Project description:While the myelin proteome is well characterized in mice, much less is known about the protein composition of myelin in non-mammalian species such as zebrafish (Danio rerio), despite its increasing popularity as model organism for myelin biology. Here, we assess the proteome of myelin biochemically purified from the brains of adult zebrafish by complementary proteomic approaches for deep qualitative and quantitative coverage. A data-independent acquisition (DIA) workflow with alternating low and elevated energy (MSE) provided the high dynamic range required for correct quantification of highest-abundance proteins. According to this analysis, the predominant Ig-CAM myelin protein zero (MPZ/P0), myelin basic protein (MBP) and the short chain dehydrogenase 36K constitute 12%, 8% and 6% of the total myelin protein, respectively. Combined with data from an ion mobility-enhanced version of the MSE mode (referred to as UDMSE) and from 1D-gel-based proteomic approaches, we provide the most comprehensive proteome resource of zebrafish myelin so far, correlate it with previously established mRNA-abundance profiles, and demonstrate both similarities and heterogeneity of myelin composition between teleost fish and rodents.
Project description:Zebrafish organ proteomics was carried out for proteogenomic analysis. High resolution mass spectrometry-based proteomic profiling of 11 adult organs (eye, brain, liver, spleen, intestine-pancreas, ovary, testes, muscle, heart and head) and two developmental stages (embryos 48 and 120 hours post-fertilization) of zebrafish (SAT line) was carried out. Protein extracts were reduced and alkylated (iodoacetamide). Off-loine fractionation was carried out by SDS-PAGE and basic RPLC. MS analysis was cariied on Agilent 6540 Q-TOF. Spleen and Testis samples were also analyzed on Thermo Orbitrap LTQ Velos.
Project description:To identify the target of the monoclonal antibody, Immuoprecipitation (IP) was performed to pull-down target protein from cell culture extraction, corn or zebrafish tissue lysate. The IP product was validated on silver-stained SDS-PAGE and Western blot. The target band was cut out and performed LC-MS/MS for target protein identification.
Project description:Identification and semi-quantitation of phosphorylation sites on Schizosaccharomyces pombe DNA-binding protein Rap1. A Rap1 construct was cultured in homozygous diploid temperature-sensitive pat1-114 mutant strain of S. pombe cells (time points: 3.5 h and 4.5h), and was purified by anti-PK immunoprecipitation. The resulting samples were run into a 1D SDS-PAGE gel with the target band excised, tryptically digested and analysed by LC-MS/MS using a Velos Orbitrap mass spectrometer.
Project description:Three types of BMs from adult human eyes, the inner limiting membrane, the retinal vascular BMs and the lens capsule, were isolated for analysis by 1D-SDS-PAGE and LC-MS/MS.