title: Targeted proteomics analysis of protein degradation in plant signaling on an LTQ-Orbitrap mass spectrometer.
abstract: Targeted proteomics has become increasingly popular recently because of its ability to precisely quantify selected proteins in complex cellular backgrounds. Here, we demonstrated the utility of an LTQ-Orbitrap Velos Pro mass spectrometer in targeted parallel reaction monitoring (PRM) despite its unconventional dual ion trap configuration. We evaluated absolute specificity (>99%) and sensitivity (100 amol on column in 1 μg of total cellular extract) using full and mass range scans as survey scans together with data-dependent (DDA) and targeted MS/MS acquisition. The instrument duty cycle was a critical parameter limiting sensitivity, necessitating peptide retention time scheduling. We assessed synthetic peptide and recombinant peptide standards to predict or experimentally determine target peptide retention times. We applied optimized PRM to protein degradation in signaling regulation, an area that is receiving increased attention in plant physiology. We quantified relative abundance of selected proteins in plants that are mutant for enzymatic components of the N-end rule degradation (NERD) pathway such as the two tRNA-arginyl-transferases ATE1 and ATE2 and the two E3 ubiquitin ligases PROTEOLYSIS1 and 6. We found a number of upregulated proteins, which might represent degradation targets. We also targeted FLAGELLIN SENSITIVE2 (FLS2), a pattern recognition receptor responsible for pathogen sensing, in ubiquitin ligase mutants to assay the attenuation of plant immunity by degradation of the receptor.
authors.affiliation: Proteome Analytics Research Group, ‡Independent Junior Research Group on Protein Recognition and Degradation, §Independent Junior Research Group Ubiquitination in Immunity, and ∥Cellular Signaling Group, Department of Stress and Developmental Biology (SEB), Leibniz Institute of Plant Biochemistry (IPB) , Halle (Saale) D-06120, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Majovsky
authors.forename: Christin
authors.initials: C
authors.lastname: Naumann
authors.forename: Chil-Woo
authors.initials: CW
authors.lastname: Lee
authors.forename: Ines
authors.initials: I
authors.lastname: Lassowskat
authors.forename: Marco
authors.initials: M
authors.lastname: Trujillo
authors.forename: Nico
authors.initials: N
authors.lastname: Dissmeyer
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hoehenwarter
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D059748:Proteolysis; D040901:Proteomics; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010940:Plant Proteins
keywords: Arabidopsis; Degradomics; N-end rule pathway; N-terminal; parallel reaction monitoring; plant−pathogen interaction; protein quantification; targeted protein degradation