Computational protocol: Identification of lipids and lipid-binding proteins in phloem exudates from Arabidopsis thaliana

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Protocol publication

[…] Phloem exudate was analysed using an Ultrafast Microprotein Analyzer system with a peptide microtrap and a C18 reverse-phase column (Michrom BioResources, Auburn, CA, USA). Samples were resuspended in 100 μl of deionized water and loaded onto the peptide microtrap. The gradient went from 5% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA) in water to 65% ACN/0.1% TFA in water in 42 min. The flow rate was 500 μl min−1. Fractions of interest were further analysed using matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) or Edman sequencing. Edman sequencing was performed by the MSU- Research Technology Support Facility.Alternatively, phloem exudates from 20 leaves per replicate (three replicates in total) were subjected to 15% SDS–PAGE. Protein bands were excised and digested with trypsin according to . The digested peptides were extracted into 60% ACN/1% TFA and the appearance of tryptic fragments was monitored using MALDI-TOF-MS. For liquid chromatography–tandem mass spectrometry (LC-MS/MS), samples were dried and resuspended into 2% ACN/0.1% formic acid. LC-MS was performed on a Capillary LC system (Waters Corp., Milford, MA, USA) coupled to an LCQ DECA ion trap mass spectrometer (Thermofinnigan, San Jose, CA, USA) equipped with a nanospray ionization source. The sample was trapped onto a Peptide Cap Trap (Michrom BioResources) and flushed onto a 5 cm×75 μm ID picofrit column packed with 5 μm ProteoPep C18 material (New Objective, Woburn, MA, USA). Peptides were eluted with a gradient of 2–95% ACN in 0.1% formic acid at a flow rate of 200 nl min−1 for 60 min. Peptides/proteins were identified using the programs SEQUEST, MASCOT, or gpm.org. Carbamidomethyl cysteine was set as the fixed modification and oxidation of methionine was allowed. Up to two missed tryptic sites were permitted. Peptide tolerance was set to 2.5 Da, and MS/MS tolerance was set to 0.8 Da. Positive identifications were confirmed by individually comparing MS/MS spectra. Positive identification required at least two unique peptides per proteins counting only peptides with significant scores (95% confidence per peptide; >2.5 for SEQUEST). have suggested that inclusion of single-hit proteins does not necessarily increase the false discovery rate. Four additional proteins with a single peptide identification were included due to the fact that their mass corresponded to the predicted size from the gel and they were seen in at least two independent preparations. One protein was submitted to Edman sequencing (Ed in Supplementary Table S1 available at JXB online) at the Michigan State University Research Technology Support Facility. Database searches using individual tryptic fragments were performed using the BLAST searches at NCBI (http://www.ncbi.nlm.nih.gov/blast). Predicted function/localization of proteins is based on the NCBI, GO (http://www.ebi.ac.uk/QuickGO/), and/or Bar (http://bar.utoronto.ca/welcome.htm) databases as well as the cited literature. […]

Pipeline specifications

Software tools Comet, QuickGO
Application MS-based untargeted proteomics
Organisms Arabidopsis thaliana, Sus scrofa, Homo sapiens
Chemicals Acyl Coenzyme A, Carbon, Fatty Acids, Phosphatidylinositols, Uridine Diphosphate, Oxylipins