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.]]>