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At least 703 records · Page 39Linked to original sources
PubMed2004

Tandem affinity purification and identification of protein complex components.

As with the budding yeast Saccharomyces cerevisiae, the completion of the Schizosaccharomyces pombe genome sequence has opened new opportunities to investigate the functional organization of a eukaryotic cell. These include analysis of gene expression patterns, comprehensive gene knockout and synthetic lethal screens, global protein localization analysis, and direct protein interaction mapping. We describe here the tandem affinity purification or TAP approach combined with DALPC mass spectrometry to identify components of protein complexes as we have applied it to S. pombe. This approach can theoretically be applied to the entire proteome as has been done in S. cerevisiae to gain insight into functional protein assemblies and to elucidate functions of uncharacterized proteins.

Multiprotein Complexes↗
PubMed2003

A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.

Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.

Epidermal Growth Factor↗
PubMed2002

RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach.

To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3' end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.

Amino Acid Sequence↗
PubMed2006

Cytosolic and nuclear protein targets of thiol-reactive electrophiles.

Reactive electrophiles formed from toxic drugs and chemicals and by endogenous oxidative stress covalently modify proteins. Although protein covalent binding is thought to initiate a variety of adaptive and toxic responses, the identities of the protein targets are generally unknown, as are protein structural features that confer susceptibility to modification. We have analyzed the protein targets in nuclear and cytoplasmic proteomes from HEK293 cells treated in vitro with two biotin-tagged, thiol-reactive electrophiles, (+)-biotinyl-iodoacetamidyl-3, 6-dioxaoctanediamine (PEO-IAB) and 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC). Biotinylated peptides were captured by affinity enrichment using neutravidin beads, and the adducted peptides were then analyzed by multidimensional liquid chromatography-tandem mass spectrometry. A total of 897 adducts were mapped to different cysteine residues in 539 proteins. Adduction was selective and reproducible, and > 90% of all adducted proteins were modified at only one or two sites. A core group of 125 cysteines (14% of the total) was consistently modified by both electrophiles. Selective modification of several protein domain structures and motifs indicates that certain protein families are particularly susceptible to alkylation. This approach can be extended to studies of other protein-damaging oxidants and electrophiles and can provide new insights into targets and consequences of protein damage in toxicity and disease.

Alkylation↗
PubMed2006

Systematic identification and functional screens of uncharacterized proteins associated with eukaryotic ribosomal complexes.

Translation regulation is a critical means by which cells control growth, division, and apoptosis. To gain further insight into translation and related processes, we performed multifaceted mass spectrometry-based proteomic screens of yeast ribosomal complexes and discovered an association of 77 uncharacterized yeast proteins with ribosomes. Immunoblotting revealed an EDTA-dependent cosedimentation with ribosomes in sucrose gradients for 11 candidate translation-machinery-associated (TMA) proteins. Tandem affinity purification linked one candidate, LSM12, to the RNA processing proteins PBP1 and PBP4. A second candidate, TMA46, interacted with RBG1, a GTPase that interacts with ribosomes. By adapting translation assays to high-throughput screening methods, we showed that null yeast strains harboring deletions for several of the TMA genes had alterations in protein synthesis rates (TMA7 and TMA19), susceptibility to drugs that inhibit translation (TMA7), translation fidelity (TMA20), and polyribosome profiles (TMA7, TMA19, and TMA20). TMA20 has significant sequence homology with the oncogene MCT-1. Expression of human MCT-1 in the Deltatma20 yeast mutant complemented translation-related defects, strongly implying that MCT-1 functions in translation-related processes. Together these findings implicate the TMA proteins and, potentially, their human homologs, in translation related processes.

Amino Acid Sequence↗
PubMed2003

An improved dual-expression concept, generating high-quality antibodies for proteomics research.

A novel, improved dual bacterial-expression system, designed for large-scale generation of high-quality polyclonal antibody preparations intended for proteomics research, is presented. The concept involves parallel expression of cDNA-encoded proteins, as a fusion with two different tags in two separate vector systems. Both systems enable convenient blotting procedures for expression screening on crude bacterial cell cultures and single-step affinity purification under denaturing conditions. One of the fusion proteins is used to elicit antibodies, and the second fusion protein is used in an immobilized form as an affinity ligand to enrich antibodies with selective reactivity to the cDNA-encoded part, common for the two fusion proteins. To evaluate the system, four cDNA clones from putative nuclear proteins from the non-biting midge Chironomus tentans were expressed. Antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting and immunofluorescence procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The four antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the four antibodies gave specific staining in Western-blot analysis of nuclear cell extracts. Furthermore, two of the antibody preparations gave specific staining in immunofluorescence analysis of C. tentans cells. We conclude that the dual-vector concept presented offers a highly stringent strategy for the generation of monospecific polyclonal antibodies, which are useful in proteomics research.

Animals↗
PubMed2002

A new method to estimate ligand-receptor energetics.

In the discovery of new drugs, lead identification and optimization have assumed critical importance given the number of drug targets generated from genetic, genomics, and proteomic technologies. High-throughput experimental screening assays have been complemented recently by "virtual screening" approaches to identify and filter potential ligands when the characteristics of a target receptor structure of interest are known. Virtual screening mandates a reliable procedure for automatic ranking of structurally distinct ligands in compound library databases. Computing a rank score requires the accurate prediction of binding affinities between these ligands and the target. Many current scoring strategies require information about the target three-dimensional structure. In this study, a new method to estimate the free binding energy between a ligand and receptor is proposed. We extend a central idea previously reported (Bock, J. R., and Gough, D. A. (2001) Predicting protein-protein interactions from primary structure. Bioinformatics 17, 455-460; Bock, J. R., and Gough, D. A. (2002) Whole-proteome interaction mining. Bioinformatics, in press) that uses simple descriptors to represent biomolecules as input examples to train a support vector machine (Smola, A. J., and Schölkopf, B. (1998) A Tutorial on Support Vector Regression, Neuro-COLT Technical Report NC-TR-98-030, Royal Holloway College, University of London, UK) and the application of the trained system to previously unseen pairs, estimating their propensity for interaction. Here we seek to learn the function that maps features of a receptor-ligand pair onto their equilibrium free binding energy. These features do not comprise any direct information about the three-dimensional structures of ligand or target. In cross-validation experiments, it is demonstrated that objective measurements of prediction error rate and rank-ordering statistics are competitive with those of several other investigations, most of which depend on three-dimensional structural data. The size of the sample (n = 2,671) indicates that this approach is robust and may have widespread applicability beyond restricted families of receptor types. It is concluded that newly sequenced proteins, or those for which three-dimensional crystal structures are not easily obtained, can be rapidly analyzed for their binding potential against a library of ligands using this methodology.

Drug Design↗
PubMed2006

Prediction of protein-protein interaction based on structure.

A great challenge in the proteomics and structural genomics era is to predict protein structure and function from sequence, including the identification of biological partners. The development of a procedure to construct position-specific scoring matrices for the prediction and identification of sequences with putative significant affinity faces this challenge. The local and web applications used for sequence and structure search, sequence alignment, protein modeling, molecule edition and modification, and scoring matrices construction are described in detail. The methodology is based on the information contained in structural databases and takes into account the subtle conformational and sequence details that characterize different structures within a family. Using the matrices, the protein sequence databases can be easily scanned to locate putative partners of biological significance. The success of this methodology opens the way for the prediction of protein-protein interaction at genome scale.

Algorithms↗
PubMed2004

Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry.

Since protein phosphorylation is a dominant mechanism of information transfer in cells, there is a great need for methods capable of accurately elucidating sites of phosphorylation. In recent years mass spectrometry has become an increasingly viable alternative to more traditional methods of phosphorylation analysis. The present study used immobilized metal affinity chromatography (IMAC) coupled with a linear ion trap mass spectrometer to analyze phosphorylated proteins in mouse liver. A total of 26 peptide sequences defining 26 sites of phosphorylation were determined. Although this number of identified phosphoproteins is not large, the approach is still of interest because a series of conservative criteria were adopted in data analysis. We note that, although the binding of non-phosphorylated peptides to the IMAC column was apparent, the improvements in high-speed scanning and quality of MS/MS spectra provided by the linear ion trap contributed to the phosphoprotein identification. Further analysis demonstrated that MS/MS/MS analysis was necessary to exclude the false-positive matches resulting from the MS/MS experiments, especially for multiphosphorylated peptides. The use of the linear ion trap considerably enabled exploitation of nanoflow-HPLC/MS/MS, and in addition MS/MS/MS has great potential in phosphoproteome research of relatively complex samples.

Amino Acid Sequence↗
PubMed2005

Defining putative T cell epitopes from PE and PPE families of proteins of Mycobacterium tuberculosis with vaccine potential.

The identification of T cell epitopes from immune relevant antigens of Mycobacterium tuberculosis is a critical step in the development of a vaccine covering diverse populations. Two multigene families, PE-PGRS and PPE make up about 10% of the M. tuberculosis genome. However, the functions of the proteins coded by these large numbers of genes are unknown. All possible nonameric peptide sequences from PE and PPE proteins were analysed in silico for their ability to bind to 33 alleles of class I HLA. These results reveal that of all PE and PPE proteins, a significant number of these peptides are predicted to be high-affinity HLA binders, irrespective of the length of the protein. The pathogen peptides that could behave as self or partially self-peptides in the host were eliminated using a comparative study with human proteome, thus reducing the number of peptides for analysis. The structural basis for recognition of the nonamers by the respective HLA molecules thus predicted was analyzed by molecular modeling. The structural analysis showed good correlation with the binding prediction. The analysis also led to an understanding of the binding profile of the peptides with respect to different alleles of class I HLA. The predicted epitopes can be tested experimentally for their inclusion in a potential vaccine against tuberculosis that is HLA haplotype-specific.

Alleles↗
PubMed2005

Finding the target after screening the phenotype.

Although most screening for new drug leads is being directed at known or emerging molecular targets, there has been a renaissance in screening based on changes in cell or organismal phenotypes. Phenotype-based screening is accompanied by the challenge of identifying the molecular target or targets bound by the drug leads and responsible for their pharmacological activity. A variety of technologies and approaches are being explored for target identification after phenotypic screening. Direct approaches employing affinity chromatography, expression cloning and protein microarrays analyze the compound bound to its target. Indirect approaches are based on comparison of the genome-wide activity profile of the compound with databases of the activity profiles of other compounds with known targets or activity profiles following specific genetic changes. This review will use case studies of target identification efforts and highlight the advantages and disadvantages of the various approaches to target identification after phenotypic screening.

Animals↗
PubMedDate not supplied

Phosphatase-directed phosphorylation-site determination: a synthesis of methods for the detection and identification of phosphopeptides.

Phosphopeptides can be difficult to detect and sequence by mass spectrometry (MS) due to low ionization efficiency and suppression effects in the MS mode, and insufficient fragmentation in the tandem MS (MS/MS) mode, respectively. To address this problem, we have developed a technique called Phosphatase-directed Phosphorylation-site Determination (PPD), which combines on-target phosphatase reactions, MALDI MS/MS of IMAC beads on target, and hypothesis-driven MS (HD-MS). In this method, on-target dephosphorylation experiments are conducted on IMAC-bound phosphopeptides, because dephosphorylated peptides have, in general, higher MS sensitivities than the corresponding phosphopeptides. The detected dephosphorylated peptides are sequenced by MS/MS, which identifies the potentially phosphorylated peptide and the total number of Ser, Thr, or Tyr residues that could hypothetically be phosphorylated within that peptide. On the basis of this information, a mass list containing every possible phosphorylation state of each observed peptide (where 1 HPO(3) = 80 Da) is used to direct MALDI-MS/MS on the phosphorylated peptides bound to IMAC beads at each theoretical mass from the list. If the peptide is present, the resulting MS/MS spectrum reveals the exact site(s) of phosphorylation in the peptide. We have demonstrated the applicability of PPD to the detection of in vivo phosphorylation sites on the Drosophila Stem Loop Binding Protein (dSLBP), and the complementarity of this new technique to conventional MS phosphorylation site mapping methods, since the phosphorylation sites in dSLBP could not be detected by other methods.

Amino Acid Sequence↗
PubMed2006

Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism.

Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in 22 phosphoproteins with a putative role in RNA metabolism, including splicing of mRNAs. Among these phosphoproteins, 12 Ser/Arg-rich (SR) splicing factors were identified. A conserved phosphorylation site was found in most of the phosphoproteins, including the SR proteins, suggesting that these proteins are targeted by the same or a highly related protein kinase. To test this hypothesis, Arabidopsis SR protein-specific kinase 4 (SRPK4) that was initially identified as an interactor of SR proteins was tested for its ability to phosphorylate the SR protein RSp31. In vitro kinase assays showed that all in vivo phosphorylation sites of RSp31 were targeted by SRPK4. These data suggest that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.

Amino Acid Motifs↗
PubMedDate not supplied

Evaluation of shotgun sequencing for proteomic analysis of human plasma using HPLC coupled with either ion trap or Fourier transform mass spectrometry.

This paper reports on studies directed to the characterization of the proteome of human plasma by the shotgun sequencing approach, namely the use of HPLC coupled to mass spectrometry (MS). The report will present data from two laboratories that allows the comparison of peptide and protein identifications by either accurate mass measurement on a Fourier transform mass spectrometry or MS/MS fragmentation on an ion trap mass spectrometer. Because the dynamic range of the protein components of plasma is one of the largest for a biological sample, the analysis of such a challenging sample was aided by the use of these two MS approaches. The major classes of proteins observed were transport proteins, enzymes, and enzyme inhibitors, blood-clotting factors, membrane-associated proteins including soluble forms of receptors, hormones, immunoglobulins, and other glycoproteins. The protein identifications were also highly consistent with results obtained from 2D gel studies, although a larger number of additional proteins were observed with the shotgun sequencing approach. The quantitation of low to medium level proteins was explored in the ion trap with an add-back of a known amount of human growth hormone (hGH) at a clinically relevant level (5 ug/L). The isotope coded affinity tag (ICAT) approach was used to quantitate successfully different levels of hGH in replicate analysis via the disulfide linked tryptic peptide (T6-T16). These studies suggest that the shotgun sequencing approach can be used to characterize part of the plasma proteome and serve as a starting point for the use of multidimensional analytical approaches for the analysis of complex biological samples.

Algorithms↗
PubMed2004

Comprehensive proteomic analysis of interphase and mitotic 14-3-3-binding proteins.

14-3-3 proteins regulate the cell division cycle and play a pivotal role in blocking cell cycle advancement after activation of the DNA replication and DNA damage checkpoints. Here we describe a global proteomics analysis to identify proteins that bind to 14-3-3s during interphase and mitosis. 14-3-3-binding proteins were purified from extracts of interphase and mitotic HeLa cells using specific peptide elution from 14-3-3 zeta affinity columns. Proteins that specifically bound and eluted from the affinity columns were identified by microcapillary high pressure liquid chromatography tandem mass spectrometry analysis. Several known and novel 14-3-3-interacting proteins were identified in this screen. Identified proteins are involved in cell cycle regulation, signaling, metabolism, protein synthesis, nucleic acid binding, chromatin structure, protein folding, proteolysis, nucleolar function, and nuclear transport as well as several other cellular processes. In some cases 14-3-3 binding was cell cycle-dependent, whereas in other cases the binding was shown to be cell cycle-independent. This study adds to the growing list of human 14-3-3-binding proteins and implicates a role for 14-3-3 proteins in a plethora of essential biological processes.

14-3-3 Proteins↗
PubMed2007

Computational proteomics of biomolecular interactions in the sequence and structure space of the tyrosine kinome: deciphering the molecular basis of the kinase inhibitors selectivity.

Understanding and predicting the molecular basis of protein kinases specificity against existing therapeutic agents remains highly challenging and deciphering this complexity presents an important problem in discovery and development of effective cancer drugs. We explore a recently introduced computational approach for in silico profiling of the tyrosine kinases binding specificity with a class of the pyrido-[2,3-d]pyrimidine kinase inhibitors. Computational proteomics analysis of the ligand-protein interactions using parallel simulated tempering with an ensemble of the tyrosine kinases crystal structures reveals an important molecular determinant of the kinase specificity. The pyrido-[2,3-d]pyrimidine inhibitors are capable of dynamically interacting with both active and inactive forms of the tyrosine kinases, accommodating structurally different kinase conformations with a similar binding affinity. Conformational tolerance of the protein tyrosine kinases binding with the pyrido[2,3-d]pyrimidine inhibitors provides the molecular basis for the broad spectrum of potent activities and agrees with the experimental inhibition profiles. The analysis of the pyrido[2,3-d]pyrimidine sensitivities against a number of clinically relevant ABL kinase mutants suggests an important role of conformational adaptability of multitargeted kinase inhibitors in developing drug resistance mechanisms. The presented computational approach may be useful in complementing proteomics technologies to characterize activity signatures of small molecules against a large number of potential kinase targets.

Amino Acid Motifs↗