DOI: 10.25345/c59w09b1z
title: MassIVE MSV000096660 - Reproductive Adaptation of Astyanax mexicanus Under Nutrient Limitation
title: Proteins were extracted from forty 2-cell-embryos each from Surface, Pachon, and Molino cavefish in six biological replicates using a Lipid Extraction Kit (ab211044) and 1.5ml BioMasher II Micro Tissue Homogenizers (749625-0010) according to manufacture protocols. Briefly, embryos were homogenized in 500ul Extraction Buffer on ice for one minute, then incubated at room temperature for 20 minutes at 1000g. After centrifugation at 10,000g for 5 minutes at 4C, pellets as non-polar coproduct was saved for proteomic analysis. Three biological replicate pellets were resuspended in 100ul of 8M Urea with 100mM of Tris, pH8.5 and reduced with 5mM tris(2-carboxyethyl) phosphine (TCEP) and alkylated with 10mM 2-chloroacetamide (CAM) for 30 minutes protected from light at room temperature. Endoproteinase Lys-C (Promega) was added at 1:1000 w/w at 37C overnight. The reactions were diluted to 2M Urea by adding 100mM of Tris, pH8.5, then Trypsin (Promega Gold) was added at 1:200 w/w at 37C overnight. The digested samples were centrifuged at 16,000g for 30 minutes and transferred supernatant to new tubes. All samples were cleaned up by Pierce Peptide Desalting Spin Columns (89851) before a colorimetric peptide assay (Pierce). To each of the TMT10plex (Thermo Fisher, 90110) 0.8mg vials, 20 ul of anhydrous acetonitrile were added, then mixed with 30ug peptides and incubated 1 hr at RT. The differentially labeled samples (40ul each) were combined, and the resulting volume was reduced using a SpeedVac (Savant) to less than 10ul. The dried TMT-labeled peptide mixture was resuspended in 300ul of 0.1% trifluoroacetic acid (TFA). One high pH fractionation cartridge (Pierce, cat. 84868) was placed on a new 2.0ml sample tube and 300ul of the TMT-labeled peptide mixture were loaded onto the column. A total of 8 HpH RP fractions were collected by sequential elution in new sample tubes using 300ul of 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25% and 50% acetonitrile in 0.1% TFA. The solvents were evaporated to dryness using vacuum centrifugation. Dried samples were resolubilized in 44ul of buffer A (5% acetonitrile in 0.1% formic acid, FA) before LC-MS analysis. TMT-labeled peptides were analyzed on Orbitrap Eclipse Tribrid Mass Spectrometer (Thermo Scientific), equipped with a Nanospray Flex Ion Source, and coupled to a Vanquish Neo System. Peptides (22ul for each HpH RP fraction) were loaded on an Acclaim PepMap 100 C18 trap cartridge (0.3mm inner diameter (i.d.), 5mm length; Thermo Fisher Scientific) with the Neo loading pump at 2ul/minute via the autosampler. A 75um i.d. analytical microcapillary column was packed in-house with 25mm of 1.9um ReproSil-Pur C18-AQ resin (Dr. Masch). AgileSLEEVE (Analytical Sales & Products) was used to maintain column temperature at 40C. The organic solvent solutions were water:acetonitrile:formic acid at 95:5:0.1 (volume ratio) for buffer A (pH 2.6) and 20:80:0.1 (volume ratio) for buffer B. The chromatography gradient was a 5 minutes column equilibration step in 1% B; a 74 minutes ramp to reach 30% B; 20 minutes from 30% to 60% B; 3 minutes to reach 90% B; a 10 minutes wash at 90% B; 0.1 minutes to 1% B; followed by a 12 minutes column re-equilibration step in 1% B. The Neo pump flow rate was set to 0.180ul/minute. The Orbitrap Eclipse was set up to run the TMTpro-SPS-MS3 method. 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. Carbamidomethyl (57.0215 Da on Cys) and TMT10plex (229.163 Da on Kn) were searched statically, while methionine oxidation (15.9949 Da) 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. The LC/MSn dataset was processed using Proteome Discoverer 3.1 (Thermo Fisher Scientific). MS/MS spectra were searched against a mexicanus protein database (NCBI 2022-07) complemented with common contaminants. 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 (+57.021 Da), and lysine and peptide N-termini with TMT tag (+229.163 Da) and dynamic oxidation of methionine (+15.995 Da). 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. Differentially enriched proteins were identified using ANOVA.