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TMT8plex_bRP_WI38_Ctr_Aging_Btz_Etp

ABSTRACT: WI-38 cells were washed three times with PBS, and cell lysis, trypsin/Lys-C digestion, and peptide purification were performed using the EasyPep Mini MS sample prep kit (Thermo Fisher Scientific). Peptides (25 μg) from each sample were labeled with 0.2 mg of TMT 10-plex reagents (Thermo Fisher Scientific) and combined and fractionated using the Pierce high pH reversed-phase peptide fractionation kit (Thermo Fisher Scientific). Peptides (1 µg) from each fraction were analyzed by MS using an EASY-nLC 1200 system connected online to an Orbitrap Fusion Lumos (Thermo Fisher Scientific) equipped with a FAIMS-Pro ion mobility interface (Thermo Fisher Scientific). Peptides were loaded onto a C18 analytical column (Ionopticks; Aurora Series Emitter Column, AUR2-25075C18A; 25 cm × 75 μm, 1.6 μm FSC C18 with nanoZero fitting) and separated using a 4-h gradient (solvent A, 0.1% FA; and solvent B, 80% ACN/0.1% FA). For data-dependent acquisition of MS/MS spectra, FAIMS-Pro was set to three phases (−40, −60, and −80 CV), and the most intense ions (cycle time: 1 s) with a charge state from +2 to +7 were selected for fragmentation by higher energy collisional dissociation. The fragment ions were detected using the Orbitrap system. Data were analyzed using SEQUEST in Proteome Discoverer (v.2.2). The mass tolerances for the precursor and fragment ions were 10 ppm and 0.02 Da, respectively, and peptide identification was filtered at a false discovery rate <0.01. TMT quantification was performed using the Reporter Ions Quantifier node in Proteome Discoverer.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Shigeo Murata

PROVIDER: PXD029271 | JPOST Repository | Fri Oct 21 00:00:00 BST 2022

REPOSITORIES: jPOST

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Project description:2-cell (E1.5) stage embryos were cultured in normal KSOM+AA conditions until E3.5 +2h and thereafter 100 embryos each were moved to control or p38-MAPKi conditions and cultured for another 7 hours (E3.5 +9h). The embryos were then washed through pre-warmed (37˚C) Hank’s balanced salt solution (HBSS, Sigma-Aldrich; Cat. No. H9269) and lysed by moving to a 1.5ml centrifuge tube containing about 15µl of SDT-lysis buffer (4% (w/v) SDS, 100 mM Tris-HCl pH 7.6, 0.1 M DTT). Cell lysis was performed by incubating the tubes in a 95˚C thermoblock for 12 minutes, brief centrifugation at 750 rpm, cooling to room temperature and storage at -80˚C. Individual protein solutions were processed by filter-aided sample preparation (FASP) method with some modifications (see the associated publication for more details). Resulting peptides were cleaned by liquid-liquid extraction (3 iterations) using water saturated ethyl acetate. 1/10th of the FASP eluate was taken out for direct LC-MS measurements, evaporated completely in SpeedVac concentrator (Thermo Fisher Scientific). Peptides were further transferred into LC-MS vials using 50μL of 2.5% formic acid (FA) in 50% acetonitrile (ACN) and 100μL of pure ACN and with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator. Phosphopeptides were enriched from the remaining 9/10th of the cleaned FASP eluate after complete solvent evaporation (SpeedVac concentrator) using High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific) according to manufacturer protocol. Phosphopeptide standards (0.1 pmol of MS PhosphoMix 1, 2, 3 Light; Sigma-Aldrich, St. Louis, Missouri, USA) was added to suspended sample in binding/equilibration buffer. Flow-through fraction was dried and used for second enrichment step using High-Select™ Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to manufacturer protocol. Resulting phosphopeptides were extracted into LC-MS vials by 2.5% FA in 50% ACN and 100% ACN with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator). LC-MS/MS analyses of all peptide mixtures (2 peptide solutions for each sample – 1) not enriched; 2) enriched on phosphopeptides using TiO2 enrichment kit) were done using RSLCnano system connected to Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Prior to LC separation, tryptic digests were online concentrated and desalted using trapping column (100 μm × 30 mm, column compartment temperature of 40˚C) filled with 3.5-μm X-Bridge BEH 130 C18 sorbent (Waters). After washing of trapping column with 0.1% FA, the peptides were eluted (flow rate - 300nl/min) from the trapping column onto an analytical column (Acclaim Pepmap100 C18, 3 µm particles, 75 μm × 500 mm; column compartment temperature of 40˚C, Thermo Fisher Scientific) by using 50 or 100 minutes long nonlinear gradient program (1-56% of mobile phase B; mobile phase A: 0.1% FA in water; mobile phase B: 0.1% FA in 80% ACN) for analysis of phosphopeptide enriched fractions or not enriched peptide mixtures, respectively. Equilibration of the trapping column and the analytical column was done prior to sample injection to sample loop. The analytical column outlet was directly connected to the Digital PicoView 550 (New Objective) ion source with sheath gas option and SilicaTip emitter (New Objective; FS360-20-15-N-20-C12) utilization. ABIRD (ESI Source Solutions) was installed. MS data were acquired in a data-dependent strategy with cycle time for 3 seconds and with survey scan (350-2000 m/z). The resolution of the survey scan was 60000 (200 m/z) with a target value of 4×105 ions and maximum injection time of 50ms. HCD MS/MS (30% relative fragmentation energy, normal mass range) spectra were acquired with a target value of 5.0x104. The MS/MS spectra were recorded in Orbitrap at resolving power of 30,000 or 15,000 (200 m/z) and the maximum injection time for MS/MS was 500 or 22 ms for analysis of phosphopeptides enriched fraction or non-enriched peptide mixture, respectively. Dynamic exclusion was enabled for 60 seconds after one MS/MS spectra acquisition. The isolation window for MS/MS fragmentation was set to 1.6 m/z.

Project description:Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Despite Zmiz1 critical role in angiogenesis, its role in lymphatic vasculature is unknown. Here, we use HDLECs cell line to profile transcriptional changes upon Zmiz1 knockdown using siRNA. Methods: Total RNA was extracted from control and Zmiz1 siRNA transfected HDLECs using GeneJET RNA Purification Kit (Thermo Fisher Scientific, K0732) following the manufacturer instructions. RNA concentration and RNA integrity (RIN) number were determined using Qubit RNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Q32852) and Bioanalyzer RNA 6000 Nano assay kit (Agilent, 5067-1511) respectively. RNA library was prepared using TruSeq RNA Library Prep Kit v2 (Illumina, RS-122-2001) according to the manufacturer instructions. mRNA library was quantified using Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Q32851) and verified using the Bioanalyzer DNA1000 assay kit (Agilent, 5067-1505). Verified samples were sequenced using the NextSeq1000/2000 P2 Reagents (200 Cycles) v3 (Illumina, 20046812) on a Nextseq1000/2000. Sequenced reads were aligned to the human (hg19) reference genome with RNA-Seq alignment tool (STAR aligner). The aligned reads were used to quantify mRNA expression and determine differentially expressed genes using the RNA-Seq Differential Expression tool (version 1.0.1). Both alignment and differential expression analysis were performed using the tools in the illumina BaseSpace Sequence Hub. Results: We found 2,228 differentially expressed genes of which 1,115 genes were upregulated while 1,113 genes were downregulated. Downregulated genes were enriched in biological processes such as lymph vessel development, cell migration and heart valve development. Conclusions: We identify Zmiz1 regulates Prox1 in lymphatic endothelial cells

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TMT8plex_bRP_WI38_Ctr_Aging_Btz_Etp

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