Reproductive Adaptation of Astyanax mexicanus Under Nutrient Limitation
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive Plus
ORGANISM(S): Astyanax Mexicanus (ncbitaxon:7994)
SUBMITTER: 
Rohner, Nicolas
PROVIDER: MSV000096792 | MassIVE | Tue Jan 07 12:38:00 GMT 2025
REPOSITORIES: MassIVE
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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.
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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.
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