Peptide identification using peptide amino acid attribute vectors

We describe the theoretical basis for a peptide identification method wherein peptides are represented as vectors based on their amino acid composition and grouped into clusters. Unknown peptides are identified by finding the database cluster and peptide entries with the shortest Euclidian distance. We demonstrate that the amino acid composition of peptides is virtually as informative as the sequence and allows rapid peptide identification more accurately than peptide mass alone.     

Structure based function prediction of proteins using fragment library frequency vectors

The function of the protein is primarily dictated by its structure. Therefore it is far more logical to find the functional clues of the protein in its overall 3-dimensional fold or its global structure. In this paper, we have developed a novel Support Vector Machines (SVM) based prediction model for functional classification and prediction of proteins using features extracted from its global structure based on fragment libraries. Fragment libraries have been previously used for abintio modelling of proteins and protein structure comparisons. The query protein structure is broken down into a collection of short contiguous backbone fragments and this collection is discretized using a library of fragments. The input feature vector is frequency vector that counts the number of each library fragment in the collection of fragments by all-to-all fragment comparisons. SVM models were trained and optimised for obtaining the best 10-fold Cross validation accuracy for classification. As an example, this method was applied for prediction and classification of Cell Adhesion molecules (CAMs). Thirty-four different fragment libraries with sizes ranging from 4 to 400 and fragment lengths ranging from 4 to 12 were used for obtaining the best prediction model. The best 10-fold CV accuracy of 95.25% was obtained for library of 400 fragments of length 10. An accuracy of 87.5% was obtained on an unseen test dataset consisting of 20 CAMs and 20 NonCAMs. This shows that protein structure can be accurately and uniquely described using 400 representative fragments of length 10.     

Species identification using a small nuclear gene fragment: application to sympatric wild carnivores from South-western Europe

Species identification is essential for non-invasive studies of elusive and rare animals, and for detecting illegal harvest or trade of wildlife species. However, most molecular tests identify only a limited number of species or require multiple laboratory steps to distinguish many taxa. Additionally, most protocols use mitochondrial DNA being, therefore, especially prone to problems such as nuclear insert copies, high intraspecific diversity or heteroplasmy. Here, we developed a molecular test based on the polymorphisms detected on a small nuclear gene fragment (221 bp of the IRBP -Interphotoreceptor Retinoid-Binding Protein- exon 1). This fragment revealed 51 variable sites (including 12 non-synonymous and 19 species-specific sites), which enabled the successful distinction of all 16 carnivore species native to South-western Europe. A SSCP (Single-Strand Conformational Polymorphism) gel electrophoresis technique was also optimized to allow the simple and inexpensive application of this molecular test. Sequences and SSCP profiles were consistent in identifying a total of 387 samples, including faeces (172) and hairs (17) collected non-invasively in the field. Due to its low cost, simplicity, and wide range of identifiable species, this test shows great promise to facilitate studies in molecular ecology, conservation genetics, and forensic analysis, as well as DNA bar-coding projects.     

Species identification in seven small millet species using polymerase chain reaction-restriction fragment length polymorphism of trnS-psbC gene region.

The chloroplast trnS-psbC gene regions from total genomic DNA of 119 accessions from seven small millet species were amplified by polymerase chain reaction (PCR) and digested with eight restriction enzymes individually as well as in combinations of two enzymes to generate restriction fragment length polymorphism (PCR-RFLP). PCR-RFLP with individual enzymes revealed polymorphism between only some species. However, all the species could be distinguished by using a combination of two enzymes, specifically HaeIII and MspI. PCR-RFLP of 11 to 20 accessions with the same enzyme combination showed no intraspecific variation, which established that the differential banding patterns were species specific. In contrast, the same enzyme combination was not useful for differentiating different species of the genera Cajanus, Rhyncosia, Abies, Rhizophora, Ceriops, and Bruguiera, and it also revealed intraspecies variation in three species of Abies. The present study indicated that digestion of trnS-psbC with two four-base recognizing enzymes reveals more variation than with either enzyme alone and that it may be a method of choice for species identification in some genera.     

Exploiting the kernel trick to correlate fragment ions for peptide identification via tandem mass spectrometry

MOTIVATION: The correlation among fragment ions in a tandem mass spectrum is crucial in reducing stochastic mismatches for peptide identification by database searching. Until now, an efficient scoring algorithm that considers the correlative information in a tunable and comprehensive manner has been lacking. RESULTS: This paper provides a promising approach to utilizing the correlative information for improving the peptide identification accuracy. The kernel trick, rooted in the statistical learning theory, is exploited to address this issue with low computational effort. The common scoring method, the tandem mass spectral dot product (SDP), is extended to the kernel SDP (KSDP). Experiments on a dataset reported previously demonstrate the effectiveness of the KSDP. The implementation on consecutive fragments shows a decrease of 10% in the error rate compared with the SDP. Our software tool, pFind, using a simple scoring function based on the KSDP, outperforms two SDP-based software tools, SEQUEST and Sonar MS/MS, in terms of identification accuracy. SUPPLEMENTARY INFORMATION: http://www.jdl.ac.cn/user/yfu/pfind/index.html     

Ultra-high intra-spectrum mass accuracy enables unambiguous identification of fragment reporter ions in isobaric multiplexed quantitative proteomics

Isobaric tagging using reagents such as tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ) have become popular tools for mass spectrometry based quantitative proteomics. Because the peptide quantification information is collected in tandem mass spectra, the accuracy and precision of this method largely depend on the resolution with which precursor ions can be selected for the fragmentation and the specificity of the generated reporter ion. The latter can constitute an issue if near isobaric ion signals are present in such spectra because they may distort quantification results. We propose a simple remedy for this problem by identifying reporter ions via the accurate mass differences within a single tandem mass spectrum instead of applying fixed mass error tolerances for all tandem mass spectra. Our results show that this leads to unambiguous reporter ion identification and complete removal of interfering signals. This mode of data processing is easily implemented in software and offers advantages for protein quantification based on few peptides.     

Peptide identification quality control

Identification of large proteomics data sets is routinely performed using sophisticated software tools called search engines. Yet despite the importance of the identification process, its configuration and execution is often performed according to established lab habits, and is mostly unsupervised by detailed quality control. In order to establish easily obtainable quality control criteria that can be broadly applied to the identification process, we here introduce several simple quality control methods. An unbiased quality control of identification parameters will be conducted using target/decoy searches providing significant improvement over identification standards. MASCOT identifications were for instance increased by 13% at a constant level of confidence. The target/decoy approach can however not be universally applied. We therefore also quality control the application of this strategy itself, providing useful and intuitive metrics for evaluating the precision and robustness of the obtained false discovery rate.     

Rapid identification of Giardia duodenalis assemblages in NSW using terminal-restriction fragment length polymorphism

Humans are infected by 2 genetic assemblages (A and B) of Giardia duodenalis, a protozoan parasite that causes gastro-intestinal disease. Sub-assemblages AI, AII, BIII and BIV are commonly identified in human cases. Detection requires amplification of G. duodenalis loci. Subsequent DNA sequencing or restriction fragment length polymorphism (RFLP) identifies sub-assemblages but is expensive (DNA sequencing) or insensitive (RFLP). This study investigated a fluorescence-based detection method, using terminal-restriction fragment length polymorphism (T-RFLP) of the glutamate dehydrogenase gene to characterize human infections. Clinical samples (n=73), positive for Giardia were collected in New South Wales, Australia, and were used to evaluate T-RFLP detection. The accuracy and sensitivity of T-RFLP detection was established by comparison to DNA sequencing and RFLP. Sub-assemblage assignment by T-RFLP identified BIV as the common subtype in N.S.W cases, whilst AI, AII and BIII were also detected. When compared to DNA sequencing and RFLP, analysis by T-RFLP was a reliable and reproducible method. Automated fluorescent detection enabled accurate sizing of restriction fragments and provided a sensitive alternative to RFLP. Discrimination of sub-assemblages by T-RFLP was comparable to DNA sequencing, but was efficient and inexpensive. The protocol described here provides a rapid and sensitive diagnostic tool for routine sample screenings in epidemiological research.     

Solution Structure of an Immunoactive Peptide fragment of Staphylococcal Protein-A

Abstract

Staphylococcal protein-A (SpA) is known to bind the Fc fragment of immunoglobin G in vitro and induce a myriad of immunogenic responses in vivo. The latter is ascribed to be due to the interaction of Fc and SpA. It has also been proposed that in vivo proteolytically cleaved fragments of SpA may be functioning in the same manner. One such fragment (EQQNAFYEILHLPNLNEEQR), fragment 8–27 of the B-domain (SpA-B), was recently shown to exhibit in vivo immunogenic response [Sinha, P., Sengupta, J., and Ray, P. K. (1999) Biochem. Biophys. Res. Commun. 258, 141–147]. As a first step towards understanding the mode of interaction of this peptide with the Fc fragment, we have studied the solution conformation of this isolated peptide by CD and NMR. The peptide, with 7 contact residues in the crystal structure of the SpA-B/Fc complex and comprising of mostly helixI and part of helixII of the 3-helix bundle of SpA-B, was found to be present predominantly in extended structure. However it showed nascent turn/helix like conformations around F14 & Y15. These two residues are known to play a vital role in SpA-B/Fc interaction as deciphered from crystal structure and NMR studies of SpA-B/Fc complex and mutational studies. The implications of our results, especially the nascent conformations found around F14 & Y15, in design of SpA-B mimetic small molecules are discussed.     

Commentary on Automatic identification of Chagas disease vectors using data mining and deep learning techniques

We appreciate Gurgel-Goncalves et al. for offering their insights and opinions on our research on the automatic identification of vectors for Chagas disease. Our reply will address the key points raised and provide clarification on the following topics: (1) expert supervision, (2) labeling of the dataset, (3) recognition of the results from benchmark methods, (4) quality of training images, and (5) balancing of data.     

Effects of cations on small fragment of DNA polymerase I using a novel FRET assay

DNA polymerase I （Poll） digested by protease produces a small fragment （SF） containing 5~--~3~ exonuclease activity. The 5~-~3＇ exonuclease activity of poll cleaves the down- stream RNA primer strands during DNA replication in vivo. Previous in vitro studies suggested its capability of cleaving duplex from 5＇ terminal and a flap-structure-spe- cific endonuclease activity. From the crystal structures of other nucleases and biochemical data, a two-metal-ion mechanism has been proposed but has not been deter- mined. In this study, we cloned, expressed, and purified the SF protein, and established a novel fluorescence resonance energy transfer （FRET） assay to analyze the catalytic activ- ity of the SF protein. The effects of several metal ions on its catalytic capability were analyzed using this FRET assay. Results showed that Mg2＋, Mn2＋, and Zn2＋ were able to activate the cleavage of SF, while Ca2＋, Ni2＋, and Co2＋ were not suitable for SF catalysis. The effects of K＋, Na＋, and dNTP were also determined.     

Misleading gene conversion frequencies due to a PCR artifact using small fragment homologous replacement

Recent studies have reported successful correction of the most common F508del mutation in cystic fibrosis (CF) airway epithelial cells by small fragment homologous replacement (SFHR). We wished to apply the SFHR methodology to our CF bronchial epithelial cells, of compound heterozygous genotype (F508del/W1282X), in which nucleic acid transfer was previously optimized by electroporation. Using a PCR-based detection methodology, with one of the primers located outside the SFHR homology region, we obtained SFHR dose-dependent F508del to wild-type CFTR gene conversion frequencies reaching 30%. However, the increased wild-type/F508del CFTR allele ratio was transient, vanishing at 5 days posttransfection. Furthermore, we have been unable to reproduce the SFHR-mediated repair of the F508del mutation in our cellular model when both detection primers were located outside the SFHR homology region. A thorough reexamination of our initial detection strategy revealed that a false positive result was originated from a PCR artifact created by the SFHR fragment itself. Thus, nonamplifiable detection methods, such as Southern blotting, protein analysis, or functional assays, should be performed, whenever possible, to correctly assess gene conversion frequencies.     

The occurrence of peptaibols and structurally related peptaibiotics in fungi and their mass spectrometric identification via diagnostic fragment ions.

Peptaibols and related peptide antibiotics (peptaibiotics) display diagnostically useful fragmentation patterns during mass spectrometry (FAB-MS, ESI-CID-MS/MS and CID-MSn]. The paper compiles fragmentation data of pseudo-molecular ions reported in the literature as a guide to the rational identification of recurrently isolated and new peptaibols and peptaibiotics. Taxonomic and ecological aspects of microorganisms producing peptaibols and peptaibiotics are discussed.     

BCG identification by DNA restriction fragment patterns

Seven daughter strains of BCG were characterized by restriction fragment analysis with the enzymes BstEII, PvuII and BclI. Comparisons of fragment patterns confirmed that BCG is correctly classified as a Mycobacterium bovis variant and suggested that the Swedish strain is most closely related to the original BCG strain.     

Non-invasive genetic identification of small mammal species using real-time polymerase chain reaction

DNA identification of non-invasive samples is a potentially useful tool for monitoring small mammal species. Here we describe a novel method for identifying five small mammal species: wood mouse, bank vole, common shrew, pygmy shrew and water shrew. Species-specific real-time polymerase chain reaction primers were designed to amplify fragments of the mitochondrial cytochrome b gene from hair and scat samples. We also amplified nuclear DNA from scats, demonstrating their potential as a source of DNA for population genetic studies.     

An algorithm for identification of CTX-M-type beta-lactamase genes using restriction fragment length polymorphism analysis of PCR-product

The algorithm of the identification of the bla(CTX-M) genes coding CTX-M-type beta-lactamases providing resistance to cephalosporins III-IV was developed. This algorithm provides identification of 49 genes of 96 genes presented in the GenBank database so far. Remaining 47 genes can be identified as consisting of small sub-groups composed of 2-6 genes with the exception of sub-group of the bla(CTX-M-14)-like genes composed of 13 genes. The identification of the bla(CTX-M) genes is based on two-step restriction fragment length polymorphism analysis of 544 bp PCR-product (PCR-RFLP). In the first step, determination of subtype (cluster) of the bla(CTX-M) gene occurred using the restriction nuclease Alu I: cluster 1, -2, -8, -9 or -25. Moreover, four genes can be identified just at this step: bla(CTX-M)-59, (cluster 2); bla(CTX-M-63) (cluster 8), bla(CTX-M-45) (cluster 9), and bla(CTX-M-78) (hybrid gene between cluster 2 and cluster 25). At the second step gene identification goes on inside of each cluster separately using a set of 26 restriction nucleases. As a result of the PCR-RFLP-analysis, 23 bla(CTX-M) genes can be identified at the cluster 1, 11 genes--at the cluster 2, 4 genes--at the cluster 8, 9 genes--at the cluster 9, 1 gene--at the cluster 25, and 2 hybrid genes: bla(CTX-M-78) (between clusters 2 and 25), and bla(CTX-M-64) (between clusters 1 and 9). The described algorithm was used for identification of the blac(CTX-M) genes (n = 585) detected in Enterobacteriaceae nosocomial isolates (n = 877), collected from Russial hospitals in 2003-2007. It was shown that major genes belonged to cluster 1 (n = 543), namely--bla(CTX-M-15) gene (n = 515), bla(CTX-M-3) (n = 25), bla(CTX-M-22) (n = 1), bla(CTX-M-23) (n = 1), and bla(CTM-34) (n = 1). Moreover, the genes atributed to cluster 2 were identified: bla(CTX-M-2) (n = 1), and bla(CTX-M-5) (n = 4); and genes belonged to cluster 9: bla(CTX-M-9) (n = 2), and bla(CTX-M-14) (n = 35).     

Identification of a network involved in thapsigargin-induced apoptosis using a library of small interfering RNA expression vectors

We describe here the construction of a library of small interfering RNA expression vectors targeted to a few hundred apoptosis-related genes and the application of this library to an investigation of thapsigargin (TG)-induced apoptosis. Thapsigargin triggers endoplasmic reticulum stress, with subsequent apoptosis, but the molecular mechanisms underlying this process are incompletely understood. Using our library, we identified three anti-apoptotic genes, namely, NOXA, E2F1, and MAPK1, in addition to already characterized genes in the apoptotic pathway. In contrast to proposals by others, our data revealed (i) that TG-induced apoptosis is associated with Apaf1 in a caspase-3- and caspase-9-independent manner; (ii) that the E2F1-PUMA pathway might be involved; and (iii) that the ERK pathway, via MAP3K8 (mitogen-activated protein kinase kinase 8), is required for the induction by TG of apoptosis. Our study demonstrates clearly that unexpected and novel genes can be identified effectively by our method, and it provides evidence for the efficacy and utility of the comprehensive analysis of signaling networks and pathways using a library of small interfering RNA expression vectors.     

不同长度茎部shRNA表达载体构建与鉴定

本研究针对同一目的基因设计构建不同茎部长度的shRNA表达载体,并对其在细胞及胚胎水平的干扰效应做一比较。以绿色荧光蛋白基因为沉默效应的靶基因,设计茎部长度分别为21bp、27bp、29bp的干扰片段,退火后连入带有H6启动子的真核表达载体psiSTRIKE中(分别命名为EGFP-21siRNA、EGFP-27siRNA和EGFP-29siRNA),将构建成功的载体以脂质体法转染小鼠胚胎成纤维细胞,利用荧光定量PCR对其荧光表达进行精确定量。不同茎部长度的shRNA载体均使绿色荧光蛋白基因表达降低,茎部为29bp时比21bp、27bp表现出更明显的沉默效应。细胞水平沉默效应的初步验证,为筛选适合小鼠个体水平的最佳发夹结构奠定了基础。