis cytosolic ribosomes: Identification of ribosomal protein paralogs and re-annotation of the ribosomal protein genes

• Published in: Journal of proteomics Vol. 128; pp. 436 - 449
• Main Authors: Hummel, Maureen, Dobrenel, Thomas, Cordewener, Jan (J.H.G.)., Davanture, Marlène, Meyer, Christian, Smeekens, Sjef (J.C.M.)., Bailey-Serres, Julia, America, Twan (A.H.P.)., Hanson, Johannes
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 14.10.2015; Elsevier
• Subjects: A. thaliana; Amino Acid Sequence; Arabidopsis - metabolism; Arabidopsis Proteins - metabolism; BIOS Applied Metabolic Systems; Chromatography, Liquid - methods; Data-independent acquisition; DIA; Gene Expression Profiling - methods; LC-MS; Life Sciences; Mass Spectrometry - methods; Molecular Sequence Data; Paralogs; PRI BIOS Applied Metabolic Systems; Proteome - chemistry; Proteome - metabolism; Ribosomal protein; Ribosomal Proteins - chemistry; Ribosomal Proteins - metabolism; Ribosomes; DDA; Paralogs; nt; RPKM; rRNA; LC–MS; Ribosomes; RPG; mRNA; r-protein; PEB; PLGS; Ribosomal protein; Data-independent acquisition; RPL; TAIR; MS medium; RPP; RPS; UPLC; A. thaliana; 60S; DIA; MSE; LC-MS
• ISSN: 1874-3919; 1876-7737; 1876-7737
• DOI: 10.1016/j.jprot.2015.07.004

Arabidopsis thaliana cytosolic ribosomes are large complexes containing eighty-one distinct ribosomal proteins (r-proteins), four ribosomal RNAs (rRNA) and a plethora of associated (non-ribosomal) proteins. In plants, r-proteins of cytosolic ribosomes are each encoded by two to seven different expressed and similar genes, forming an r-protein family. Distinctions in the r-protein coding sequences of gene family members are a source of variation between ribosomes. We performed proteomic investigation of actively translating cytosolic ribosomes purified using both immunopurification and a classic sucrose cushion centrifugation-based protocol from plants of different developmental stages. Both 1D and 2D LC–MSE with data-independent acquisition as well as conventional data-dependent MS/MS procedures were applied. This approach provided detailed identification of 165 r-protein paralogs with high coverage based on proteotypic peptides. The detected r-proteins were the products of the majority (68%) of the 242 cytosolic r-protein genes encoded by the genome. A total of 70 distinct r-proteins were identified. Based on these results and information from DNA microarray and ribosome footprint profiling studies a re-annotation of Arabidopsis r-proteins and genes is proposed. This compendium of the cytosolic r-protein proteome will serve as a template for future investigations on the dynamic structure and function of plant ribosomes. Translation is one of the most energy demanding processes in a living cell and is therefore carefully regulated. Translational activity is tightly linked to growth control and growth regulating mechanism. Recently established translational profiling technologies, including the profiling of mRNAs associated with polysomes and the mapping of ribosome footprints on mRNAs, have revealed that the expression of gene expression is often fine-tuned by differential translation of gene transcripts. The eukaryotic ribosome, the hub of these important processes, consists of close to eighty different proteins (depending on species) and four large RNAs assembled into two highly conserved subunits. In plants and to lesser extent in yeast, the r-proteins are encoded by more than one actively transcribed gene. As r-protein gene paralogs frequently do not encode identical proteins and are regulated by growth conditions and development, in vivo ribosomes are heterogeneous in their protein content. The regulatory and physiological importance of this heterogeneity is unknown. Here, an improved annotation of the more than two hundred r-protein genes of Arabidopsis is presented that combines proteomic and advanced mRNA expression data. This proteomic investigation and re-annotation of Arabidopsis ribosomes establish a base for future investigations of translational control in plants. [Display omitted] •Arabidopsis cytosolic ribosomes were isolated using two orthogonal methods for proteomic profiling.•Analysis with three different MS methodologies showed the synergistic value in ribosomal protein identification.•Proteomic data combined with ribosome footprint mRNA-sequencing data further improved the annotation of the ribo-proteome.•The combined proteomic methodology detected the 83% of the cytosolic r-protein genes encoded by the Arabidopsis genome.