| Metadata |
| datasetIdentifier | PASS01237 |
| datasetType | Other |
| submitter | Peter Blattmann <blattmann@imsb.biol.ethz.ch> |
| submitter_organization | ETH Zurich |
| lab_head_full_name | Prof. Dr. Ruedi Aebersold |
| lab_head_email | aebersold@imsb.biol.ethz.ch |
| lab_head_organization | ETH Zurich |
| lab_head_country | Switzerland |
| datasetTag | ZebrafishSWATHLib |
| datasetTitle | Zebrafish SWATH-MS spectral library |
| publicReleaseDate | 2018-12-19 00:00:00 |
| finalizedDate | 2018-12-18 06:14:06 |
| summary | Zebrafish spectral library covering 9 different zebrafish tissues (brain, eye, heart, intestine, liver, muscle, ovaries, spleen, and testes). |
| contributors | Peter Blattmann, Vivienne Stutz, Giulia Lizzo, Joy Richard, Philipp Gut, Ruedi Aebersold |
| publication | in preparation |
| growth | Adult AB zebrafish were raised at 28°C under standard husbandry conditions. |
| treatment | Brain, eyes, testes, and ovaries from male or female zebrafish, and the muscle from male zebrafish were cut into sections or grinded while cooling with liquid nitrogen. From the grinded tissues 3 mg were transferred into pressure cycling technology (PCT) Microtubes. For spleen, liver, heart and intestine of male or female zebrafish, a piece of 0.9 – 3.8 mg of tissue was transferred into PCT Microtubes for subsequent processing. |
| extraction | For all samples, lysis and digestion were performed based on a protocol described in Guo et al. (Guo et al., 2015). Lysis buffer, pH 8.0 (6 M urea, 2 M thiourea, 100 mM ammonium bicarbonate, 5 mM EDTA, cOmplete protease inhibitor (1:50)) was added to the tissue sections, and lysis and digestion of the samples was performed with the Barocycler NEP2320EXT (Pressure BioSciences) at 31°C. Lysis was conducted using the PCT-MicroPestle with 60 cycles consisting of 50 s at 45 kpsi followed by 10 s at atmospheric pressure. For reduction and alkylation, the buffer was diluted to 3.75 M urea with 100 mM ammonium bicarbonate, before peptides were simultaneously reduced with 9 mM tris(2-carboxyethyl)phosphine (TCEP) and alkylated with 35 mM iodoacetamide for 30 min in the dark at 25°C. |
| separation | Samples for high-pH RP-HPLC fractionation were resuspended in Buffer A (20 mM ammoniumformate and 0.1 % ammonia solution in water, pH 10) and 80 µg of peptides were injected into an Agilent Infinity 1260 (HP Degasser, Vial Sampler, Cap Pump) and 1290 (Thermostat, FC-µS) system. The peptides were separated at 30°C on an YMC-Triart C18 Reversed Phase Column with diameter of 0.5 mm, length of 250 mm, particle size of 3 µm, and pore size of 12 nm. At a flow of 11 µL/min the peptides were separated by a linear 56 min gradient from 5 % to 35 % Buffer B (20 mM ammoniumformate, 0.1 % ammonia solution, 90 % acetonitrile in water, pH10) against Buffer A followed by a linear 4 min gradient from 35 % to 90 % Buffer B against Buffer A and 6 min at 90 % Buffer B. The resulting 36 fractions per organ were pooled based on the collection order from fraction 3 to fraction 33 or 34 (depending on the UV profile) into 8 samples by the following scheme: fraction x was pooled with fractions x+8, x+16, and x+24. The buffer of the pooled samples was evaporated using vacuum centrifugation at 45°C. As described above, the peptides were dissolved in 2% (v/v) acetonitrile and 0.1% (v/v) FA in water and iRT peptides were added (1:20). |
| digestion | The first digestion step was performed using LysC (estimated enzyme/protein ratio of 1:100), and was carried out in the barocycler using 45 cycles of 50 s at 20 kpsi followed by 10 s at atmospheric pressure. For the second digestion, samples were diluted to 2 M Urea with 100 mM ammonium bicarbonate and digested using Trypsin (estimated enzyme/protein ratio of 1:75) for 90 cycles consisting of 50 s at 20 kpsi followed by 10 s at atmospheric pressure. The digestion was quenched by acidifying samples to pH 1.5 with trifluoroacetic acid (TFA). The peptides were desalted using C18-columns (The Nest Group Inc.) and 2% (v/v) acetonitrile and 0.1% (v/v) TFA in water and eluted with 50% (v/v) acetonitrile and 0.1% (v/v) TFA in water. The buffer was evaporated using vacuum centrifugation at 45°C. Dried peptides were either dissolved in 2% (v/v) acetonitrile and 0.1% (v/v) formic acid (FA) in water and iRT peptides (Biognosys) added (1:20), or they were prepared for high-pH RP-HPLC fractionation. |
| acquisition | The peptides were quantified on an ABSciex TripleTOF 5600 instrument after separating 0.9 – 3 µg by nano-flow liquid chromatography (NanoLC Ultra 2D, Eksigent). The peptides were separated by reverse-phase chromatography on a fused silica PicoTip™ Emitter (inner diameter 75 mm) (New Objective, Woburn, USA) manually packed column with C18 beads (MAGIC, 3 µm, 200 Å, BISCHOFF, Leonberg, Germany) to a length of 20 cm for the whole lysate samples, and 30 cm for the pooled fractions. A flow of 300 nl/min and a linear 120 min gradient from 2% to 35% Buffer B (98% acetonitrile and 0.1% formic acid in H2O) in Buffer A (2% acetonitrile and 0.1% formic acid in H2O) was used to separate the peptides. Precursor selection on the MS1 level was performed with a Top20 method using an accumulation time of 250 ms and a dynamic exclusion time of 20 s. The MS1 spectra were obtained in an m/z range from 360 to 1460. Fragmentation of the precursor peptides was achieved by Collision induced dissociation (CID) with rolling collision energy for peptides with charge 2+ adding a spread of 15eV. For MS2 spectra, only fragments with a charge state from 2 to 5 were selected using an accumulation time of 150 ms. |
| informatics | The SWATH spectral library was built using the previously published workflow (Schubert et al., 2015) with some modifications concerning the search engines, number of missed cleavages, mass errors, and selection of proteins and peptides by the iProphet cutoffs. First, raw files were converted into centroided mzXML files with ProteoWizard version 3.0.8851. The spectra were then searched using an in-house pipeline using the search engines X!Tandem with k-score plugin (2013.06.15.1) and Comet (2016.01 rev.3) and a protein sequence database. The protein sequence database was obtained from the Ensembl Release 91 (dec2017.archive.ensembl.org; Danio_rerio.GRCz10.pep.all.fa) and further processed using an R script to select only the longest protein-coding transcript for each protein-coding gene. The search was conducted on an in-house platform (Kunszt et al., 2015) using as search parameters a parent mass error of ± 25 ppm, a fragment mass error of ± 0.05 Da, trypsin digestion allowing for 2 missed cleavages, carbamidomethyl (C) as a fixed modification, and oxidation (M) as a variable modification. After combining the searches, only proteins passing an iProphet probability corresponding to a Mayu (Reiter et al., 2009) protein-FDR of 0.010 were selected. For these proteins all peptides passing an iProphet peptide-FDR < 0.0100 were selected using SpectraST (v.5.0) followed by generating a consensus spectral library with a retention time normalization using iRT peptides. This spectral library was then used to generate the SWATH spectral library (Schubert et al., 2015). |
| instruments | AB SCIEX TripleTOF 5600 |
| species | Danio rerio |
| massModifications | static: C+57.0215, variable: M+15.994915 |
