MPIPZ - ASY1 in vitro phosphorylation

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ASY1 in vitro phosphorylation.sky.zip2018-10-10 09:47:211810772
In vitro phosphorylation of ASY1 by CDKA;1

  • Organism: Arabidopsis thaliana
  • Instrument: Q Exactive Plus
  • SpikeIn: No
  • Keywords: phosphorylation, PRM, Arabidopsis thaliana, meiosis
  • Lab head: Hirofumi Nakagami
Abstract
Meiosis is a complex cell division program reducing chromosome number as a prerequisite for sexual reproduction and at the same time leading to a new combination of parental alleles contributing to biodiversity. Little is known about how the distinct order of the molecular processes of meiosis are orchestrated. Here we show that the Arabidopsis Cdk1/Cdk2 homolog CDKA;1 is a master regulator of meiosis needed for several aspects of meiosis such as chromosome synapsis. We identify the chromosome axis protein ASYNAPTIC 1 (ASY1), the Arabidopsis Hop1 homolog, which is required for synaptonemal complex formation, as a phospho-target of CDKA;1. Live cell imaging together with biochemical assays shows that CDK-dependent phosphorylation of ASY1 is required for its recruitment to the chromosome axis via ASYNAPTIC 3 (ASY3), the Arabidopsis Red1 homolog, counteracting the disassembly activity of the AAA+ ATPase PACHYTENE CHECKPOINT 2 (PCH2). Furthermore, we have mapped the closure motif of ASY1, typical for HORMA domain proteins, and provide evidence that the Cdk-dependent phosphorylation of ASY1 regulates the self-polymerization of ASY1 along the chromosome axis during early meiosis. Hence, the phosphorylation of ASY1 by CDKA;1 appears to be a two-pronged mechanism to initiate chromosome axis formation in meiosis.
Experiment Description
Sample preparation and LC-MS/MS data acquisition. The protein mixtures were reduced with dithiothreitol, alkylated with chloroacetamide, and digested with trypsin. These digested samples were desalted using StageTips with C18 Empore disk membranes (3 M) (Rappsilber et al, 2003), dried in a vacuum evaporator, and dissolved in 2% ACN, 0.1% TFA. Samples were analysed using an EASY-nLC 1200 (Thermo Fisher) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher). Peptides were separated on 16 cm frit-less silica emitters (New Objective, 0.75 µm inner diameter), packed in-house with reversed-phase ReproSil-Pur C18 AQ 1.9 µm resin (Dr. Maisch). Peptides were loaded on the column and eluted for 50 min using a segmented linear gradient of 5% to 95% solvent B (0 min : 5%B; 0-5 min -> 5%B; 5-25 min -> 20%B; 25-35 min ->35%B; 35-40 min -> 95%B; 40-50 min ->95%B) (solvent A 0% ACN, 0.1% FA; solvent B 80% ACN, 0.1%FA) at a flow rate of 300 nL/min. Mass spectra were acquired in data-dependent acquisition mode with a TOP15 method. MS spectra were acquired in the Orbitrap analyzer with a mass range of 300–1500 m/z at a resolution of 70,000 FWHM and a target value of 3×106 ions. Precursors were selected with an isolation window of 1.3 m/z. HCD fragmentation was performed at a normalized collision energy of 25. MS/MS spectra were acquired with a target value of 5x105 ions at a resolution of 17,500 FWHM, a maximum injection time of 120 ms and a fixed first mass of m/z 100. Peptides with a charge of 1, greater than 6, or with unassigned charge state were excluded from fragmentation for MS2; dynamic exclusion for 20s prevented repeated selection of precursors. For the targeted analysis samples were resolved using the above-mentioned segmented linear gradient. The acquisition method consisted of a full scan method combined with a non-scheduled PRM method. The 16 targeted precursor ions were selected based on the results of DDA peptide search in Skyline. MS spectra were acquired in the Orbitrap analyzer with a mass range of 300–2000 m/z at a resolution of 70,000 FWHM and a target value of 3×10^6 ions, followed by MS/MS acquisition for the 16 targeted precursors. Precursors were selected with an isolation window of 2.0 m/z. HCD fragmentation was performed at a normalized collision energy of 27. MS/MS spectra were acquired with a target value of 2x10^5 ions at a resolution of 17,500 FWHM, a maximum injection time of 120 ms and a fixed first mass of m/z 100. Data processing. Raw data from DDA acquisition were processed using MaxQuant software (version 1.5.7.4, http://www.maxquant.org/) {Cox et al., Nat. Biotechnol. 2008, 26, 1367.}. MS/MS spectra were searched by the Andromeda search engine against a database containing the respective proteins used for the in vitro reaction. Trypsin specificity was required and a maximum of two missed cleavages allowed. Minimal peptide length was set to seven amino acids. Carbamidomethylation of cysteine residues was set as fixed, phosphorylation of serine, threonine and tyrosine, oxidation of methionine and protein N-terminal acetylation as variable modifications. The match between runs option was disabled. Peptide-spectrum-matches and proteins were retained if they were below a false discovery rate of 1% in both cases. The DDA approach only enabled the identification of T142. To analyze the putative phosphorylation sites at T184 and T535 a targeted approach was employed. Raw data from the DDA acquisition were analyzed on MS1 level using Skyline (Version 4.1.0.18169, https://skyline.ms) {McLean et al., Bioinformatics, 2010, 26, 966.} and database containing the respective proteins used for the in vitro reaction. Trypsin specificity was required and a maximum of two missed cleavages allowed. Minimal peptide length was set to seven maximum length to 25 amino acids. Carbamidomethylation of cysteine, phosphorylation of serine, threonine and tyrosine, oxidation of methionine and protein N-terminal acetylation were set as modifications. Results were filtered for precursor charges of 2, 3 and 4. For each phosphorylated precursor ion a respective non-phosphorylated precursor ion was targeted as a control, furthermore several precursor ions from the backbone of ASY were chosen as controls between the samples. In total 16 precursors were chosen to be targeted with a PRM approach. After acquisition of PRM data the raw data were again processed using MaxQuant software, with above-mentioned parameters and phosphorylation of serine, threonine and tyrosine as variable modification. Peptide search results were analyzed using Skyline using above-mentioned parameters, additionally data were filtered for b- and y-ions and ion charges +1 and +2.
Sample Description
+ CDKA,1: His-MBP-ASY full length (16 ng/uL), His-MBP-SDS/CDKA, 1 (150 ug/uL) - CDKA,1: His-MBP-ASY full length (16 ng/uL) buffer composition: 50mM Tris-HCl, pH 7.5, 10mM MgCl2, 5mM DTT, 1mM ATP
Created on 10/10/18, 9:47 AM