Dataset Information

Analysis of protein phosphorylation in response to the ATP treatment in Arabidopsis thaliana

ABSTRACT: The project intended to reveal protein phosphorylation patterns in Arabidopsis thaliana in response to ATP. For this purpose, Arabidopsis thaliana plants, including WT, ATP receptor mutants (p2k1, p2k2, and double mutant p2k1/p2k2), and P2K1 overexpression plants, were treated with ATP or buffer (as the negative control). Crude membrane proteins were then extracted, reduced with DTT, alkylated with iodoacetamide, and digested with Lys-C/trypsin. The digested peptides were then acidified with formic acid, desalted with C18 SPE columns, and concentrated in a Speed-Vac concentrator. The Phosphopeptides were enriched from the above digested peptide samples using IMAC and then analyzed with LC-MS/MS. Data was searched with MaxQuant (ver. 2.0.1.0), which identified and quantified peptides and proteins across all of with Arabidopsis thaliana data set (Uniprot.2020.11.02).

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Arabidopsis Thaliana (ncbitaxon:3702)

SUBMITTER: Gary Stacey

PROVIDER: MSV000095888 | MassIVE |

REPOSITORIES: MassIVE

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Publications

Identifying receptor kinase substrates using an 8,000 peptide kinase client library enriched for conserved phosphorylation sites.

Kim Daewon D Jorge Gabriel Lemes GL Xu Chunhui C Su Lingtao L Cho Sung-Hwan SH Ahsan Nagib N Chen Dongqin D Zhou Lijuan L Gritsenko Marina A MA Zhou Mowei M Wan Jinrong J Pasa-Tolic Ljiljana L Xu Dong D Bartley Laura E LE Thelen Jay J JJ Stacey Gary G

Molecular & cellular proteomics : MCP 20250207

In eukaryotic organisms, protein kinases regulate diverse protein activities and signaling pathways through phosphorylation of specific protein substrates. Isolating and characterizing kinase substrates is vital for defining downstream signaling pathways. The Kinase Client (KiC) assay is an in vitro synthetic peptide LC-MS/MS phosphorylation assay that has enabled identification of protein substrates (i.e., clients) for various protein kinases. For example, previous use of a 2,100-member (2k) pe ...[more]

PMID: 39923935

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Project description:Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using Acyl-ATP (AcATP) probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Protein extracts were labelled with NHS-LC-Biotin, purified over neutravidin and eluted with formic acid. Eluted proteins were separated by 1-D SDS Gel and digested with Trypsin. Peptide mixtures were analysed by LC-MS/MS on an LTQ Velos. Spectra were searched using Mascot 2.3 using a fixed modification of cysteine (57.02 Da for carbamidomethyl), and variable modifications of methionine (15.99 Da for oxidation) and lysine (339.16 and 355.16 Da for BHAc and OBHAc labeling, respectively). A mass tolerance was set to 0.3 Da for the precursor ions and 0.4 Da for fragment ions. Up to two mis-cleavages for trypsin were permitted since the labeling would prevent cleavage at the labeled lysine. The MS2 spectra were searched against the TAIR10_pep_2012 (version November 2012) containing 35386 proteins of Arabidopsis thaliana, supplemented with a small database with 1095 artefact proteins and a reversed decoy database of the same proteins (totals 72962 entries). An oxidized version of the modifier was observed. Alternatively Arabidopsis leaf proteome was labeled with desthiobiotin-acyl ATP and digested with trypsin. Labeled peptides were purified on streptavidin matrix and sequenced by LC-MS/MS on an LTQ linear ion trap. Here the MS2 spectra were searched using SEQUEST against the TAIR9_pep_20090619 database (version June 2009) containing 32,769 proteins of Arabidopsis thaliana. MS2 searches included fixed iodoacetamide modification of cysteine (57 Da for alkylation), and variable modifications of methionine (16 Da for oxidation) and lysine (196 Da for DBAcATP labeling). Up to three mis-cleavages with trypsin were allowed and non-tryptic or half-tryptic peptides were excluded. A mass tolerance was set to 3 Da for the precursor ions and 1 Da for fragment ions.

Project description:Pilotexp_IMAC_TiO2_eluate_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The enriched phosphopeptides were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID (MSA) and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. IMAC_TiO2_eluate_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The enriched phosphopeptides were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID (MSA) and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. IMAC_TiO2_FT_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The unphosphorylated peptides found in the flowthrough of the TiO2 column were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. The iTRAQ ratio obtained for the unmodified peptides from this flowthrough sample was used to calculate protein ratios for the 4 different conditions.