Computational protocol: Extracellular ATP elicits DORN1-mediated RBOHD phosphorylation to regulate stomatal aperture

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Protocol publication

[…] Mapping of the DORN1 autophosphorylation sites was done using a bottom-up proteomics approach. The purified, recombinant DORN1 kinase was phosphorylated in vitro in the presence of 5 mM MgCl2, MnCl2, and/or both MgCl2 and MnCl2 with or without ATP. Proteins were subsequently digested in-solution with trypsin. The tryptic peptides were fragmented by using either collision-induced dissociation (CID) or by using a “decision tree” method, which utilizes both CID and ETD during a single sample analysis and analyzed by using a LTQ Orbitrap XL ETD mass spectrometer (Thermo Fisher, San Jose, CA). Acquired MS/MS spectra were searched against the entire Arabidopsis protein sequence database obtained from TAIR database (TAIR10) and concatenated to a randomized version of TAIR10 (i.e., decoy) generated using an in-house developed program (DecoyDB Creator, available at Peptide spectral matches were evaluated primarily using the XCorr scoring function of SEQUEST employing a 1% false discovery rate. Phosphorylation site localization was performed using phosphoRS (Proteome Discoverer, version 1.0.3, Thermo Fisher). Each phosphopeptide spectrum was inspected manually and accepted only when the phosphopeptide had the highest pRS site probability, pRS score, XCorr value, and site-determining fragment ions allowed unambiguous localization of the phosphorylation site. This peptide score is based on the cumulative binomial probability that the observed match is a random event. The value of the pRS score strongly depends on the data scored and scores ≥50 was considered as a potential phosphopeptide. On the other hand, pRS site probability of each phosphorylation site is an estimation of the probability (0–100%) for the respective site being truly phosphorylated. pRS site probabilities above ≥95% are good evidence that the respective site is truly phosphorylated. The spectral counting method was employed to demonstrate the phosphorylation status in response to the given treatments. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD006678. […]

Pipeline specifications

Software tools Comet, ptmRS, Proteome Discoverer
Databases TAIR ProteomeXchange
Application MS-based untargeted proteomics
Organisms Bacteria
Chemicals Adenosine Triphosphate, NADP, Oxygen