Post-transcriptional and translational regulation of mRNA-like long non-coding RNAs by microRNAs in early developmental stages of zebrafish embryos.
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ABSTRACT:
SUBMITTER: Lee KT
PROVIDER: S-EPMC5437968 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Zebrafish is a well-recognized organism for investigating vertebrate development and human diseases. However, the data on zebrafish proteome are scarce, particularly during embryogenesis. This is mostly due to the overwhelming abundance of egg yolk proteins, which tend to mask the detectable presence of less abundant proteins. We developed an efficient procedure to reduce the amount of yolk in zebrafish early embryos to improve the Liquid chromatography-tandem mass spectrometry (LC-MS)-based shotgun proteomics analysis. We demonstrated that the deyolking procedure resulted in a greater number of proteins being identified. This protocol resulted in approximately 2-fold increase in the number of proteins identified in deyolked samples at cleavage stages, and the number of identified proteins increased greatly by 3-4 times compared to non-deyolked samples in both oblong and bud stages. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed a high number of functional proteins differentially accumulated in the deyolked versus non-deyolked samples. The most prominent enrichments after the deyolking procedure included processes, functions, and components related to cellular organization, cell cycle, control of replication and translation, and mitochondrial functions. This deyolking procedure improves both qualitative and quantitative proteome analyses and provides an innovative tool in molecular embryogenesis of polylecithal animals, such as fish, amphibians, reptiles, or birds.
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Project description:Sample Preparation | Four biological replicate of 25 dechorionated, but non-deyolked, zebrafish embryos were collected at 4 time points of embryonic development: 1-cell stage (0-hpf, hour post fertilization), 2-hph, 4-hpf, and 6-hpf. All 16 pooled embryo samples were lysed independently by adding 100 ul of Mammalian lysis buffer and 1x Protease Inhibitor cocktail.
Digestion | Samples were reduced and alkylated with TCEP and 2-chloroacetamide. Six volumes of pre-chilled acetone were added, and the precipitation was let to proceed overnight. The precipitated proteins were dissolved in 100 ul of 50 mM TEAB, mass spectrometry grade trypsin (Promega Gold) was added at 1:40 w/w and the digestion was let to proceed at 37oC overnight. Pierce fluorometric peptide assay was performed on 10 ul of each digested sample.
Tandem Mass Tag (TMT) Labeling | 20 ul of anhydrous acetonitrile were added to each of the 0.5mg TMTpro 16plex reagents. For each sample, 20 ug of peptides were measured out and adjusted to 100 ul with 100 mM TEAB, then mixed with the TMT reagents. The replicate samples for each of the time points were labeled for 1 hr with TMTpro-126, 127N, 127C, -128N (0-hpf, replicates 1-4); 128C, 129N, 129C, 130C (2-hpf, 1-4); 130C, 131N, 131C, 132N (4-hpf, 1-4), 132C, 133N, 133C, 134N (6-hpf, 1-4), respectively. After checking labeling efficiency (>99%), 30 ul each of the 16 differentially labeled samples were combined in a new tube and the resulting volume was reduced using a SpeedVac concentrator to less than 10 ul.
High pH Reverse Phase Fractionation | 300 ul of the TMT-labeled peptide mixture (in 0.1% TFA) were loaded onto a Pierce high pH fractionation cartridge placed on a 2.0 ml sample tube. After centrifuging at 3000 x g for 2 minutes, the eluate was collected as the flow-through fraction. The loaded cartridge was placed on a new sample tube, washed with 300 ul of ddH20, and the eluate collected as wash fraction. An additional round of washing was performed using 300 ul of 5% acetonitrile in 0.1% TFA to remove unreacted TMT reagent. A total of 9 HpH RP fractions (E1-E9) were collected by sequential elution in new sample tubes using 300 ul of 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 50%, and 80% acetonitrile in 0.1% TFA. Dried samples were resolubilized in 44 ul of buffer A (5% acetonitrile in 0.1% formic acid, FA) before LC-MS analysis.
Multiplexed Mass Spectrometry Analysis | TMT-labeled peptides were analyzed on an Orbitrap Eclipse Tribrid Mass Spectrometer with a FAIMS Pro interface, equipped with a Nanospray Flex Ion Source, and coupled to a Dionex UltiMate 3000 RSCLnano System. Peptides were loaded on an Acclaim PepMap 100 C18 trap cartridge (0.3 mm i.d., 5 mm length) with the U3000 loading pump via the autosampler. A 75 um i.d. analytical microcapillary column was packed in-house with 250 mm of 1.9 um ReproSil-Pur C18-AQ resin (Dr. Masch). The Orbitrap Eclipse was set up to run the TMT-SPS-MS3 method with three FAIMS compensation voltages (CV) at -40V, -55V, and -70V and a cycle time of 1 sec. Briefly, peptides were scanned from 400-1600 m/z in the Orbitrap at 120,000 resolving power before MS2 fragmentation by CID at 35% NCE and detection in the ion trap set to turbo detection. Dynamic exclusion was enabled for 45s. Real time search (RTS) was performed during the ddMS2 IT CID step against a non-redundant Danio rerio sequence database downloaded from UniProtKB 2021-03 and complemented with common contaminants. Carbamidomethyl and TMTpro16plex (304.2071 Da on Kn) were searched statically, while methionine oxidation was searched as a variable modification. Synchronous precursor scanning (SPS) selected the top 10 MS2 peptides for TMT reporter ion detection in the Orbitrap using HCD fragmentation at 65% NCE at 50,000 resolving power. In addition, RTS was also set up to reject further fragmentation of peptides mapping to contaminants and yolk proteins using an exclusion list with the keywords Contaminant, Phosvitin, and Vitellogenin.
MS/MS Data Processing | The LC/MSn dataset was searched using Proteome Discoverer 2.4 against the same protein database used for RTS. SEQUEST-HT implemented through Proteome Discoverer was set up as: precursor ion mass tolerance 10 ppm, fragment mass tolerance 0.6 Dalton, up to two missed cleavage sites, static modification of cysteine, and lysine and peptide N-termini with TMT tag (+229.163 Da) and dynamic oxidation of methionine. Results were filtered to a 1% FDR at peptides levels using Percolator through Proteome Discoverer. MS3 spectra were processed to extract intensity for each reporter ion. Proteins were quantified by summing reporter ion intensities across all matching PSMs.
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