DOI: 10.5281/zenodo.17735079
title: Proteomic analysis of zebrafish spinal cord regeneration (2 dpl)
title: unlesioned vs.2 day post-injury

description: ***Part 2/2***

 

Description

Label-free mass spectrometry-based quantitative proteomics was applied to a larval zebrafish spinal cord injury model, which allows axon regeneration and functional recovery within two days (days post lesion; dpl) after a spinal cord transection in 3 day-old larvae (dpf). Proteomic profiling of the lesion site was performed at 2 dpl and corresponding age-matched unlesioned control tissue (5 dpf).

Sample Processing Protocol





For each sample, proteins were isolated from trunk tissue of 75 pooled larvae spanning approximately three somites in length and containing the lesion site (2 dpl) or corresponding unlesioned age-matched control tissue (5 dpf). Mass spectrometry analysis was performed on three biological replicates for each condition. Isolation of proteins was done as described in Schiller et al. Mol Syst Biol. 2015 with some modification. Proteins were extracted in three distinct fractions, and each fraction was analyzed separately by LC-MS/MS. Zebrafish tissue was homogenized in PBS for 10x 30 s at high intensity and 10x 30 s pauses in between, using the Bioruptor Plus sonication system (Diogenode). After centrifugation at 16,000 g for 2 min, the supernatant containing the soluble proteins was collected (fraction A). The pellet was resuspended in detergent-containing buffer (50 mM Tris, 5% Glycerin, 500 mM NaCl, 1% NP40, 2% SDC, 1% SDS, 1% DNAse I, 1 mM MgCl2, followed by incubation at 0°C for 20 min and homogenization using the Bioruptor Plus sonication system. After centrifugation at 16,000 g for 2 min, the supernatant containing the detergent-soluble proteins (fraction B) and the detergent-insoluble protein pellet (fraction C) were collected. Proteins of fraction 1 and fraction 2 were precipitated by incubation with acetone at -20°C for overnight. The pellets of all three fractions were dissolved in reduction and alkylation buffer (6M guandinium hydrochloride, 100 mM Tris-HCl pH 8.5, 10 mM TCEP, 50 mM CAA), followed by incubation at 99°C for 15 min and sonification in the Bioruptor Plus sonication system. Thereafter, the proteins were diluted 1:3 with Urea-buffer (4.5 mM Urea, 10 mM Tris-HCl, 3% Acetonitrile, 1 µg of LysC and incubated at 37°C for 3 h. Thereafter, the digestion mixture was diluted 1:3 with 10% Acetonitrile in MS grade water, followed by sonification in the Bioruptor Plus sonication system and incubation with 1 µg of LysC and 2 µg of Trypsin at 37°C for overnight. Acetonitrile was removed using a SpeedVac (Christ) and peptides were further purified using in-house produced 3 plug SCX stage tips (EmporeTM Cation Solid Phase Extraktion Disks). After elution with 60 µL of 5% ammonia solution in 80% acetonitrile samples were vacuum dried in the SpeedVac. Peptides were solubilized in 6 µL Buffer A (100 % MS-LC grade water and 0.1% Formic acid) and a total volume of 3 µL were loaded onto a 30 cm-long column (75 µm inner diameter (Polymicro); packed in-house with ReproSil-Pur 120 C18-AQ 1.9-micron beads, Dr. Maisch GmbH) via the autosampler of the Thermo Scientific Easy-nLC 1200 (Thermo Fisher Scientific) at 60°C. Using the nanoelectrospray interface, eluting peptides were directly sprayed onto the Q Exactive HF Orbitrap LC-MS/MS system (Thermo Fisher Scientific). As gradient, the following steps were programmed with increasing addition of buffer B (80% Acetonitrile, 0.1% Formic acid): linear increase from 30% over 120 min, followed by a linear increase to 60% over 10 min, followed by a linear increase to 95% over the next 5 min, and finally buffer B was maintained at 95% for another 5 min. The mass spectrometer was operated in a data-dependent mode with survey scans from 300 to 1750 m/z (resolution of 60000 at m/z = 200), and up to 15 of the top precursors were selected and fragmented using higher energy collisional dissociation (HCD with a normalized collision energy of value of 28). The MS2 spectra were recorded at a resolution of 15k (at m/z = 200). AGC target for MS1 and MS2 scans were set to 3E6 and 1E5 respectively within a maximum injection time of 100 ms for MS1 and 25 ms for MS2. Dynamic exclusion was set to 16 ms.

Data Processing Protocol

Raw data were processed using the MaxQuant computational platform (version 1.6.7.0) (Cox and Mann. Nat Biotechnol. 2008) with standard settings applied. Briefly, the peak list was searched against the zebrafish (Danio rerio) proteome database (SwissProt and TrEMBL, 46847 entries) with an allowed precursor mass deviation of 4.5 ppm and an allowed fragment mass deviation of 20 ppm. MaxQuant enables individual peptide mass tolerances by default, which was used in the search. Cysteine carbamidomethylation was set as static modification, and methionine oxidation and N-terminal acetylation as variable modifications. Proteins were quantified across samples using the label-free quantification algorithm in MaxQuant generating label-free quantification (LFQ) intensities. The match-between-runs option was enabled.

Sample description

unlesioned: samples 7, 8, 9

2 dpl: samples 10, 11, 12