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PubMed2007

Methods for peptide identification by spectral comparison.

BACKGROUND: Tandem mass spectrometry followed by database search is currently the predominant technology for peptide sequencing in shotgun proteomics experiments. Most methods compare experimentally observed spectra to the theoretical spectra predicted from the sequences in protein databases. There is a growing interest, however, in comparing unknown experimental spectra to a library of previously identified spectra. This approach has the advantage of taking into account instrument-dependent factors and peptide-specific differences in fragmentation probabilities. It is also computationally more efficient for high-throughput proteomics studies. RESULTS: This paper investigates computational issues related to this spectral comparison approach. Different methods have been empirically evaluated over several large sets of spectra. First, we illustrate that the peak intensities follow a Poisson distribution. This implies that applying a square root transform will optimally stabilize the peak intensity variance. Our results show that the square root did indeed outperform other transforms, resulting in improved accuracy of spectral matching. Second, different measures of spectral similarity were compared, and the results illustrated that the correlation coefficient was most robust. Finally, we examine how to assemble multiple spectra associated with the same peptide to generate a synthetic reference spectrum. Ensemble averaging is shown to provide the best combination of accuracy and efficiency. CONCLUSION: Our results demonstrate that when combined, these methods can boost the sensitivity and specificity of spectral comparison. Therefore they are capable of enhancing and complementing existing tools for consistent and accurate peptide identification.

Journal Article↗

PubMed2001

Back to the future: the human protein index (HPI) and the agenda for post-proteomic biology.

The effort to produce an index of all human proteins (the human protein index, or HPI) began twenty years ago, before the initiation of the human genome program. Because DNA sequencing technology is inherently simpler and more scalable than protein analytical technology, and because the finiteness of genomes invited a spirit of rapid conquest, the notion of genome sequencing has displaced that of protein databases in the minds of most molecular biologists for the last decade. However, now that the human genome sequence is nearing completion, a major realignment is under way that brings proteins back to the center of biological thinking. Using an influx of new and improved protein technologies--from mass spectrometry to re-engineered two-dimensional (2-D) gel systems, the original objectives of the HPI have been expanded and the time frame for its execution radically shortened. Several additional large scale technology efforts flowing from the HPI are also described.

Biotechnology↗

PubMed2004

Towards a comprehensive view of the primary structure of venom proteins from the parasitoid wasp Pimpla hypochondriaca.

Venom from the parasitoid wasp Pimpla hypochondriaca has potent in vivo activity against insect haemocytes and disrupts host immune responses. Using hybridisation techniques, and more recently random sequence analysis, we had previously identified cDNAs encoding 10 venom proteins from this wasp and deduced their primary structures. We have now extended the random sequence analysis and discovered a further nine cDNAs encoding proteins with predicted signal sequences. The mature proteins were calculated to have masses of between 4 and 22 kDa. Post-signal sequence residues predicted from the cDNAs matched those derived by Edman degradation from venom proteins separated using gel filtration and reverse phase chromatography, confirming that the cloned cDNAs encode proteins which are secreted into the venom sac. Proteins containing at least six cysteine residues were abundant and seven of these cysteine-rich venom proteins, cvp1-7, were identified. The sequences of some of these proteins were similar, or contained similar cysteine arrangements, to Kunitz type protease inhibitors, pacifastin, the trypsin inhibitor domain protein family, atracotoxin and omega-conotoxin, respectively, which occur in a diverse range of animals including spiders, molluscs, humans and grasshoppers. Two small venom proteins, svp1 and svp2, as well as cvp7 did not have similar sequences to proteins in the GenBank protein database suggesting they may be highly specialised venom components. The random sequencing approach has provided a rapid means of determining the primary structure of the majority of Pimpla hypochondriaca venom proteins.

Amino Acid Sequence↗

PubMed1997

A primer pair for amplifying part of the genome of all potyvirids by RT-PCR.

Sequence analysis was used to design a pair of degenerate oligonucleotide primers that amplified a 1.6-2.1 kbp fragment from the 3' end of the genome (virion protein gene and part of the NIb gene) of 17 species of the Potyviridae ('potyvirids'); 11 potyviruses, 2 bymoviruses, 2 macluraviruses, an ipomovirus and a rymovirus. The 'potyvirid primer 1' hybridizes to the 3' terminal poly-A region of the genome, and 'potyvirid primer 2' to the genomic region encoding the-GNNSGQ-motif of the NIb protein. Database searches showed that the potyvirid 2 primer is specific for potyvirids. Associated analyses indicated that the published amino acid sequence of part of the wheat streak mosaic rymovirus NIb protein is probably incorrect in part.

Amino Acid Sequence↗

PubMed2000

An on-line protein digestion device for rapid peptide mapping by electrospray mass spectrometry.

A simple laboratory-constructed device has been developed for fast on-line protein digestion followed by peptide mapping by use of electrospray mass spectrometry. Taking advantage of its nonsolubility properties at near-neutral pH values, pepsin could be nonpermanently attached to the PEEK tube commonly employed as transfer capillary between the syringe and the electrospray ion source. After optimization of experimental conditions such as pH, solvent, and exposure time, efficient digestion of several model proteins of molecular weights between 14,000 and 66,000, some having disulfide bridges, was successfully carried out. This technique provided reliable and reproducible sequence information by means of C-terminal-specific cleavages of aromatic and hydrophobic residues. As an application, protein identification could be achieved using a protein database search software.

Amino Acid Sequence↗

PubMed2000

Indentification of venom proteins of spider S. huwena on two-dimensional electrophoresis gel by N-terminal microsequencing and mass spectrometric peptide mapping.

Venom proteins of the spider Selenocosmia huwena were separated by two-dimensional gel electrophoresis, with the separation in the first dimension on a wide range of immobilized pH (3-10) gradients. Over 300 protein spots were presented on a silver-stained 2D gel. The protein spots with molecular weight >10 kDa were analyzed, after electrotransferring to polyvinyldene difluoride (PVDF) membrane, by N-terminal microseqencing. Some of the silver-stained protein spots with molecular weight over 10 kDa were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including (1) in-gel reduction, alkylation, and enzymatic digestion; (2) extraction and desalting by using the pipette tip containing a small C18 microcolumn (Ziptip); and (3) direct MAIDI-TOF mass analysis and protein database searching. Several known toxins such as HWTX-I, HWTX-II, HWTX-IV, and SHL-I were identified and some new components were found among these protein spots.

Animals↗

PubMed2001

Helicobacter pylori CagA: analysis of sequence diversity in relation to phosphorylation motifs and implications for the role of CagA as a virulence factor.

CagA is transported into host target cells and subsequently phosphorylated. Clearly this is a mechanism by which Helicobacter pylori could take control of one or more host cell signal transduction pathways. Presumably the end result of this interaction favors survival of H. pylori, irrespective of eventual damage to the host cell. CagA is noted for its amino acid (AA) sequence diversity, both within and outside the variable region of the molecule. The primary purpose of this review is to examine how variation in the type and number of CagA phosphorylation sites might determine the outcome of infection by different strains of H. pylori. The answer to this question could help to explain the widely disparate results obtained when H. pylori CagA status has been compared to type and severity of disease outcome in different populations, that is in different countries. Analysis of all available CagA sequences revealed that CagA contains both tyrosine phosphorylation motifs (TPMs) and cyclic-AMP-dependent phosphorylation motifs (CPMs). There are two potential CPMs near the N-terminus of CagA and at least two in the repeat region; these are not all equally well conserved. We also defined a 48-residue AA sequence, which includes the N-terminal TPM at tyrosine (Y)-122, which distinguishes between Eastern (Hong Kong-Taiwan-Japan-Thailand) H. pylori isolates and those from the West (Europe-Africa-the Americas-Australia). All 28 of the Eastern type CagA proteins have a functional N-terminal TPM whereas 11 of 47 (23.4%) of the Western type contain an inactive motif, with threonine (T) replacing the critical aspartic acid (D) residue. Only 13 of 24 (54%) known CagA sequences have an active TPM in the repeat region and only one has two TPMs in this region. The potential TPM near the C-terminus of CagA is not likely to be important since only 3 of 24 (12.5%) sequences were found to be intact. Protein database searches revealed that the AA sequence immediately following the TPM at Y-122 in CagA is homologous with a pair of PDZ domains which are common in signal transducing proteins, particularly tyrosine phosphatases. This provides a theoretical link between CagA and many of the observed responses of host cells to H. pylori. In summary, not all CagA proteins are equal in their potential for initiating host cell responses via signal transduction pathways. The degree of functional diversity of this protein depends upon which phosphorylation motifs are critical to the biological activity of CagA.

Amino Acid Motifs↗

PubMed2001

Exploring the transcriptome of the malaria sporozoite stage.

Most studies of gene expression in Plasmodium have been concerned with asexual and/or sexual erythrocytic stages. Identification and cloning of genes expressed in the preerythrocytic stages lag far behind. We have constructed a high quality cDNA library of the Plasmodium sporozoite stage by using the rodent malaria parasite P. yoelii, an important model for malaria vaccine development. The technical obstacles associated with limited amounts of RNA material were overcome by PCR-amplifying the transcriptome before cloning. Contamination with mosquito RNA was negligible. Generation of 1,972 expressed sequence tags (EST) resulted in a total of 1,547 unique sequences, allowing insight into sporozoite gene expression. The circumsporozoite protein (CS) and the sporozoite surface protein 2 (SSP2) are well represented in the data set. A BLASTX search with all tags of the nonredundant protein database gave only 161 unique significant matches (P(N) < or = 10(-4)), whereas 1,386 of the unique sequences represented novel sporozoite-expressed genes. We identified ESTs for three proteins that may be involved in host cell invasion and documented their expression in sporozoites. These data should facilitate our understanding of the preerythrocytic Plasmodium life cycle stages and the development of preerythrocytic vaccines.

Amino Acid Motifs↗

PubMed2005

BioIE: extracting informative sentences from the biomedical literature.

SUMMARY: BioIE is a rule-based system that extracts informative sentences relating to protein families, their structures, functions and diseases from the biomedical literaturE. Based on manual definition of templates and rules, it aims at precise sentence extraction rather than wide recall. After uploading source text or retrieving abstracts from MEDLINE, users can extract sentences based on predefined or user-defined template categories. BioIE also provides a brief insight into the syntactic and semantic context of the source-text by looking at word, N-gram and MeSH-term distributions. Important Applications of BioIE are in, for example, annotation of microarray data and of protein databases. AVAILABILITY: http://umber.sbs.man.ac.uk/dbbrowser/bioie/

Database Management Systems↗

PubMed1998

Computer sequence analysis of human highly conserved zinc finger modules.

We defined a sub-family of zinc finger proteins by computer analyses and comparisons of five new finger domains against protein databases. This subclass of the cysteine-cysteine/histidine-histidine motif shows additional well conserved amino acid patterns and belongs to the human kox and gli-Kruppel gene family, sharing also the same stretches of regulatory zinc finger-containing proteins of mouse and Xenopus. We particularly describe ZF6 cDNA which contains the most interesting sequence, encoding a putative multi-domain regulatory protein.

Amino Acid Sequence↗

PubMed1995

Nonreducing two-dimensional polyacrylamide gel electrophoretic analysis of human colonic proteins.

Immunochemical detection of proteins with antigenic determinants that are dependent on the native spatial conformation of the protein can often pose problems with conventional two-dimensional polyacrylamide gel electrophoresis (2-DE). For example, many antigenic determinants are readily destroyed by reducing agents and/or urea, reagents which are critical components of many of the conventional isoelectric focusing and immobilized-pH-gradient (IPG) protocols used in the first electrophoretic dimension. Here we describe the use of commercially available precast 2-DE gels for performing nonreducing/non-urea 2-DE of proteins extracted from the human colon cancer cell line LIM 1215 with 0.3% Triton X-100 that permit the identification of antigens with conformational determinants by immunoblot analysis. Previous, related studies demonstrated the usefulness of peptide-mass fingerprinting for identifying 2-DE resolved proteins. Here we show how partial protein sequence data obtained by rapid peptide mapping, using capillary column liquid chromatography directly coupled with electrospray ionization tandem mass spectrometric methodologies, enhances the usefulness of this approach for identifying incompletely resolved proteins. The nonreducing 2-DE gel images reported in this study, along with our master 2-DE gel protein database for both normal human colonic crypts and several colon-cancer-derived cell lines, and information regarding microtechniques employed in this laboratory for obtaining structural data on 2-DE resolved proteins can be accessed over the Internet using World Wide Web (URL address: http:@www.ludwig.edu.au).

Amino Acid Sequence↗

PubMed1993

Structural similarity of DNA-binding domains of bacteriophage repressors and the globin core.

BACKGROUND: In recent years, the determination of large numbers of protein structures has created a need for automatic and objective methods for the comparison of structures or conformations. Many protein structures show similarities of conformation that are undetectable by comparing their sequences. Comparison of structures can reveal similarities between proteins thought to be unrelated, providing new insight into the interrelationships of sequence, structure and function. RESULTS: Using a new tool that we have developed to perform rapid structural alignment, we present the highlights of an exhaustive comparison of all pairs of protein structures in the Brookhaven protein database. Notably, we find that the DNA-binding domain of the bacteriophage repressor family is almost completely embedded in the larger eight-helix fold of the globin family of proteins. The significant match of specific residues is correlated with functional, structural and evolutionary information. CONCLUSION: Our method can help to identify structurally similar folds rapidly and with high-sensitivity, providing a powerful tool for analyzing the ever-increasing number of protein structures being elucidated.

Journal Article↗

PubMed2005

Immunochemical and proteomic analysis of covalent adducts formed by quinone methide tumor promoters in mouse lung epithelial cell lines.

Two quinone methide (QM) metabolites of the phenolic antioxidant butylated hydroxytoluene (BHT), 2,6-di-tert-butyl-4-methylenecyclohexa-2,5-dienone (BHT-QM) and the tert-butyl-hydroxylated derivative (BHTOH-QM), are believed to be responsible for promoting lung tumor formation in mice treated with BHT. QMs are strongly electrophilic and undergo Michael type additions with nucleophiles at the exocyclic methylene to form benzylic thioether adducts. Our goal was to identify intracellular protein targets of these QMs in order to gain insight into their effects on tumorigenesis. Cell line E10 of mouse lung epithelial origin and its spontaneous transformant, the tumorigenic E9 cell line, were treated with BHT-QM or BHTOH-QM, and cellular proteins were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Adducted proteins were detected on western blots with polyclonal antibodies developed to a conjugate of BHTOH-QM that recognized adducts of both QMs bound to thiol groups of Cys and side chain amino groups of Lys and His residues. Tryptic digests of immunoreactive proteins were analyzed by HPLC mass spectrometry (LC/MS) and identified by searching protein databases using MS/MS data. In a few cases, adducted peptides in these digests were detected by matrix-assisted laser desorption/ionization time-of-flight MS. A total of 37 immunoreactive proteins were identified including proteins involved in carbohydrate metabolism, nucleic acid synthesis, and RNA and protein processing, in addition to several cytoskeletal and stress-related proteins. About half of the protein adducts were found in both cell lines. Adducts detected only in transformed E9 cells include glutathione S-transferase P1, peroxiredoxin 2, nucleoside diphosphate kinase, and vinculin, whereas several alkylated cytoskeletal proteins such as tubulins, vimentin, calvasculin, and calcyclin were detected exclusively in E10 cells. Several of the proteins modified by BHT-derived QMs have been implicated in various aspects of tumorigenesis and are excellent candidates for further study into the consequences of alkylation on cellular transformation.

Animals↗

PubMed2002

Global analysis of outer membrane proteins from Leptospira interrogans serovar Lai.

Recombinant leptospiral outer membrane proteins (OMPs) can elicit immunity to leptospirosis in a hamster infection model. Previously characterized OMPs appear highly conserved, and thus their potential to stimulate heterologous immunity is of critical importance. In this study we undertook a global analysis of leptospiral OMPs, which were obtained by Triton X-114 extraction and phase partitioning. Outer membrane fractions were isolated from Leptospira interrogans serovar Lai grown at 20, 30, and 37 degrees C with or without 10% fetal calf serum and, finally, in iron-depleted medium. The OMPs were separated by two-dimensional gel electrophoresis. Gel patterns from each of the five conditions were compared via image analysis, and 37 gel-purified proteins were tryptically digested and characterized by mass spectrometry (MS). Matrix-assisted laser desorption ionization-time-of-flight MS was used to rapidly identify leptospiral OMPs present in sequence databases. Proteins identified by this approach included the outer membrane lipoproteins LipL32, LipL36, LipL41, and LipL48. No known proteins from any cellular location other than the outer membrane were identified. Tandem electrospray MS was used to obtain peptide sequence information from eight novel proteins designated pL18, pL21, pL22, pL24, pL45, pL47/49, pL50, and pL55. The expression of LipL36 and pL50 was not apparent at temperatures above 30 degrees C or under iron-depleted conditions. The expression of pL24 was also downregulated after iron depletion. The leptospiral major OMP LipL32 was observed to undergo substantial cleavage under all conditions except iron depletion. Additionally, significant downregulation of these mass forms was observed under iron limitation at 30 degrees C, but not at 30 degrees C alone, suggesting that LipL32 processing is dependent on iron-regulated extracellular proteases. However, separate cleavage products responded differently to changes in growth temperature and medium constituents, indicating that more than one process may be involved in LipL32 processing. Furthermore, under iron-depleted conditions there was no concomitant increase in the levels of the intact form of LipL32. The temperature- and iron-regulated expression of LipL36 and the iron-dependent cleavage of LipL32 were confirmed by immunoblotting with specific antisera. Global analysis of the cellular location and expression of leptospiral proteins will be useful in the annotation of genomic sequence data and in providing insight into the biology of Leptospira.

Amino Acid Sequence↗

PubMed1988

Myristoyl CoA:protein N-myristoyltransferase activities from rat liver and yeast possess overlapping yet distinct peptide substrate specificities.

A variety of eukaryotic viral and cellular proteins possesses an NH2-terminal N-myristoylglycine residue important for their biological functions. Recent studies of the primary structural requirements for peptide substrates of the enzyme responsible for this modification in yeast demonstrated that residues 1, 2, and 5 play a critical role in enzyme: ligand interactions (Towler, D. A., Adams, S. P., Eubanks, S. R., Towery, D. S., Jackson-Machelski, E., Glaser, L., and Gordon J. I. (1987b) Proc. Natl. Acad. Sci. U. S. A. 84, 2708-2812). This was determined by examining as substrates a series of synthetic peptides whose sequences were systematically altered from a "parental" peptide derived from the known N-myristoylprotein bovine heart cyclic AMP-dependent protein kinase (A kinase) catalytic subunit. We have now extended these studies in order to examine structure/activity relationships in the COOH-terminal regions of octapeptide substrates of yeast N-myristoyltransferase (NMT). The interaction between yeast NMT and the side chain of residue 5 in peptide ligands is apparently sterically constrained, since Thr5 is unable to promote the very high affinity binding observed with a Ser5 substitution. A substrate hexapeptide core has been defined which contains much of the information necessary for recognition by this lower eukaryotic NMT. Addition of COOH-terminal basic residues to this hexapeptide enhances peptide binding, while COOH-terminal acidic residues destabilize NMT: ligand interactions. Based on the results obtained from our in vitro studies of over 80 synthetic peptides and yeast NMT, we have identified a number of potential N-myristoylproteins from searches of available protein databases. These include hepatitis B virus pre-S1, human SYN-kinase, rodent Gi alpha, and bovine transducin-alpha. Peptides corresponding to the NH2-terminal sequences of these proteins and several known N-myristoylproteins were assayed using yeast NMT as well as partially purified rat liver NMT. While a number of the synthetic peptides exhibited similar catalytic properties with the yeast and mammalian enzymes, surprisingly, the SYN-kinase, Gi alpha, and transducin-alpha peptides were N-myristoylated by rat NMT but not by yeast NMT. This suggests that either multiple NMT activities exist in rat liver or the yeast and rodent enzymes have similar but distinct peptide substrate specificities.

Acyl Coenzyme A↗

PubMed1999

Comparative modeling of CASP3 targets using PSI-BLAST and SCWRL.

We present results of comparative modeling on 11 targets from the CASP3 experiment. Our methods comprise the following steps: first, PSI-BLAST is used to find homologues of the target sequence in the nonredundant GenBank protein sequence database; second, after several iterations of PSI-BLAST, the resulting profile or position-specific similarity matrix is used to search a database of Protein Databank (PDB) sequences; third, from the list of hits resulting from the PDB search, a parent structure is chosen on the basis of the quality of the alignment and the quality of the experimental structure; fourth, this alignment is adjusted manually whenever insertions or deletions take place in secondary structure regions of the parent; fifth, the backbone is modeled from the parent structure and the alignment; and finally, the program SCWRL is used to replace nonconserved side chains onto the parent backbone given the target sequence. For comparison, we also produced structural models from the unaltered PSI-BLAST alignment, from an alignment from the nonprofile version of BLAST, and from the global sequence alignment program CLUSTAL W. Our results indicate that PSI-BLAST produced considerably better alignments than would be possible with either global or local pairwise sequence alignment algorithms and that manual adjustments were helpful. SCWRL, which uses a backbone-dependent rotamer library to predict side-chain conformations, did well in comparison with other methods used in CASP3.

Algorithms↗

PubMed1999

A large scale structural analysis of cDNAs in a unicellular green alga, Chlamydomonas reinhardtii. I. Generation of 3433 non-redundant expressed sequence tags.

To understand genetic information carried in a unicellular green alga, Chlamydomonas reinhardtii, normalized and size-selected cDNA libraries were constructed from cells at photoautotrophic growth, and a total of 11,571 5'-end sequence tags were established. These sequences were grouped into 3433 independent EST species. Similarity search against the public non-redundant protein database indicated that 817 groups showed significant similarity to registered sequences, of which 140 were of previously identified C. reinhardtii genes, but the remaining 2616 species were novel sequences. The coverage of full-length protein coding regions was estimated to be over 60%. These cDNA clones and EST sequence information will provide a powerful source for the future genome-wide functional analysis of uncharacterized genes.

Animals↗

PubMed2004

Computational identification and characterization of novel genes from legumes.

The Fabaceae, the third largest family of plants and the source of many crops, has been the target of many genomic studies. Currently, only the grasses surpass the legumes for the number of publicly available expressed sequence tags (ESTs). The quantity of sequences from diverse plants enables the use of computational approaches to identify novel genes in specific taxa. We used BLAST algorithms to compare unigene sets from Medicago truncatula, Lotus japonicus, and soybean (Glycine max and Glycine soja) to nonlegume unigene sets, to GenBank's nonredundant and EST databases, and to the genomic sequences of rice (Oryza sativa) and Arabidopsis. As a working definition, putatively legume-specific genes had no sequence homology, below a specified threshold, to publicly available sequences of nonlegumes. Using this approach, 2,525 legume-specific EST contigs were identified, of which less than three percent had clear homology to previously characterized legume genes. As a first step toward predicting function, related sequences were clustered to build motifs that could be searched against protein databases. Three families of interest were more deeply characterized: F-box related proteins, Pro-rich proteins, and Cys cluster proteins (CCPs). Of particular interest were the >300 CCPs, primarily from nodules or seeds, with predicted similarity to defensins. Motif searching also identified several previously unknown CCP-like open reading frames in Arabidopsis. Evolutionary analyses of the genomic sequences of several CCPs in M. truncatula suggest that this family has evolved by local duplications and divergent selection.

Algorithms↗