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PubMed2003

The application of new software tools to quantitative protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry: I. Statistically annotated datasets for peptide sequences and proteins identified via the application of ICAT and tandem mass spectrometry to proteins copurifying with T cell lipid rafts.

Lipid rafts were prepared according to standard protocols from Jurkat T cells stimulated via T cell receptor/CD28 cross-linking and from control (unstimulated) cells. Co-isolating proteins from the control and stimulated cell preparations were labeled with isotopically normal (d0) and heavy (d8) versions of the same isotope-coded affinity tag (ICAT) reagent, respectively. Samples were combined, proteolyzed, and resultant peptides fractionated via cation exchange chromatography. Cysteine-containing (ICAT-labeled) peptides were recovered via the biotin tag component of the ICAT reagents by avidin-affinity chromatography. On-line micro-capillary liquid chromatography tandem mass spectrometry was performed on both avidin-affinity (ICAT-labeled) and flow-through (unlabeled) fractions. Initial peptide sequence identification was by searching recorded tandem mass spectrometry spectra against a human sequence data base using SEQUEST software. New statistical data modeling algorithms were then applied to the SEQUEST search results. These allowed for discrimination between likely "correct" and "incorrect" peptide assignments, and from these the inferred proteins that they collectively represented, by calculating estimated probabilities that each peptide assignment and subsequent protein identification was a member of the "correct" population. For convenience, the resultant lists of peptide sequences assigned and the proteins to which they corresponded were filtered at an arbitrarily set cut-off of 0.5 (i.e. 50% likely to be "correct") and above and compiled into two separate datasets. In total, these data sets contained 7667 individual peptide identifications, which represented 2669 unique peptide sequences, corresponding to 685 proteins and related protein groups.

Amino Acid Sequence↗
PubMed2006

Quantitative proteomic analysis of post-translational modifications of human histones.

Histone proteins are subject to a range of post-transcriptional modifications in living cells. The combinatorial nature of these modifications constitutes the "histone code" that dictates chromatin structure and function during development, growth, differentiation, and homeostasis of cells. Deciphering of the histone code is hampered by the lack of analytical methods for monitoring the combinatorial complexity of reversible multisite modifications of histones, including acetylation and methylation. To address this problem, we used LC-MSMS technology and Virtual Expert Mass Spectrometrist software for qualitative and quantitative proteomic analysis of histones extracted from human small cell lung cancer cells. A total of 32 acetylations, methylations, and ubiquitinations were located in the human histones H2A, H2B, H3, and H4, including seven novel modifications. An LC-MSMS-based method was applied in a quantitative proteomic study of the dose-response effect of the histone deacetylase inhibitor (HDACi) PXD101 on histone acetylation in human cell cultures. Triplicate LC-MSMS runs at six different HDACi concentrations demonstrated that PXD101 affects acetylation of histones H2A, H2B, H3, and H4 in a site-specific and dose-dependent manner. This unbiased analysis revealed that a relative increase in acetylated peptide from the histone variants H2A, H2B, and H4 was accompanied by a relative decrease of dimethylated Lys(57) from histone H2B. The dose-response results obtained by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post-translational modifications is a viable approach for functional analysis of candidate drugs, such as HDAC inhibitors.

Acetylation↗
PubMed2003

Early drug safety evaluation: biomarkers, signatures, and fingerprints.

When target organ toxicity arises in animal models during routine drug safety evaluation, it raises several key questions: Is this target organ toxicity related to the pharmacology? What is the mode of action (MOA)? Is the target organ toxicity relevant to humans? Pathology or prior knowledge of the compound class may provide clues on a possible MOA for toxicity. However, if this deductive approach yields no results, the inductive approach offered by new technologies can generate novel research leads. For example, toxicogenomics can generate a gene expression profile of the toxicity that can be compared with reference compounds or with other candidate drugs. Similarly, proteomic analysis of the protein profile at the toxic vs. the efficacious dose can provide clues on MOA for the toxicity and may allow differentiation of the pathways of the toxic response from those required for pharmacological activity.

Animals↗
PubMed2005

Differential immunogenicity of HIV-1 clade C proteins in eliciting CD8+ and CD4+ cell responses.

BACKGROUND: The relative immunogenicity of human immunodeficiency virus type 1 (HIV-1) proteins for CD8+ and CD4+ cell responses has not been defined. METHODS: HIV-1-specific T cell responses were evaluated in 65 chronically HIV-1-infected untreated subjects by interferon- gamma flow cytometry with peptides spanning the clade C consensus sequence. RESULTS: The magnitude of HIV-1-specific CD8+ T cell responses correlated significantly with CD4+ cell responses, but the percentage of CD8+ T cells directed against HIV-1 (median, 2.76%) was always greater than that of CD4+ cells (median, 0.24%). Although CD8+ T cell responses were equally distributed among Gag, Pol, and the regulatory and accessory proteins, Gag was the dominant target for CD4+ cell responses. There was no consistent relationship between virus-specific CD8+ or CD4+ cell response and viral load. However, the median viral load in subjects in whom Gag was the dominant CD8+ T cell target was significantly lower than that in subjects in whom non-Gag proteins were the main target (P=.007). CONCLUSIONS: Gag-specific responses dominate the CD4+ T cell response to HIV, whereas CD8+ T cell responses are broadly distributed, which indicates differential immunogenicity of these cells against HIV-1. The preferential targeting of Gag by CD8+ T cells is associated with enhanced control of viral load.

CD4-Positive T-Lymphocytes↗
PubMed2006

Variation among species in proteomic sulphur content is related to environmental conditions.

The elemental composition of proteins influences the quantities of different elements required by organisms. Here, we considered variation in the sulphur content of whole proteomes among 19 Archaea, 122 Eubacteria and 10 eukaryotes whose genomes have been fully sequenced. We found that different species vary greatly in the sulphur content of their proteins, and that average sulphur content of proteomes and genome base composition are related. Forces contributing to variation in proteomic sulphur content appear to operate quite uniformly across the proteins of different species. In particular, the sulphur content of orthologous proteins was frequently correlated with mean proteomic sulphur contents. Among prokaryotes, proteomic sulphur content tended to be greater in anaerobes, relative to non-anaerobes. Thermophiles tended to have lower proteomic sulphur content than non-thermophiles, consistent with the thermolability of cysteine and methionine residues. This work suggests that persistent environmental growth conditions can influence the evolution of elemental composition of whole proteomes in a manner that may have important implications for the amount of sulphur used by living organisms to build proteins. It extends previous studies that demonstrated links between transient changes in environmental conditions and the elemental composition of subsets of proteins expressed under these conditions.

Adaptation, Physiological↗
PubMed2003

Continued discovery of transcriptional units expressed in cells of the mouse mononuclear phagocyte lineage.

The current RIKEN transcript set represents a significant proportion of the mouse transcriptome but transcripts expressed in the innate and acquired immune systems are poorly represented. In the present study we have assessed the complexity of the transcriptome expressed in mouse macrophages before and after treatment with lipopolysaccharide, a global regulator of macrophage gene expression, using existing RIKEN 19K arrays. By comparison to array profiles of other cells and tissues, we identify a large set of macrophage-enriched genes, many of which have obvious functions in endocytosis and phagocytosis. In addition, a significant number of LPS-inducible genes were identified. The data suggest that macrophages are a complex source of mRNA for transcriptome studies. To assess complexity and identify additional macrophage expressed genes, cDNA libraries were created from purified populations of macrophage and dendritic cells, a functionally related cell type. Sequence analysis revealed a high incidence of novel mRNAs within these cDNA libraries. These studies provide insights into the depths of transcriptional complexity still untapped amongst products of inducible genes, and identify macrophage and dendritic cell populations as a starting point for sampling the inducible mammalian transcriptome.

Animals↗
PubMed2001

Whole proteome pI values correlate with subcellular localizations of proteins for organisms within the three domains of life.

Isoelectric point (pI) values have long been a standard measure for distinguishing between proteins. This article analyzes distributions of pI values estimated computationally for all predicted ORFs in a selection of fully sequenced genomes. Histograms of pI values confirm the bimodality that has been observed previously for bacterial and archaeal genomes () and reveal a trimodality in eukaryotic genomes. A similar analysis on subsets of a nonredundant protein sequence database generated from the full database by selecting on subcellular localization shows that sequences annotated as corresponding to cytosolic and integral membrane proteins have pI distributions that appear to correspond with the two observed modes of bacteria and archaea. Furthermore, nuclear proteins have a broader distribution that may account for the third mode observed in eukaryotes. On the basis of this association between pI and subcellular localization, we conclude that the bimodal character of whole proteome pI values in bacteria and archaea and the trimodal character in eukaryotes are likely to be general properties of proteomes and are associated with the need for different pI values depending on subcellular localization. Our analyses also suggest that the proportions of proteomes consisting of membrane-associated proteins may be currently underestimated.

Animals↗
PubMed2003

Experimental analysis of the Arabidopsis mitochondrial proteome highlights signaling and regulatory components, provides assessment of targeting prediction programs, and indicates plant-specific mitochondrial proteins.

A novel insight into Arabidopsis mitochondrial function was revealed from a large experimental proteome derived by liquid chromatography-tandem mass spectrometry. Within the experimental set of 416 identified proteins, a significant number of low-abundance proteins involved in DNA synthesis, transcriptional regulation, protein complex assembly, and cellular signaling were discovered. Nearly 20% of the experimentally identified proteins are of unknown function, suggesting a wealth of undiscovered mitochondrial functions in plants. Only approximately half of the experimental set is predicted to be mitochondrial by targeting prediction programs, allowing an assessment of the benefits and limitations of these programs in determining plant mitochondrial proteomes. Maps of putative orthology networks between yeast, human, and Arabidopsis mitochondrial proteomes and the Rickettsia prowazekii proteome provide detailed insights into the divergence of the plant mitochondrial proteome from those of other eukaryotes. These show a clear set of putative cross-species orthologs in the core metabolic functions of mitochondria, whereas considerable diversity exists in many signaling and regulatory functions.

Arabidopsis↗
PubMed2001

Identification of four proteins from the small subunit of the mammalian mitochondrial ribosome using a proteomics approach.

Proteins in the small subunit of the mammalian mitochondrial ribosome were separated by two-dimensional polyacrylamide gel electrophoresis. Four individual proteins were subjected to in-gel Endoprotease Lys-C digestion. The sequences of selected proteolytic peptides were obtained by electrospray tandem mass spectrometry. Peptide sequences obtained from in-gel digestion of individual spots were used to screen human, mouse, and rat expressed sequence tag databases, and complete consensus cDNAs for these species were deduced in silico. The corresponding protein sequences were characterized by comparison to known ribosomal proteins in protein databases. Four different classes of mammalian mitochondrial small subunit ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins are homologs to Escherichia coli S9 and S5 proteins. The presence of these newly identified mitochondrial ribosomal proteins are also investigated in the Drosophila melanogaster, Caenorhabditis elegans, and in the genomes of several fungi.

Amino Acid Sequence↗
PubMed2001

Genomic style of proteins: concepts, methods and analyses of ribosomal proteins from 16 microbial species.

We have introduced the concept of genomic 'style' of proteins. By style we understand those properties of a large set of proteins which are specific to the genome of one species (species primary-self) and different from the genome of another species (species contrasted-self). To characterise the style, we took advantage of the frequencies of amino acids and dipeptides present in non-identical segments of the complete set of orthologous ribosomal proteins encoded by 16 microbial species. We confirm the dependence of the overall amino acid composition on the genomic (G+C) content, and introduce a rectification procedure making it possible to extricate appropriate species-specific characteristics, which are no longer related to this content. The rectified frequencies are used to calculate inter-species distance matrices, and to build genomic evolutionary trees. Remarkably, the phylograms derived from the frequencies of non-identical residues in proteins closely resemble the classical phylograms based upon the conservation of identical residues in ribosomal RNAs. We believe that the concept of genomic style of proteins can be a useful tool for the study of evolution.

Amino Acid Sequence↗
PubMed2006

Proteomic study of peptide deformylase inhibition in Streptococcus pneumoniae and Staphylococcus aureus.

Peptide deformylase (PDF) is an essential enzyme in both gram-negative and gram-positive bacteria. It hydrolyzes formylated N-terminal peptides to generate free N-terminal peptides during the process of protein maturation. Inhibition of this enzyme results in cessation of bacterial growth. We have examined the effect of a potent PDF inhibitor, LBM-415 (also known as VIC-104959), on the proteomes of Staphylococcus aureus and Streptococcus pneumoniae using two-dimensional electrophoresis. Both S. aureus and S. pneumoniae showed accumulation of many N-terminal formylated peptides/proteins upon PDF inhibition. In S. pneumoniae, formylated peptide/protein accumulation was time dependent. Following inhibition, subsequent removal of the inhibitor resulted in deformylation of formylated peptides/proteins; this recovery process was also time dependent. If instead the inhibited cells were maintained in the presence of sub-MIC levels of the PDF inhibitor, the formylated peptides/proteins remained for a much longer time, which correlated with a prolonged postantibiotic effect in vitro. These observations may have broader implications for the application of this class of antibiotics in vivo.

Amidohydrolases↗
PubMed2006

PRIMA: peptide robust identification from MS/MS spectra.

In proteomics, tandem mass spectrometry is the key technology for peptide sequencing. However, partially due to the deficiency of peptide identification software, a large portion of the tandem mass spectra are discarded in almost all proteomics centers because they are not interpretable. The problem is more acute with the lower quality data from low end but more popular devices such as the ion trap instruments. In order to deal with the noisy and low quality data, this paper develops a systematic machine learning approach to construct a robust linear scoring function, whose coefficients are determined by a linear programming. A prototype, PRIMA, was implemented. When tested with large benchmarks of varying qualities, PRIMA consistently has higher accuracy than commonly used software MASCOT, SEQUEST and X! Tandem.

Amino Acid Sequence↗
PubMed2005

Functional clustering algorithm for high-dimensional proteomics data.

Clustering proteomics data is a challenging problem for any traditional clustering algorithm. Usually, the number of samples is largely smaller than the number of protein peaks. The use of a clustering algorithm which does not take into consideration the number of features of variables (here the number of peaks) is needed. An innovative hierarchical clustering algorithm may be a good approach. We propose here a new dissimilarity measure for the hierarchical clustering combined with a functional data analysis. We present a specific application of functional data analysis (FDA) to a high-throughput proteomics study. The high performance of the proposed algorithm is compared to two popular dissimilarity measures in the clustering of normal and human T-cell leukemia virus type 1 (HTLV-1)-infected patients samples.

Journal Article↗
PubMed2004

Advances in pituitary pathology: use of novel techniques.

Many new techniques are rapidly being developed and applied to the study of normal and neoplastic pituitary tissues. These range from techniques used for the past few decades such as in situ hybridization to more recent developments such as comparative genomic hybridization, laser capture microdissection, DNA and tissue arrays, proteomics, and RNA interference technology. These approaches have been applied to answer many challenging questions about pituitary function and pathophysiology. This chapter reviews many of these recent developments and shows their applications to pituitary biology in order to demonstrate how these new techniques are providing insights about basic aspects, clinical, and pharmacologic knowledge of the pituitary gland and of pituitary tumors.

Endocrinology↗
PubMed2002

RIO: analyzing proteomes by automated phylogenomics using resampled inference of orthologs.

BACKGROUND: When analyzing protein sequences using sequence similarity searches, orthologous sequences (that diverged by speciation) are more reliable predictors of a new protein's function than paralogous sequences (that diverged by gene duplication). The utility of phylogenetic information in high-throughput genome annotation ("phylogenomics") is widely recognized, but existing approaches are either manual or not explicitly based on phylogenetic trees. RESULTS: Here we present RIO (Resampled Inference of Orthologs), a procedure for automated phylogenomics using explicit phylogenetic inference. RIO analyses are performed over bootstrap resampled phylogenetic trees to estimate the reliability of orthology assignments. We also introduce supplementary concepts that are helpful for functional inference. RIO has been implemented as Perl pipeline connecting several C and Java programs. It is available at http://www.genetics.wustl.edu/eddy/forester/. A web server is at http://www.rio.wustl.edu/. RIO was tested on the Arabidopsis thaliana and Caenorhabditis elegans proteomes. CONCLUSION: The RIO procedure is particularly useful for the automated detection of first representatives of novel protein subfamilies. We also describe how some orthologies can be misleading for functional inference.

Animals↗
PubMed2006

An application of statistics to comparative metagenomics.

BACKGROUND: Metagenomics, sequence analyses of genomic DNA isolated directly from the environments, can be used to identify organisms and model community dynamics of a particular ecosystem. Metagenomics also has the potential to identify significantly different metabolic potential in different environments. RESULTS: Here we use a statistical method to compare curated subsystems, to predict the physiology, metabolism, and ecology from metagenomes. This approach can be used to identify those subsystems that are significantly different between metagenome sequences. Subsystems that were overrepresented in the Sargasso Sea and Acid Mine Drainage metagenome when compared to non-redundant databases were identified. CONCLUSION: The methodology described herein applies statistics to the comparisons of metabolic potential in metagenomes. This analysis reveals those subsystems that are more, or less, represented in the different environments that are compared. These differences in metabolic potential lead to several testable hypotheses about physiology and metabolism of microbes from these ecosystems.

Algorithms↗