Online automated in vivo zebrafish phosphoproteomics: from large-scale analysis down to a single embryo.
In the developing embryo, as in many other biological processes, complex signaling pathways are under tight control of reversible phosphorylation, guiding cell proliferation, differentiation, and growth. Therefore the large-scale identification of signaling proteins and their post-translational modifications is crucial to understand the proteome biology of the developing zebrafish embryo. Here, we used an automated, robust, and sensitive online TiO 2-based LC-MS/MS setup to enrich for phosphorylated peptides from 1 day old zebrafish embryos. We identified, with high confidence, 1067 endogenous phosphorylation sites in a sample taken from 60 embryos (approximately 180 microg), 321 from 10 embryos, and 47 phosphorylation sites from a single embryo, illustrating the sensitivity of the method. This data set, representing by far the largest for zebrafish, was further exploited by searching for serine/threonine or tyrosine kinase motifs using Scansite. For one-third of the identified phosphopeptides a potential kinase motif could be predicted, where it appeared that Cdk5 kinase, p38MAPK, PKA, and Casein Kinase 2 substrates were the most predominant motifs present, underpinning the importance of these kinases in signaling pathways in embryonic development. The phosphopeptide data set was further interrogated using alignments with phosphopeptides identified in recent large-scale phosphoproteomics screens in human and mouse samples. These alignments revealed conservation of phosphorylation sites in several proteins suggesting preserved function in embryonic development.
Lemeer
Simone
S
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands. a.j.r.heck@uu.nl
Pinkse
Martijn W H
MW
Mohammed
Shabaz
S
van Breukelen
Bas
B
den Hertog
Jeroen
J
Slijper
Monique
M
Heck
Albert J R
AJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D016384:Consensus Sequence; D017124:Conserved Sequence; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D014025:Titanium; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D010750:Phosphoproteins; D029961:Zebrafish Proteins; C009495:titanium dioxide; D014025:Titanium