Evaluation du couplage entre la chromatographie en phase liquide et la spectrometrie de masse en tandem pour les analyses protéomiques

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Organelle Proteomics

Proteomic analyses have proven to be an invaluable tool in obtaining high-throughput protein identification from low-abundance, complex biological samples. In Organelle Proteomics, detailed protocols provide step-by-step instructions to successfully study organelle proteomes by performing the purification of the various organelles present in eukaryotic cells, as well as by preparing certain sub-fractions of organelles. A series of chapters cover the whole analytical procedure of organelle characterization, from its purification starting with whole cells up to protein identification using mass spectrometry. Devoted to methods enabling a global estimate of the reliability of the protein list assigned to an organelle, Organelle Proteomics allows scientists to gain a vital and important understanding of organelle study

Characterization of E. coli ribosomal particles : combined analysis of whole proteins by mass spectrometry and of proteolytic digests by liquid chromatography-tandem mass spectrometry.

This chapter describes the purification of ribosomal particles from a mutant strain of Escherichia coli using sucrose gradients and the characterization of their protein composition by a combination of mass spectrometry (MS) techniques. The main objective is to identify the ribosomal proteins that are missing in an aberrant ribosomal particle corresponding to a defective large subunit. To address this question, the tryptic digests of the purified ribosomal particles are analyzed by the coupling between liquid chromatography and tandem MS. The presence or absence of a given ribosomal protein in the defective particle is determined by comparing the MS intensities of its identified tryptic peptides with that of the mature large subunit. These analyses also allow identification of proteins copurifying with the ribosomal particles. To detect low-mass proteins escaping identification by the above method, intact proteins are also analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and nano-ESI-QqTOF MS

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

International audienceIn eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin‐associated proteins contribute to DNA‐related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation‐dependent regulation of plant chromatin and chromatin‐associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation

Proteomic Analysis of Histone Variants and Their PTMs: Strategies and Pitfalls

International audienc

Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana

International audienceThe nucleus is the organelle where basically all DNA-related processes take place in eukaryotes, such as replication, transcription, and splicing as well as epigenetic regulation. The identification and description of the nuclear proteins is one of the requisites toward a comprehensive understanding of the biological functions accomplished in the nucleus. Many of the regulatory mechanisms of protein functions rely on their PTMs among which phosphorylation is probably one of the most important properties affecting enzymatic activity, interaction with other molecules, localization, or stability. So far, the nuclear and subnuclear proteome and phosphoproteome of the model plant Arabidopsis thaliana have been the subject of very few studies. In this work, we developed a purification protocol of Arabidopsis chromatin-associated proteins and performed proteomic and phosphoproteomic analyses identifying a total of 879 proteins of which 198 were phosphoproteins that were mainly involved in chromatin remodeling, transcriptional regulation, and RNA processing. From 230 precisely localized phosphorylation sites (phosphosites), 52 correspond to hitherto unidentified sites. This protocol and data thereby obtained should be a valuable resource for many domains of plant research