Dataset Information

LC/MS/MS Analyses of enriched ECM-enriched preparations from planaria

ABSTRACT: ECM isolation - Whole-mount planarian decellularization has been optimized based on a previous publication (Sonpho et al. 2020). We optimized three protocols for decellularization, which we refer to as No Pretreatment ECM (NP-ECM), Formaldehyde ECM (FA-ECM), and N-Acetyl Cysteine pretreatment (NAC-ECM). All worms were collected from static culture. All three protocols were designed for 20 planarians. Proteomics analysis - All decellularized samples were centrifuged at 16,900 g at 4C to recover the ECM pellets. Non-decellularized planarians (whole animal, WA) were prepared for comparison by freezing them in liquid nitrogen. ECM and WA pellets were resuspended in 120 uL of 100 mM Tris-HCl, pH 8.5, and 8 M Urea. The samples were vortexed vigorously to dissolve pellets. To reduce disulfide bonds, Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Pierce) was added to 5 mM and incubated at RT for 30 min. Reduced cysteines were alkylated by adding 2.4 uL of a freshly made 0.5 M chloroacetamide stock solution (Sigma) and incubating at RT in the dark for 30 min. Samples were deglycosylated with PNGase F by adding 5ul of a solution consisting of 18uL of Glycobuffer 2 (10x) and 27uL of PNGaseF (NEB, P0708S). Endoproteinase Lys-C (Promega), 5 uL at 1 ug/uL, was added and samples were incubated at 37C overnight while shaking. Solutions were diluted to 2 M urea by adding 360 uL of 100 mM Tris-HCl and 2 uL of CaCl2. For trypsin digestion, 10 uL of Trypsin (Promega) at 0.1 ug/uL was added and samples were incubated at 37C overnight while shaking. Reactions were quenched by adding 90% formic acid to a final concentration of 5%. Peptides were diluted with Buffer A (5% acetonitrile (ACN), 0.1% formic acid (FA)). Then, samples were injected on an in-house packed 50 um inner diameter microcapillary column packed with 10 cm of 1.9 um Aqua C18 resin (Dr. Maisch GmbH). The samples were injected in 100% Buffer A and subsequently eluted using an Ultimate 3000 UPLC (Dionex) at a flow rate of 0.120 uL/min for 240 min from 10-40% Buffer B (80% ACN, 0.1% FA) before ramping to 95% B in 25 min. The high organic concentration was maintained for 15 min before re-equilibration and the reinjection with the next sample. Peptides were ionized by the application of a 2.5 kV positive voltage applied distally to the peptides eluting into a Q-Exactive Plus (QE+) mass spectrometer (Thermo Scientific, Waltham, MA). Full MS spectra were recorded on the eluting peptides over a 350 to 1700 m/z range at 70,000 resolving power. The AGC (automated gain control) target was 1x106 with a max injection time set to 50 ms. The top 15 peptides with charges 2-5 were fragmented by High energy Collision Dissociation (HCD) at 27% normalized collision energy (NCE) and the MS/MS fragmentation was collected at 17,500 resolving power, with an AGC target of 1x105 and a maximum injection time set to 150 ms. Dynamic exclusion was enabled for 30 seconds. Mass spectrometer scan functions and HPLC solvent gradients were controlled by the XCalibur data system (Thermo Scientific, Waltham, MA). MS/MS spectra were first searched using ProLuCID v. 1.3.3 with a peptide mass tolerance of 10 ppm and 25 ppm for peptide and fragment ions, respectively. Trypsin specificity was imposed on both ends of candidate peptides during the search against a protein database containing 30,863 S. mediterranea predicted protein sequences as well as 483 usual contaminants such as human keratins, IgGs and proteolytic enzymes. To estimate false discovery rates (FDR), each protein sequence was randomized (keeping the same amino acid composition and length) and the resulting "shuffled" sequences were added to the database, for a total search space of 62,564 amino acid sequences. A mass shift of 57.0215 Da was statically added to cysteine residues to account for alkylation by CAM, mass shifts of +15.9949 was differentially added to methionine, lysine, and proline residues to account for oxidation and hydroxylation, and -0.98401 Da was differentially added to asparagine residues to account for deglycosylation by PNGaseF. DTASelect v.1.9 was used in combination with an in-house software, swallow v1.0 to select and sort peptide/spectrum matches (PSMs) to FDRs of less than 1% at the peptide and protein levels.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Schmidtea Mediterranea (ncbitaxon:79327)

SUBMITTER: Laurence Florens

PROVIDER: MSV000087054 | MassIVE | Mon Mar 15 16:09:00 GMT 2021

SECONDARY ACCESSION(S): PXD024753

REPOSITORIES: MassIVE

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Publications

Decellularization Enables Characterization and Functional Analysis of Extracellular Matrix in Planarian Regeneration.

Sonpho Ekasit E Mann Frederick G FG Levy Michaella M Ross Eric J EJ Guerrero-Hernández Carlos C Florens Laurence L Saraf Anita A Doddihal Viraj V Ounjai Puey P Sánchez Alvarado Alejandro A

Molecular & cellular proteomics : MCP 20210818

The extracellular matrix (ECM) is a three-dimensional network of macromolecules that provides a microenvironment capable of supporting and regulating cell functions. However, only a few research organisms are available for the systematic dissection of the composition and functions of the ECM, particularly during regeneration. We utilized the free-living flatworm Schmidtea mediterranea to develop an integrative approach consisting of decellularization, proteomics, and RNAi to characterize and inv ...[more]

PMID: 34416386

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Project description:Sample preparation: P-AMPK+AMP, P-AMPK+ATPγS, and AMPK+ATPγS protein samples were crosslinked in HBST buffer (25 mM HEPES pH 7.5 at room temperature, 200 mM NaCL, and 1 mM TCEP). Non-crosslinked negative controls were generated using the same procedure without the addition of DSSO. Reactions were initiated by spiking 1 uL of 75 mM DSSO (Thermo-Fisher) in DMSO into a 50 uL solution containing 10 uM protein and mixing. The final molar ratio of crosslinker:protein was 150:1. The reactions were incubated at 25°C for 45 minutes prior to being quenched by the addition of Tris pH 8.0 to a final concentration of 50 mM. Each crosslink reaction was done in triplicate and the reaction replicates were pooled after quenching. 10 μL samples of the pooled samples were loaded for SDS-PAGE analysis with Coomassie staining to confirm the presence of crosslinked protein. The remaining 140 μL of crosslinked and non-crosslinked samples were then acetone precipitated at -20°C overnight and the protein was pelleted by centrifugation at 16,000 rcf for 5 minutes at 4°C. After decanting, the pellets were dried for 15 minutes at room temperature before being resuspended in 12.5 uL of resuspension buffer (50 mM ammonium bicarbonate, 8M urea, pH 8.0). ProteaseMAX (Promega) was added to 0.02% and the solutions were mixed on an orbital shaker operating at 400 RPM for 5 minutes. After resuspension, 87.5 uL of digestion buffer (50 mM ammonium bicarbonate, pH 8.0) was added. Cysteines in the protein samples were reduced and akylated as follows. DTT was added to 5 mM final concentration and the protein solutions were incubated at 56°C in an orbital shaker operating at 400 RPM and for 20 minutes. After reduction, 2.7 uL of 550 mM iodoacetamide was added and the solutions were incubated in the dark at room temperature for 15 minutes. Reduced and alkylated protein solutions were digested using trypsin at a ratio of 1:200 (w/w trypsin:protein) and incubating at 37°C. After overnight incubation at 37°C, the samples were acidified by the addition of trifluoroacetic acid (TFA, Thermo-Fischer) to 1%. Samples were then frozen and stored at -20°C until analysis.

Project description:Approximately 1 x 10^8 GFP-HA-KERP2 expressing Entamoeba histolytica cells were cross-linked in 0.5% formaldehyde (in PBS) for 10 minutes at room temperature. Cross-linking was quenched with 125 mM glycine for 5 minutes. Cells were washed once with PBS and subjected to nuclear fractionation using nuclei isolation buffer (10 mM Hepes-KOH pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 2% NP-40, 1 uM E-64, 0.5 mM PMSF, and 1x cOmplete protease inhibitor cocktail). Nuclear pellets were resuspended in sonication buffer (50 mM Tris-HCl pH 8.0, 140 mM NaCl2, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 5 mM DTT, 1 uM E-64, 0.5 mM PMSF, 1x cOmplete) and sonicated using a Covaris instrument. Sonicated samples were centrifuged at 16000 x g for 20 minutes. A 200 uL aliquot of supernatant was transferred to a 1.5 mL Protein LoBind tube. Remaining sample was stored as input control. For immunoprecipitation, 1 mL of ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 0.5% BSA, 1x cOmplete) containing 50 uL of GFP(ab290)-antibody conjugated Dynabeads was added and rotated at 4 C for 12 hours. Beads were washed five times with low-salt wash buffer (20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), followed by two washes with high-salt wash buffer (same as above, but 500 mM NaCl). DNA-protein complexes were eluted with 200 uL of ChIP elution buffer (10 mM Tris-HCl pH 8.0, 1% SDS, 5 mM EDTA, 300 mM NaCl) at room temperature for 20 minutes. Input samples were thawed and diluted with 190 uL of elution buffer. All samples were incubated at 65 C for 8 hours to reverse cross-links, followed by RNase A treatment (1 uL) at 37 C for 1 hour and Proteinase K digestion (5 uL) at 55 C for 2 hours. DNA was purified by phenol/chloroform extraction with 1 uL glycogen as carrier, followed by ethanol precipitation overnight at -30 C. DNA pellets were washed with 80% ethanol, air-dried, and resuspended in 20 uL TE (ChIP) or 40 uL TE (input). DNA concentrations were quantified using a Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific). Purified DNA was submitted to Rhelixa for ChIP-seq.

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