Abstract
The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis).
Highlights
From the ‡Institute of Molecular Pathology (IMP), Vienna, Austria; §Department of Chromosome Biology, Max F
We found many factors directly involved in DNA repair and chromatin remodeling
Many of the proteins have already been known for their importance to maintain genome integrity, we identified several of them as direct or indirect targets of ataxia telangiectasia-mutated (ATM) and ATR
Summary
Plant Material—Arabidopsis thaliana wild type plants and mutant lines (double homozygous offspring of a double heterozygous atm atr line (atm-2: Salk_006953; [8]; atr-2: Salk_032841; [12]) were in ecotype Columbia-0 (Col-0). Peptides generated by the FASP protocol were eluted at a flow rate of 275 nl/min applying a linear gradient from 0 to 100% B (30% ACN, 0.08% FA) in 60 min, followed by a step to 85% B and 15% C (80% ACN, 10% trifluoroethanol (Sigma-Aldrich), 0.08% FA) in 1 min, and another steep gradient to 10% B and 90% C in 4 min. Samples from the protein precipitation experiments and the TiO2 flow-through were analyzed in a slightly modified MS/MS set-up. For the sample of the protein precipitation experiment only a dataset containing the phosphopeptide-enriched sample was generated This dataset was filtered to 0.5% FDR for PSMs. The Mascot ion score threshold was set to 23 for CID spectra and to 13 for HCD spectra. To perform normalization on the protein level for the sample from the protein precipitation experiment, the nonphosphorylated peptides, which had been co-isolated in the phosphopeptide-enriched sample were used to calculate a protein ratio. A Pfam domain search was performed using Pfam 27.0 applying the standard settings
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