Detection of Hypothetical Proteins in Human Fetal Perireticular Nucleus Felix Hepner,† Jae-Kyung Myung,† Norbert Ulfig,‡ Arnold Pollak,† and Gert Lubec*,† Department of Pediatrics, Medical University of Vienna, Vienna, Austria, and Neuroembryonic Research Laboratory, Faculty of Medicine, University of Rostock, Germany Received May 9, 2005
Abstract: There is a legion of hypothetical proteins (HP) in prokaryotic and eukaryotic proteomes and the aim of this study was to describe HP in the perireticular nucleus (PN), a key structure in human brain development. Tissue from four PNs was homogenized and extracted proteins were run on two-dimensional gel electrophoresis followed by in-gel digestion and mass spectrometrical identification of proteins. Several databases were used for obtaining bioinformatic information and searching for functional and structural domains. Five spots represented HP: KIAA0423 protein (Q9Y4F4), hypothetical protein KIAA0153 (Q14166), hypothetical protein DKFZp564A2416 (Q9NTW4),hypotheticalproteinDKFZp564H1122(Q9H0W9), and hypothetical protein DKFZp564D1378 (Q9H0R4). These structures were predicted to serve in cell cycle, DNAcondensation, neurogenesis, or apoptosis. The existence of formerly HP proteins in the PN of human fetal brain is shown, thus extending knowledge of the brain proteome and proposing the method used as a suitable analytical tool for searching HP. Keywords: hypothetical proteins • proteomics • fetal brain • development • two-dimensional electrophoresis • mass spectrometry
Introduction Neither genomes nor proteomes are completely analyzed in pro- and eukaryotes and many unknown and hypothetical genes and proteins still remain uncharacterized.1,2 Performing protein hunting in order to generate an atlas of the chemical neuroanatomy of the human fetal and adult brain using protein profiling by proteomics techniques, we identified a series of so-called hypothetical proteins by two-dimensional gel-electrophoresis followed by mass spectrometrical identification of proteins.3,4 These hypothetical proteins included as yet unknown proteins, hypothetical proteins, or conserved hypothetical proteins,5 as well as proteins with unknown function. No generally accepted glossary and terminology for classification * To whom correspondence should be addressed. CChem, FRSC (UK), Medical University of Vienna, Department of Pediatrics, Wa¨hringer Gu ¨ rtel 18, A 1090 Vienna, Austria. Tel.: +43-1-40400-3215. Fax: +43-1-40400-3194. E-mail address:
[email protected]. † Medical University of Vienna. ‡ University of Rostock. 10.1021/pr050133p CCC: $30.25
2005 American Chemical Society
of these proteins has been described so far, but hypothetical proteins (HP) are understood as either proteins predicted from nucleic acid sequences or proteins without documented experimental evidence for their existence. Apart from the technical problems with annotation, sequencing errors, and protein sequence alignments,6,7 some proteins remain “unknown”, as they are not listed appropriately or are absent in protein databases.8-10 In our experience, about 2-3% of proteins identified in a standard two-dimensional gel of brain proteins can be assigned to the categories of unknown or HP.11 These are the major interesting structures, as most of the other proteins of the proteomes, e.g., metabolic, cytoskeleton, chaperone, signaling, and antioxidant proteins, have enjoyed manifold reporting. As at least 1/3 of genes from genomic databases and many more proteins from protein databases can be regarded as unknown or hypothetical proteins, protein hunting is challenging, thus providing experimental evidence for their existence. It was the aim of this study to systematically search for HP in several areas of the human brain and herein, of fetal nucleus perireticularis. We intended to fairly identify the observed HP structures by a robust protein chemical approach and to categorize the individual HP accordingly. Finally, we used bioinformatics tools to assign a putative function to the HP. Another goal was to provide an analytical tool in order to find and localize HP spots in a two-dimensional gel. Five HP were observed in this developmentally important brain area that were not detected so far at the protein level, neither in the literature nor in own brain proteome studies.12
Experimental Section Sample Preparation. Conceptus abortus fetal brain samples (PN; n ) 4, male) with mean gestation age of 21 ( 3 weeks were obtained from Norbert Ulfig from the Neuroembryonic Research Laboratory, Faculty of Medicine, University of Rostock, Germany. Clinical information on patients is presented in Table 1. Brain samples were taken in accordance to the local Ethical Committee in Rostock. All samples were stored at -80 °C, and the freezing chain was never interrupted. Homogenized samples were suspended in 1,8 mL of sample buffer consisting of 8 M urea (Merck, Darmstadt, Germany), 2 M thiourea (Sigma, St. Louis, MO), 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate) (Sigma, St. Louis, MO), 65 mM 1,4-dithioerythritol (Merck, Germany), 1 mM EDTA (ethylenediamintetraacetic acid), 1 mM PMSF and IPG Buffer, pH 3-10 NL (Amersham Biosciences, Upsala, Journal of Proteome Research 2005, 4, 2379-2385
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Published on Web 10/25/2005
Hypothetical Proteins in the Perireticular Nucleus
technical notes
Table 1. Clinical Characterization of Fetuses Whose Perireticular Nuclei Were Used for Post Mortem (PM) Studiesa
GWb
PM interval (h)
sex
causes of death
21 19 20 24
