Proteomics Analysis of Early Developmental Stages of Zebrafish Embryos

Abstract

Zebrafish is a well-recognized organism for investigating vertebrate development and human diseases. However, the data on zebrafish proteome are scarce, particularly during embryogenesis. This is mostly due to the overwhelming abundance of egg yolk proteins, which tend to mask the detectable presence of less abundant proteins. We developed an efficient procedure to reduce the amount of yolk in zebrafish early embryos to improve the Liquid chromatography–tandem mass spectrometry (LC–MS)-based shotgun proteomics analysis. We demonstrated that the deyolking procedure resulted in a greater number of proteins being identified. This protocol resulted in approximately 2-fold increase in the number of proteins identified in deyolked samples at cleavage stages, and the number of identified proteins increased greatly by 3–4 times compared to non-deyolked samples in both oblong and bud stages. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed a high number of functional proteins differentially accumulated in the deyolked versus non-deyolked samples. The most prominent enrichments after the deyolking procedure included processes, functions, and components related to cellular organization, cell cycle, control of replication and translation, and mitochondrial functions. This deyolking procedure improves both qualitative and quantitative proteome analyses and provides an innovative tool in molecular embryogenesis of polylecithal animals, such as fish, amphibians, reptiles, or birds.

Organ-Based Proteome and Post-Translational Modification Profiling of a Widely Cultivated Tropical Water Fish, Labeo rohita

“…In 96 h post fertilization embryo, we were able to detect fatty acid binding proteins (FABPs) that have been reported to play an important role in yolk-sac absorption. 40 Proteins in zebrafish such as adenylate kinase isoenzyme 1, ATP synthase, calsequestrin, creatine kinase Btype, creatine kinase M-type, beta crystallin A2, beta-crystallin B1, periostin isoform X1, actin cytoplasmic 1, tubulin alpha, and tubulin beta that had been reported in early embryonic stages in zebrafish 41,42 were also detected in this study. A panel of proteins was mapped exclusively in gonads of either sex (Figure 3c and Figure S6).…”

“…Organ-wise distribution of methylated peptides for lysine and arginine and n-terminus can be seen in Figure 4b. However, the number of phosphorylated peptides and canonical phosphoproteins was found highest in the brain (227, 139) followed by the spinal cord (132, 69), heart (115, 81), male gonad (103, 52), kidney (102, 77), eye (91,76), muscle (89,40), liver (87,63), skin (54,42), female gonad (54,33), air bladder (53,27), gill (38,35), gut (34,28), gall bladder (16,18), spleen (15,13), fin (13,11), embryo (11,10), plasma (5,2), and scales (4,3). Overall, serine phosphorylation was found to be predominant across the organs (Figure 4c).…”

The Proteome Map and PeptideAtlas of a widely cultivated tropical water fish Labeo rohita: A resource for the Aquaculture Community

“…Few more proteins detected in rohu embryo which were previously reported in early embryonic stages in zebrafish such as adenylate kinase isoenzyme 1, ATP synthase, calsequestrin, creatine kinase B-type, creatine kinase M-type, beta crystallin A2, beta-crystallin B1, periostin isoform X1, actin cytoplasmic 1, Tubulin alpha and Tubulin beta 32,33 .…”

“…However, number of phosphorylated peptides and canonical phosphoproteins were found highest in brain (227, 139) followed by spinal cord (132, 69), heart (115, 81), male gonad (103, 52), kidney (102,77), eye (91, 76), muscle (89,40), liver (87, 63), skin (54,42), female gonad (54,33), air bladder (53,27), gill (38,35), gut (34,28), gall bladder (16,18), spleen (15, 13), fin (13, 11), embryo (11,10), plasma (5, 2) and scales (4, 3). Overall, Serine phosphorylation was found to be predominant across the organs (Fig.…”

Application of Safirinium N-Hydroxysuccinimide Esters to Derivatization of Peptides for High-Resolution Mass Spectrometry, Tandem Mass Spectrometry, and Fluorescent Labeling of Bacterial Cells

“…This approach allows the detection of protein modifications such as phosphorylation [ 8 , 9 ], oxidation [ 6 ], acetylation [ 10 ], and glycosylation [ 7 ]. Tandem mass spectrometry (MS/MS) is extensively employed in the analysis of peptides and proteins [ 11 , 12 , 13 ], often with the use of isotopic [ 14 ] as well as isobaric [ 9 , 15 , 16 , 17 ] tagging. More advanced techniques frequently utilized in proteomic studies involve isotopic dilution [ 5 , 18 , 19 , 20 , 21 ], hydrogen–deuterium exchange (HDX) [ 8 , 22 ] and multiple reaction monitoring (MRM) [ 7 ].…”