Computational protocol: The Transcriptome and Proteome of the Diatom Thalassiosira pseudonana Reveal a Diverse Phosphorus Stress Response

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Protocol publication

[…] Biomass from frozen filter samples was scraped into tubes with a clean spatula then resuspended in 600 µL B-PER reagent (Thermo Scientific, Rockford, IL) supplemented with 0.5 mM EDTA and protease inhibitor 1mMphenylmethanesulfonylfluoride. Samples were vortexed for 1 minute and incubated at room temperature for 20 minutes then chilled on ice for 10 minutes. The cells were then sonicated with a microtip on ice for 1 minute at constant duty cycle, with the temperature not exceeding 28°C. Samples were centrifuged for 30 minutes at 14,100 RCF and 4°C, and supernatants were transferred and precipitated overnight in 50% acetone 50% methanol with 0.5 mM HCl at −20°C. Precipitated protein was collected by centrifugation at 14,100 RCF for 30 minutes at 4°C and dried by speed vacuum at room temperature. Protein was resuspended in B-Per/EDTA/PMSF extraction buffer, gently mixed and incubated for 30 minutes at room temperature and splits were combined. Aliquots were taken for protein determination by DC assay using bovine serum albumin as a protein standard (BioRad Inc., Hercules CA). Proteins were stored at −80°C until digestion.Protein was then digested following the tube gel digestion procedure with some modifications. Briefly, samples were immobilized in 15% acrylamide in pH 7.5 Tris buffer, incubated twice with 10% acetic acid and 50% ethanol for 20 min and 1 h, then with 10% acetic acid and 50% methanol for 2 h at room temperature and mixing at 350 RPM, decanting between. Gel samples were cut into ∼1 mm3 pieces and incubated twice with 50% acetonitrile 50% 25 mM ammonium bicarbonate solution for 1 h and again overnight, shaking at 350 RPM at 16°C and decanting between. Proteins immobilized within the gel were successively reduced with 10 mM dithiothreitol (DTT) at 56°C for 1 h, decanted, and alkyated with 30 mM iodoacetamide for 1 h at room temperature, washed in 25 mM ammonium bicarbonate for 20 minutes and twice with 100% acetonitrile for 10 minutes, and dried for 20 minutes by speed vacuum. Gel pieces were rehydrated and digested with a solution of 10 µg trypsin in 25 mM ammonium bicarbonate for 16 h at 37°C (1∶20 ratio trypsin to total protein, Promega Gold Mass Spectrometry Grade, Promega Inc., Madison WI). The peptides were extracted by successive additions of 50 percent acetonitrile (Fisher Optima) with 5% formic acid (Michrom Ultra Pure). The extracted peptides were combined and concentrated by speed vacuum for about three hours to less than 20 µL, diluted with 2 percent acetonitrile and 0.1 percent formic acid in water (Fisher Optima) and stored at −80°C.The protein digestions were analyzed using a peptide Cap Trap in-line with a reversed phase Magic C18 AQ column (0.2×150 mm, 3 µm particle size, 200 Å pore size, Michrom Bioresources Inc. Auburn CA) on a Paradigm MS4 HPLC system (Michrom Bioresources Inc.) at a flow rate of 2 µl minute−1. A LTQ linear ion trap mass spectrometer (Thermo Scientific Inc. San Jose CA) was used with an ADVANCE electrospray source (Michrom Bioresources Inc.). The chromatography consisted of a hyperbolic gradient from 5% buffer A to 95% buffer B for 300 minutes, where A was 0.1% formic acid (Michrom Ultra Pure) in water (Fisher Optima) and B was 0.1% formic acid in acetonitrile (Fisher Optima). The mass spectrometer was set to perform MS/MS on the top 7 ions using data-dependent settings and a dynamic exclusion window of 30 s. Ions were monitored over the range of 400–2000 m/z.The mass spectra collected in this study were searched using SEQUEST (Bioworks version 3.3, Thermo Inc., San Jose CA) using an in silico tryptic peptide database assembled from the JGI modeled gene set (Thaps 3.0) (Thaps3_bd_unmapped_GeneModels_FilteredModels1_aa.fasta.gz and Thaps3_chromosomes_geneModels_FilteredModels2_aa.fasta.gz) with the addition of NCBI T. pseudonana chloroplast (EF067921) and mitochondria (DQ186202) genomes and a reversed ‘decoy’ version of these databases for false discovery rate analysis. SEQUEST parameters were set at 30% ions required per peptide, DCN of 0.1, Xcorr vs CS 1.9, 2.4, 2.9, and 1e-3 protein probability, with a false positive rate of 0.96% . Changes in each protein's relative abundances across treatments were calculated using normalized spectral counts within Scaffold (Proteome Software V3.0; protein identification probabilities of 99.0% and requiring at least two tryptic peptide identifications per protein for 1264 protein identifications) (). Database search results were further processed using the PeptideProphet statistical model within Scaffold 3.0 (Proteome Software Inc., Portland OR). Relative protein abundance was determined using Scaffold 3.0 for normalized spectral counting software operating on a 64bit Ubuntu Linux workstation. Spectral counts are normalized across samples in each experiment in Scaffold, including technical replicates, to allow comparison of relative protein abundance. Comparison of technical triplicate injections on the LC-MS demonstrates robust relative quantitation of the abundant proteins in the T. pseudonana proteome, with repeat injections showing similar spectral count values for each protein (). Proteins discussed as ‘differentially expressed’ were determined by the Fisher exact test with p<0.05 (). […]

Pipeline specifications

Software tools Comet, PeptideProphet
Application MS-based untargeted proteomics
Organisms Thalassiosira pseudonana
Diseases Phosphorus Metabolism Disorders
Chemicals Carbon, Iron, Nitrogen, Phosphorus