Targeted proteomics analysis of protein degradation in plant signaling on an LTQ-Orbitrap mass spectrometer.

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.

[1]  A. Bachmair,et al.  PRT1 of Arabidopsis thaliana encodes a component of the plant N-end rule pathway. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[2]  R. Aebersold,et al.  Selected reaction monitoring–based proteomics: workflows, potential, pitfalls and future directions , 2012, Nature Methods.

[3]  B. Domon,et al.  Targeted Proteomic Quantification on Quadrupole-Orbitrap Mass Spectrometer* , 2012, Molecular & Cellular Proteomics.

[4]  A. Varshavsky.  The N‐end rule pathway and regulation by proteolysis , 2011, Protein science : a publication of the Protein Society.

[5]  J. Callis,et al.  A delayed leaf senescence mutant is defective in arginyl-tRNA:protein arginyltransferase, a component of the N-end rule pathway in Arabidopsis. , 2002, The Plant journal : for cell and molecular biology.

[6]  S. Emr,et al.  Ubiquitin and membrane protein turnover: from cradle to grave. , 2012, Annual review of biochemistry.

[7]  T. Boller,et al.  Ligand-induced endocytosis of the pattern recognition receptor FLS2 in Arabidopsis. , 2006, Genes & development.

[8]  S. Elledge,et al.  Identification of SCF Ubiquitin Ligase Substrates by Global Protein Stability Profiling , 2008, Science.

[9]  M. Mann,et al.  Mass Spectrometry-based Proteomics Using Q Exactive, a High-performance Benchtop Quadrupole Orbitrap Mass Spectrometer* , 2011, Molecular & Cellular Proteomics.

[10]  B. Usadel,et al.  Quantitation in mass-spectrometry-based proteomics. , 2010, Annual review of plant biology.

[11]  Jonathan D. G. Jones,et al.  A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence , 2007, Nature.

[12]  Ping He,et al.  A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity , 2009, Proceedings of the National Academy of Sciences.

[13]  M. McKay,et al.  Unique Ion Signature Mass Spectrometry, a Deterministic Method to Assign Peptide Identity , 2009, Molecular & Cellular Proteomics.

[14]  Yuan-Qing Xia,et al.  It is time for a paradigm shift in drug discovery bioanalysis: from SRM to HRMS. , 2011, Journal of mass spectrometry : JMS.

[15]  Alexandra M. E. Jones,et al.  Phosphorylation-Dependent Differential Regulation of Plant Growth, Cell Death, and Innate Immunity by the Regulatory Receptor-Like Kinase BAK1 , 2011, PLoS genetics.

[16]  Scott Peterman,et al.  Highly multiplexed targeted proteomics using precise control of peptide retention time , 2012, Proteomics.

[17]  Prisca Viehoever,et al.  GABI-Kat SimpleSearch: new features of the Arabidopsis thaliana T-DNA mutant database , 2011, Nucleic Acids Res..

[18]  Ryan G. Anderson,et al.  The Ubiquitin Ligase PUB22 Targets a Subunit of the Exocyst Complex Required for PAMP-Triggered Responses in Arabidopsis [C][W] , 2012, Plant Cell.

[19]  R. Aebersold,et al.  Selected reaction monitoring for quantitative proteomics: a tutorial , 2008, Molecular systems biology.

[20]  Yong Tae Kwon,et al.  The N-end rule pathway. , 2012, Annual review of biochemistry.

[21]  L. Voesenek,et al.  Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization , 2011, Nature.

[22]  A. Varshavsky,et al.  The N-end rule pathway controls multiple functions during Arabidopsis shoot and leaf development , 2009, Proceedings of the National Academy of Sciences.

[23]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[24]  R. Vierstra.  The ubiquitin–26S proteasome system at the nexus of plant biology , 2009, Nature Reviews Molecular Cell Biology.

[25]  P. He,et al.  Direct Ubiquitination of Pattern Recognition Receptor FLS2 Attenuates Plant Innate Immunity , 2011, Science.

[26]  T. Boller,et al.  Molecular identification and characterization of the tomato flagellin receptor LeFLS2, an orthologue of Arabidopsis FLS2 exhibiting characteristically different perception specificities , 2007, Plant Molecular Biology.

[27]  Brendan MacLean,et al.  Bioinformatics Applications Note Gene Expression Skyline: an Open Source Document Editor for Creating and Analyzing Targeted Proteomics Experiments , 2022 .

[28]  V. Spicer,et al.  Peptide retention standards and hydrophobicity indexes in reversed-phase high-performance liquid chromatography of peptides. , 2009, Analytical chemistry.

[29]  K. Resing,et al.  Comparison of Label-free Methods for Quantifying Human Proteins by Shotgun Proteomics*S , 2005, Molecular & Cellular Proteomics.

[30]  M. Trujillo,et al.  Regulation of plant immune receptors by ubiquitination , 2012, Front. Plant Sci..

[31]  John M Smith,et al.  Sensitivity to Flg22 Is Modulated by Ligand-Induced Degradation and de Novo Synthesis of the Endogenous Flagellin-Receptor FLAGELLIN-SENSING2[W][OPEN] , 2013, Plant Physiology.

[32]  K. Hofmann,et al.  PRT6/At5g02310 encodes an Arabidopsis ubiquitin ligase of the N‐end rule pathway with arginine specificity and is not the CER3 locus , 2007, FEBS letters.

[33]  S. Robatzek,et al.  Mapping FLS2 function to structure: LRRs, kinase and its working bits , 2012, Protoplasma.

[34]  Julietta Marquez,et al.  The N-end rule pathway promotes seed germination and establishment through removal of ABA sensitivity in Arabidopsis , 2009, Proceedings of the National Academy of Sciences.

[35]  F. Wellmer,et al.  The plant N-end rule pathway: structure and functions. , 2010, Trends in plant science.

[36]  Derek J. Bailey,et al.  Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics* , 2012, Molecular & Cellular Proteomics.

[37]  G. Bassel,et al.  Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants , 2011, Nature.

[38]  Jonathan D. G. Jones,et al.  The plant immune system , 2006, Nature.

[39]  T. Boller,et al.  Sensitivity of Different Ecotypes and Mutants ofArabidopsis thaliana toward the Bacterial Elicitor Flagellin Correlates with the Presence of Receptor-binding Sites* , 2001, The Journal of Biological Chemistry.

[40]  Xiaojun Ding,et al.  Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a Pseudomonas syringae effector. , 2010, Cell host & microbe.

[41]  PRT1 of Arabidopsis Is a Ubiquitin Protein Ligase of the Plant N-End Rule Pathway with Specificity for Aromatic Amino-Terminal Residues1 , 2003, Plant Physiology.

[42]  D. N. Perkins,et al.  Probability‐based protein identification by searching sequence databases using mass spectrometry data , 1999, Electrophoresis.

[43]  Qikai Xu,et al.  Global Protein Stability Profiling in Mammalian Cells , 2008, Science.

[44]  K. Shirasu,et al.  Negative Regulation of PAMP-Triggered Immunity by an E3 Ubiquitin Ligase Triplet in Arabidopsis , 2008, Current Biology.