Proteome analysis of silkworm 1. Fat body

Abstract

SUMMARYProteome analysis of silkworm (Bombyx mori L.) fat bodies was performed on the fifthinstar day-3 larva stage and the expression of proteins separated on two-dimensional polyacrylamide gels was tracked from the fourth-instar day-1 larva stage to the stage before adult moth. Proteins on the 2D-gels were excised manually and treated with 4-vinylpyridine for alkylation. Each spot was analyzed by capillary high-performance liquid chromatography coupled with ion-trap mass spectrometry after proteolysis using trypsin. In the previous report 1) , the amino acid sequence database obtained from the Drosophila genome was used for the protein identification. This analysis used the amino acid sequence data elucidated from the silkworm genome. Additionally, protein expression in fat bodies was compared with that at other stages and in other tissues described in the silkworm proteome database (http://kaiko2ddb.dna.affrc.go.jp/cgi-bin/search_2DDB.cgi). Several unidentified proteins from the previous report 1) were identified with high Mascot scores. Induction and reduction of proteins during metamorphosis were observed as changes in spot concentration on sequential 2D-gels on the web.

A Hox Gene, Antennapedia, Regulates Expression of Multiple Major Silk Protein Genes in the Silkworm Bombyx mori

“…Sample preparation and electrophoresis were conducted as described previously (28). Briefly, the PSG was dissociated in lysis buffer (10 mg of tissue/300 l of buffer) containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% Nonidet P-40, 5% 2-mercaptoethanol, 0.2% Ampholine (pH 3.5-10), and EDTA-free protease inhibitor.…”

“…Mass Spectrometry and Data Analysis-Mass spectrometry and data analysis were performed largely as described previously (28). Spots were excised from five or six gels, and amaZon SL (Bruker Daltonics, Bremen, Germany) was used for the MS analysis.…”

Loss of chloroplast‐localized protein phosphatase 2Cs in Arabidopsis thaliana leads to enhancement of plant immunity and resistance to Xanthomonas campestris pv. campestris infection

“…The two-dimensional polyacrylamide gel electrophoresis (PAGE) gels were stained with CBB or Pro-Q Diamond Phosphoprotein Gel Stain (Thermo Fisher Scientific) following the manufacturer's instructions. MS and data analysis were performed largely as described previously (Kajiwara et al, 2009). Spots were excised from five or six gels, and amaZon SL (Bruker Daltonics, Bremen, Germany) was used for MS analysis.…”

Proteome analysis of silkworm 2. Hemolymph

“…Silkworms (Bombyx mori L., p50) were nurtured on mulberry leaves at 26°C, and the hemolymph of females from the fourth-instar day-1 larva stage to the stage before adult moth was used. Chemicals and details of the experimental procedure were described previously 7) . Hemolymph from fourth-instar day-1 (after about three weeks since oviposition) larva to the stage before adult moth was collected by cutting the posterior part of silkworm bodies 8) and immediately mixing with an equal volume of lysis buffer.…”