Shrimp protein identification by LC-nanoESI-MS/MS

Description

This technique is used to identify protein spots from the results of 1D/2D protein electrophoresis.

Brief protocol

In-gel protein digestion and protein identification

Protein were manually excised from the gels, washed twice with 25 mM ammonium bicarbonate buffer (pH 8.5) in 50% acetonitrile, for 15 min each time, dehydrated with 100% acetonitrile for 5 min, vacuum dried, and rehydrated with 100 ng of sequencing-grade, modified trypsin (Promega) in 25 mM ammonium bicarbonate, pH 8.5, at 37 °C for 16 h. Following digestion, tryptic peptides were extracted twice with 5% formic acid in 50% acetonitrile for 15 min each time with sonication. The extracted solutions were pooled and evaporated to dryness under vacuum. Samples were dissolved in 0.1% formic acid in 50% acetonitrile and analyzed by LC-nanoESI-MS/MS. Proteins were identified by MS/MS ion search using the search program MASCOT and the NCBI protein and EST sequence databases.For MS/MS ion search, the mass tolerance parameter was 0.25Da, MS/MS ion mass tolerance was 0.25 Da, and up to one missed cleavage was allowed. Variable modifications considered were methionine oxidation and cysteine carboxyamidomethylation. Significant hits (as defined by Mascot probability analysis) were regarded as positive identification.

References

1. H.C. Wang, H.C. Wang, J.H. Leu, G.H. Kou, Andrew H.-J. Wang and C.F. Lo. (2007) Protein expression profiling of the shrimp cellular response to white spot syndrome virus infection. Developmental & Comparative Immunology 31: 672-686.

Some of our publications that have used this platform

1. Wang HC, Wang HC, Kou GH, Lo CF, Huang WP. (2007). Identification of icp11, the most highly expressed gene of shrimp white spot syndrome virus (WSSV). Dis. Aquat. Org. 74:179-189.
2. H.C. Wang, H.C. Wang, J.H. Leu, G.H. Kou, Andrew H.-J. Wang and C.F. Lo. (2007) Protein expression profiling of the shrimp cellular response to white spot syndrome virus infection. Developmental & Comparative Immunology 31: 672-686.