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Tissue-specific Proteogenomic Analysis of Plutella xylostella Larval Midgut Using a Multialgorithm Pipeline*

https://doi.org/10.1074/mcp.M115.050989Get rights and content
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The diamondback moth, Plutella xylostella (L.), is the major cosmopolitan pest of brassica and other cruciferous crops. Its larval midgut is a dynamic tissue that interfaces with a wide variety of toxicological and physiological processes. The draft sequence of the P. xylostella genome was recently released, but its annotation remains challenging because of the low sequence coverage of this branch of life and the poor description of exon/intron splicing rules for these insects. Peptide sequencing by computational assignment of tandem mass spectra to genome sequence information provides an experimental independent approach for confirming or refuting protein predictions, a concept that has been termed proteogenomics. In this study, we carried out an in-depth proteogenomic analysis to complement genome annotation of P. xylostella larval midgut based on shotgun HPLC-ESI-MS/MS data by means of a multialgorithm pipeline. A total of 876,341 tandem mass spectra were searched against the predicted P. xylostella protein sequences and a whole-genome six-frame translation database. Based on a data set comprising 2694 novel genome search specific peptides, we discovered 439 novel protein-coding genes and corrected 128 existing gene models. To get the most accurate data to seed further insect genome annotation, more than half of the novel protein-coding genes, i.e. 235 over 439, were further validated after RT-PCR amplification and sequencing of the corresponding transcripts. Furthermore, we validated 53 novel alternative splicings. Finally, a total of 6764 proteins were identified, resulting in one of the most comprehensive proteogenomic study of a nonmodel animal. As the first tissue-specific proteogenomics analysis of P. xylostella, this study provides the fundamental basis for high-throughput proteomics and functional genomics approaches aimed at deciphering the molecular mechanisms of resistance and controlling this pest.

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Author contribution: XZ, SX and YZ designed the study; XZ, ZG, JX and SK performed the experiments; XZ and SX analyzed the data; XZ wrote the manuscript; QW, SW, WX, JA and RH revised the manuscript. All authors read and approved the final version of manuscript.

Authors declare no conflicts of interests exist.

*

This research was supported by the Agricultural Science and Technology Innovation Program (ASTIP), the National Natural Science Foundation of China (30871659), the 863 Program (2012AA101502), the Special Fund for Agro-scientific Research in the Public Interest (201203038) and Beijing Key Laboratory for Pest Control and Sustainable Cultivation of Vegetables.

This article contains supplemental material.

1

The abbreviations used are:

    EST

    expressed sequence tag

    FDR

    False discovery rate

    MS/MS

    Tandem mass spectrometry

    PSM

    Peptide spectrum match

    GSSP

    Genome search specific peptide.

**

These authors contributed equally to this article.