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Nilsson C.L.,University of Texas Medical Branch | Mostovenko E.,University of Texas Medical Branch | Lichti C.F.,University of Texas Medical Branch | Ruggles K.,University of California at Santa Cruz | And 20 more authors.
Journal of Proteome Research | Year: 2015

We describe the utility of integrated strategies that employ both translation of ENCODE data and major proteomic technology pillars to improve the identification of the missing proteins', novel proteoforms, and PTMs. On one hand, databases in combination with bioinformatic tools are efficiently utilized to establish microarray-based transcript analysis and supply rapid protein identifications in clinical samples. On the other hand, sequence libraries are the foundation of targeted protein identification and quantification using mass spectrometric and immunoaffinity techniques. The results from combining proteoENCODEdb searches with experimental mass spectral data indicate that some alternative splicing forms detected at the transcript level are in fact translated to proteins. Our results provide a step toward the directives of the C-HPP initiative and related biomedical research. © 2015 American Chemical Society.


Jeong S.-K.,Yonsei Proteome Research Center | Hancock W.S.,Northeastern University | Paik Y.-K.,Yonsei Proteome Research Center | Paik Y.-K.,Yonsei University
Journal of Proteome Research | Year: 2015

Since the launch of the Chromosome-centric Human Proteome Project (C-HPP) in 2012, the number of "missing" proteins has fallen to 2932, down from ∼5932 since the number was first counted in 2011. We compared the characteristics of missing proteins with those of already annotated proteins with respect to transcriptional expression pattern and the time periods in which newly identified proteins were annotated. We learned that missing proteins commonly exhibit lower levels of transcriptional expression and less tissue-specific expression compared with already annotated proteins. This makes it more difficult to identify missing proteins as time goes on. One of the C-HPP goals is to identify alternative spliced product of proteins (ASPs), which are usually difficult to find by shot-gun proteomic methods due to their sequence similarities with the representative proteins. To resolve this problem, it may be necessary to use a targeted proteomics approach (e.g., selected and multiple reaction monitoring [S/MRM] assays) and an innovative bioinformatics platform that enables the selection of target peptides for rarely expressed missing proteins or ASPs. Given that the success of efforts to identify missing proteins may rely on more informative public databases, it was necessary to upgrade the available integrative databases. To this end, we attempted to improve the features and utility of GenomewidePDB by integrating transcriptomic information (e.g., alternatively spliced transcripts), annotated peptide information, and an advanced search interface that can find proteins of interest when applying a targeted proteomics strategy. This upgraded version of the database, GenomewidePDB 2.0, may not only expedite identification of the remaining missing proteins but also enhance the exchange of information among the proteome community. GenomewidePDB 2.0 is available publicly at http://genomewidepdb.proteomix.org/. © 2015 American Chemical Society.


Jeong S.-K.,Yonsei Proteome Research Center | Lee H.-J.,Yonsei Proteome Research Center | Na K.,Yonsei Proteome Research Center | Cho J.-Y.,Korea University | And 9 more authors.
Journal of Proteome Research | Year: 2013

In an effort to map the human proteome, the Chromosome-centric Human Proteome Project (C-HPP) was recently initiated. As a member of the international consortium working on this project, our laboratory developed a gene-centric proteomic database called GenomewidePDB, which integrates proteomic data for proteins encoded by chromosomes with transcriptomic data and other information from public databases. As an example case, we chose chromosome 13, which is the largest acrocentric human chromosome with the lowest gene density and contains 326 predicted proteins. All proteins stored in GenomewidePDB are linked to other resources, including neXtProt and Ensembl for protein and gene information, respectively. The Global Proteome Machine database (GPMdb) and the PeptideAtlas are also accessed for observed mass spectrometry (MS) information, while Human Protein Atlas is used for information regarding antibody availability and tissue expression, respectively. Gene ontology disease information is also included. As a pilot work, we constructed this GenomewidePDB with the identified 3615 proteins including 53 chromosome 13-origin proteins that are present in normal human placenta tissue. Thus, developing a comprehensive database containing actual experimental proteomics data will provide a valuable resource for cross chromosomal comparison in the C-HPP community. © 2012 American Chemical Society.


Kim K.-Y.,Yonsei Proteome Research Center | Joo H.-J.,Yonsei Proteome Research Center | Joo H.-J.,University of Texas M. D. Anderson Cancer Center | Kwon H.-W.,Yonsei University | And 5 more authors.
Analytical Chemistry | Year: 2013

Pheromones produced by Caenorhabditis elegans are considered key regulators of development, mating, and social behaviors in this organism. Here, we present a rapid mass spectrometry-based method (PheroQu) for absolute quantitation of nematode pheromones (e.g., daumone 1, 2, and 3) both in C. elegans worm bodies (as few as 20 worms) and in liquid culture medium. Pheromones were separated by ultra performance liquid chromatography and monitored by a positive electrospray ionization detector in the multiple-reaction monitoring mode. The daf-22 mutant worms were used as surrogate matrix for calibration, and stable deuterated isotope-containing pheromone was used as internal standard for measuring changes in pheromones in N2 wild-type and other strains under different growth conditions. The worm-body pheromones were extracted by acidified acetonitrile solvent, and the secreted pheromones were extracted from culture medium with solid-phase extraction cartridges. The run time was achieved in less than 2 min. The method was validated for specificity, linearity, accuracy, precision, recovery, and stability. The assay was linear over an amount range of 2-250 fmol, and the limit of quantitation was 2 fmol amounts for daumone 1, 2, and 3 in both worm bodies and culture medium. With the PheroQu method, we were able to identify the location of pheromone biosynthesis and determine the changes in different pheromone types synthesized, according to developmental stages and aging process. This method, which is simple, rapid, sensitive, and specific, will be useful for the study of small-molecule metabolism during developmental stages of C. elegans. © 2013 American Chemical Society.


PubMed | Yonsei University, Yonsei Proteome Research Center and Korea Basic Science Institute
Type: Journal Article | Journal: Journal of proteome research | Year: 2016

Glycoproteins influence numerous indispensable biological functions, and changes in protein glycosylation have been observed in various diseases. The identification and characterization of glycoprotein and glycosylation sites by mass spectrometry (MS) remain challenging tasks, and great efforts have been devoted to the development of proteome informatics tools that facilitate the MS analysis of glycans and glycopeptides. Here we report on the development of gFinder, a web-based bioinformatics tool that analyzes mixtures of native N-glycopeptides that have been profiled by tandem MS. gFinder not only enables the simultaneous integration of collision-induced dissociation (CID) and high-energy collisional dissociation (HCD) fragmentation but also merges the spectra for high-throughput analysis. These merged spectra expedite the identification of both glycans and N-glycopeptide backbones in tandem MS data using the glycan database and a proteomic search tool (e.g., Mascot). These data can be used to simultaneously characterize peptide backbone sequences and possible N-glycan structures using assigned scores. gFinder also provides many convenient functions that make it easy to perform manual calculations while viewing the spectrum on-screen. We used gFinder to detect an additional protein (Q8N9B8) that was missed from the previously published data set containing N-linked glycosylation. For N-glycan analysis, we used the GlycomeDB glycan structure database, which integrates the structural and taxonomic data from all of the major carbohydrate databases available in the public domain. Thus, gFinder is a convenient, high-throughput analytical tool for interpreting the tandem mass spectra of N-glycopeptides, which can then be used for identification of potential missing proteins having glycans. gFinder is available publicly at http://gFinder.proteomix.org/ .


Joo H.-J.,Yonsei Proteome Research Center | Joo H.-J.,Yonsei University | Kim K.-Y.,Yonsei Proteome Research Center | Kim K.-Y.,Yonsei University | And 8 more authors.
Journal of Biological Chemistry | Year: 2010

Dauer pheromones or daumones, which are signaling molecules that interrupt development and reproduction (dauer larvae) during unfavorable growth conditions, are essential for cellular homeostasis in Caenorhabditis elegans. According to earlier studies, dauer larva formation in strain N2 is enhanced by a temperature increase, suggesting the involvement of a temperature-dependent component in dauer pheromone biosynthesis or sensing. Several naturally occurring daumone analogs (e.g. daumones 1-3) have been identified, and these molecules are predicted to be synthesized in different physiological settings in this nematode. To elucidate the molecular regulatory system that may influence the dynamic balance of specific daumone production in response to sudden temperature changes, we characterized the peroxisomal acox gene encoding acyl-CoA oxidase, which is predicted to catalyze the first reaction during biosynthesis of the fatty acid component of daumones. Using acox-1(ok2257) mutants and a new, robust analytical method, we quantified the three most abundant daumones in worm bodies and showed that acox likely contributes to the dynamic production of various quantities of three different daumones in response to temperature increase, changes that are critical in C. elegans for coping with the natural environmental changes it faces. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Hahm J.-H.,Yonsei Proteome Research Center | Kim S.,Yonsei Proteome Research Center | Kim S.,Yonsei University | Paik Y.-K.,Yonsei Proteome Research Center | Paik Y.-K.,Yonsei University
Aging Cell | Year: 2011

Innate immune responses to pathogens are governed by the nervous system. Here, we investigated the molecular mechanism underlying innate immunity in Caenorhabditis elegans against Escherichia coli OP50, a standard laboratory C. elegans food. Longevity was compared in worms fed live or UV-killed OP50 at low or high density food condition (HDF). Expression of the antimicrobial gene lys-8 was approximately 5-fold higher in worms fed live OP50, suggesting activation of innate immunity upon recognition of OP50 metabolites. Lifespan was extended and SOD-3 mRNA levels were increased in gpa-9-overexpressing gpa-9XS worms under HDF in association with robust induction of insulin/IGF-1 signaling (IIS). Expression of ins-7 and daf-28 that control lys-8 expression was reduced in gpa-9XS, indicating that GPA-9-mediated immunity is due in part to ins-7 and daf-28 downregulation. Our results suggest that OP50 metabolites in amphid neurons elicit innate immunity through the IIS pathway, and identify GPA-9 as a novel regulator of both the immune system and aging in C. elegans. © 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

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