title: Use of phosphoproteomics to study posttranslational protein modifications in Arabidopsis chloroplasts.
abstract: The posttranslational modification of proteins is important for the regulation of enzymatic activity, protein half-life, and interaction with other molecules. One of the best understood posttranslational modifications is the reversible phosphorylation of proteins at serine, threonine, or tyrosine residues. These phosphoamino acids are relatively stable in acidic solutions, and their comprehensive identification by mass spectrometry is, therefore, feasible. Phosphoproteomics-type experiments require some modifications in the sample preparation, mass spectrometry setup, and software-based data interpretation compared to standard proteomics workflows. Furthermore, phosphoproteome analyses are incompatible with long organelle isolation procedures prior to analysis, because of the highly dynamic nature of regulatory phosphorylations. In this chapter, we provide a detailed step-by-step overview of the complex experimental setup required for successful chloroplast phosphoproteome analysis, report our experience with existing methods, and comment on their application in the field.
authors.affiliation: Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Halle, Saale, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Endler
authors.forename: Sacha
authors.initials: S
authors.lastname: Baginsky
mesh_terms: D017360:Arabidopsis / Q000166:cytology* / Q000378:metabolism; D060365:Chloroplast Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D056148:Chromatography, Reverse-Phase; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018520:Plant Shoots / Q000166:cytology / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D012995:Solubility; D014025:Titanium / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
chemical_list: D060365:Chloroplast Proteins; D008565:Membrane Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; C009495:titanium dioxide; D014025:Titanium; D014357:Trypsin