Proteomics 2012, 12, 1879–1882 1879 DOI 10.1002/pmic.201100576 DATASET BRIEF Expanding the zebrafish embryo proteome using multiple fractionation approaches and tandem mass spectrometry Christopher L ¨ oßner ∗ , Sheena Wee ∗ , Siok Ghee Ler, Rachel H. X. Li, Tom Carney, Walter Blackstock and Jayantha Gunaratne Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore The proteome of zebrafish, Danio rerio, embryos has not been studied in great detail mainly due to the presence of high abundance yolk proteins in embryos. Here we report the highest number of the zebrafish embryo proteins identified so far to our knowledge, through a combi- nation of a protein-level fractionation approach (1D SDS-PAGE) and two different peptide-level fractionation approaches (IEF and strong anion exchange (SAX)) of deyolked zebrafish em- bryos followed by LC-MS/MS. We detected 5267 proteins in total of which 3464 proteins were identified with at least two peptides (less than 1% peptide false discovery rate). The analysis of proteome coverage from each method showed that 56% of detected proteins were com- mon to all approaches and 95% of the detected proteome was obtained from 1D SDS-PAGE approach alone. Bioinformatics analysis of the detected proteome demonstrated that nucleo- cytoplasmic transport (biological process) and ribosomal proteins (cellular component) were the most over-represented proteins, whereas cell–cell signaling (biological process) and extra- cellular space proteins (cellular component) were the most under-represented proteins in the identified proteome. Keywords: Animal proteomics / Deyolking / Fractionation / Gene ontology / Whole proteome / Zebrafish embryo Received: November 7, 2011 Revised: February 17, 2012 Accepted: February 21, 2012 The Zebrafish (Danio rerio) is a well-established and powerful vertebrate model organism, especially in the research areas of vertebrate embryogenesis and organ development [1]. The optical transparency together with rapid development allows in vivo visualization of cellular behavior during organogene- sis. Transgenic and forward and reverse genetic strategies are well established, facilitated by the availability of comprehen- sive genomic information [2]. However, accurate and com- plete pictures of physiological processes cannot be achieved through genomic information alone as proteins are the ex- Correspondence: Dr. Jayantha Gunaratne, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Singapore 138673, Singapore E-mail: jayanthag@imcb.a-star.edu.sg Fax: +65-67791117 Abbreviations: FASP, filter-aided sample preparation; FDR, false discovery rate; GO, gene ontology; SAX, strong anion exchange ecutors of most of the biological functions. Thus, the study of the proteome, which provides temporal and spatial informa- tion of the translated genome, is imperative for understand- ing of underlying biological mechanisms. The proteome of zebrafish, especially their embryos, has not been studied in great detail, mainly due to the presence of high abundance yolk proteins in embryos [3–7]. To circumvent this, the yolk proteins have to be depleted [8]. Although deyolking facilitates the analysis of the proteome the sample still consists of a com- plex mixture of proteins. It has been shown that combination of multiple fractionation approaches enhances proteome cov- erage of complex samples [9, 10]. We therefore used multiple fractionation strategies to expand the proteome of zebrafish embryos. We carried out 1D SDS-PAGE for protein sepa- ration, followed by in-gel tryptic digestion. For peptide-level ∗ These authors contributed equally to this work. C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com