Shotgun Identification of the Structural Proteome of Shrimp White Spot Syndrome Virus and iTRAQ Differentiation of Envelope and Nucleocapsid Subproteomes

Zhengjun Li,Qingsong Lin,Jing Chen,Jin Lu Wu,Teck Kwang Lim,Siew See Loh,Xuhua Tang,Choy-Leong Hew

doi:10.1074/mcp.m600327-mcp200

Zhengjun Li, Qingsong Lin + Show 6 more

Open Access

https://doi.org/10.1074/mcp.m600327-mcp200

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Abstract

White spot syndrome virus (WSSV) is a major pathogen that causes severe mortality and economic losses to shrimp cultivation worldwide. The genome of WSSV contains a 305-kb double-stranded circular DNA, which encodes 181 predicted ORFs. Previous gel-based proteomics studies on WSSV have identified 38 structural proteins. In this study, we applied shotgun proteomics using off-line coupling of an LC system with MALDI-TOF/TOF MS/MS as a complementary and comprehensive approach to investigate the WSSV proteome. This approach led to the identification of 45 viral proteins; 13 of them are reported for the first time. Seven viral proteins were found to have acetylated N termini. RT-PCR confirmed the mRNA expression of these 13 newly identified viral proteins. Furthermore iTRAQ (isobaric tags for relative and absolute quantification), a quantitative proteomics strategy, was used to distinguish envelope proteins and nucleocapsid proteins of WSSV. Based on iTRAQ ratios, we successfully identified 23 envelope proteins and six nucleocapsid proteins. Our results validated 15 structural proteins with previously known localization in the virion. Furthermore the localization of an additional 12 envelope proteins and two nucleocapsid proteins was determined. We demonstrated that iTRAQ is an effective approach for high throughput viral protein localization determination. Altogether WSSV is assembled by at least 58 structural proteins, including 13 proteins newly identified by shotgun proteomics and one identified by iTRAQ. The localization of 42 structural proteins was determined; 33 are envelope proteins, and nine are nucleocapsid proteins. A comprehensive identification of WSSV structural proteins and their localization should facilitate the studies of its assembly and mechanism of infection.

Highlights

White spot syndrome virus (WSSV) is a major pathogen that causes severe mortality and economic losses to shrimp cultivation worldwide
Identification of Virion-associated Proteins by Shotgun Proteomics—The purity of isolated WSSV virions using sucrose gradient separation was confirmed by electron microscopy
(wsv198, wsv216, wsv230, wsv238, wsv242, wsv256, and wsv289). Because these proteins have been confidently identified as viral structural proteins by the shotgun proteomics study and the peptides matched were all top ranked in the iTRAQ study, these protein assignments should be reliable

Summary

EXPERIMENTAL PROCEDURES

Proliferation and Isolation of WSSV Virions—WSSV used in this study originated from WSSV-infected Penaeus chinensis (China isolate). One portion was set aside as a control for total viral proteins, whereas the other portion was separated into two fractions, supernatant and pellet, by centrifugation at 200,000 ⫻ g for 1 h at 4 °C. The LC fractions were mixed with MALDI matrix solution in a flow rate of 5.4 ␮l/min through a 25-nl mixing tee (Upchurch Scientific, Oak Harbor, WA) and spotted onto 192-well MALDI target plates (Applied Biosystems) with a Probot. GPS Explorer software version 3.5 (Applied Biosystems) using the MASCOT search engine (version 2.1, Matrix Science) was used for peptide and protein identifications and iTRAQ quantification. Proteins from the virion, the envelope, and the nucleocapsid were resolved by SDS-PAGE and subjected to Western blot analysis as described above.

RESULTS

DISCUSSION

Localization methods