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The development of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has made it possible to characterize phosphopeptides in an increasingly large-scale and high-throughput fashion. However, extracting confident phosphopeptide identifications from the resulting large data sets in a similar high-throughput fashion remains difficult, as does rigorously estimating the false discovery rate (FDR) of a set of phosphopeptide identifications. This article describes a data analysis pipeline designed to address these issues. The first step is to reanalyze phosphopeptide identifications that contain ambiguous assignments for the incorporated phosphate(s) to determine the most likely arrangement of the phosphate(s). The next step is to employ an expectation maximization algorithm to estimate the joint distribution of the peptide scores. A linear discriminant analysis is then performed to determine how to optimally combine peptide scores (in this case, from SEQUEST) into a discriminant score that possesses the maximum discriminating power. Based on this discriminant score, the p- and q-values for each phosphopeptide identification are calculated, and the phosphopeptide identification FDR is then estimated. This data analysis approach was applied to data from a study of irradiated human skin fibroblasts to provide a robust estimate of FDR for phosphopeptides. The Phosphopeptide FDR Estimator software is freely available for download at http://ncrr.pnl.gov/software/.  相似文献   
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Background

Recent advances in DNA sequencing techniques resulted in more than forty sequenced plant genomes representing a diverse set of taxa of agricultural, energy, medicinal and ecological importance. However, gene family curation is often only inferred from DNA sequence homology and lacks insights into evolutionary processes contributing to gene family dynamics. In a comparative genomics framework, we integrated multiple lines of evidence provided by gene synteny, sequence homology and protein-based Hidden Markov Modelling to extract homologous super-clusters composed of multi-domain resistance (R)-proteins of the NB-LRR type (for NUCLEOTIDE BINDING/LEUCINE-RICH REPEATS), that are involved in plant innate immunity.

Results

To assess the diversity of R-proteins within and between species, we screened twelve eudicot plant genomes including six major crops and found a total of 2,363 NB-LRR genes. Our curated R-proteins set shows a 50% average for tandem duplicates and a 22% fraction of gene copies retained from ancient polyploidy events (ohnologs). We provide evidence for strong positive selection and show significant differences in molecular evolution rates (Ka/Ks-ratio) among tandem- (mean = 1.59), ohnolog (mean = 1.36) and singleton (mean = 1.22) R-gene duplicates. To foster the process of gene-edited plant breeding, we report species-specific presence/absence of all 140 NB-LRR genes present in the model plant Arabidopsis and describe four distinct clusters of NB-LRR “gatekeeper” loci sharing syntenic orthologs across all analyzed genomes.

Conclusion

By curating a near-complete set of multi-domain R-protein clusters in an eudicot-wide scale, our analysis offers significant insight into evolutionary dynamics underlying diversification of the plant innate immune system. Furthermore, our methods provide a blueprint for future efforts to identify and more rapidly clone functional NB-LRR genes from any plant species.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-966) contains supplementary material, which is available to authorized users.  相似文献   
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