Proteome analysis of silkworm 2. Hemolymph

Kajiwara, Hideyuki; Imamaki, Atsue; Nakamura, Masaru; Mita, Kazuei; Xia, Qingyu; Ishizaka, Masumi

doi:10.2198/jelectroph.53.27

Cited by 7 publications

(5 citation statements)

References 12 publications

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“…In 2009, Kajiwara investigated the proteome of silkworm hemolymph by 2D-mapping and ion-trap mass spectrometry, and identified 67 proteins in a hemolymph map of the 3 rd day of fifth instar [ 13 ]. After comparing the 56 proteins identified in this work with those 67 proteins, we found only 5 that were identical, including juvenile hormone-binding proteins (spots H36-38); aldose reductase (spots H21, H22); low molecular-weight lipid proteins (spots H27, H28 and H29); and CI8 (spot H12).…”

Section: Discussionmentioning

confidence: 99%

Comparative analysis of proteome maps of silkworm hemolymph during different developmental stages

et al. 2010

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BackgroundThe silkworm Bombyx mori is a lepidopteran insect with four developmental stages: egg, larva (caterpillar), pupa, and adult. The hemolymph of the silkworm is in an open system that circulates among all organs, and functions in nutrient and hormone transport, injury, and immunity. To understand the intricate developmental mechanisms of metamorphosis, silkworm hemolymph from different developmental stages, including the 3rd day of fifth instar, the 6th day of fifth instar, the 3rd day of pupation, the 8th day of pupal stage and the first day of the moth stage, was investigated by two-dimensional electrophoresis and mass spectrometry.ResultsTwo-dimensional polyacrylamide gel electrophoresis showed that from the larval to moth stages, silkworm hemolymph proteins changed markedly. Not only did major proteins such as SP1, SP2, and the 30 K lipoprotein change, but other proteins varied greatly at different stages. To understand the functions of these proteins in silkworm development, 56 spots were excised from gels for analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We identified 34 proteins involved in metamorphosis, programmed cell death, food digestion, metabolism, and nutrient storage and transport. Most proteins showed different expression at different stages, suggesting functions in development and metamorphosis. An abundance of proteins related to immunity were found, including hemolin, prophenoloxidase, serine proteinase-like protein, paralytic peptide-binding protein, and protease inhibitor.ConclusionsProteomics research not only provides the opportunity for direct investigation of protein expression patterns, but also identifies many attractive candidates for further study. Two-dimensional maps of hemolymph proteins expressed during the growth and metamorphosis of the silkworm offer important insights into hemolymph function and insect metamorphosis.

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Section: Discussionmentioning

confidence: 99%

Comparative analysis of proteome maps of silkworm hemolymph during different developmental stages

et al. 2010

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“…We used males and females from the fourth-instar day-1 larva stage to the stage before adult moth. Chemicals and details of the experimental procedure were described previously [2][3][4][5][6][7] . MTs were collected from both males and females from the fifth-instar day-1 larva stage to the stage before adult moth.…”

Section: Methodsmentioning

confidence: 99%

“…All information on the identified proteins and all gel images of MTs have been uploaded to the silkworm proteome database (http://kaiko2ddb.dna. affrc.go.jp/cgi-bin/search_2DDB.cgi) [1][2][3] with other tissues.…”

Section: Introductionmentioning

confidence: 99%

Proteome analysis of silkworm 3. Malpighian tube

et al. 2009

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Proteins in the Malpighian tubes of silkworms were proteomically analyzed by combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Several proteins were identified using peptide mass fingerprinting by mass spectrometry and subsequent MS/MS analysis. In this analysis, the electrophoresis patterns of the Malpighian tubes were compared from the fourth-instar day-1 larva stage to the stage before adult moth. The results of mass spectrometry analysis and changes in gel images are summarized in the silkworm proteome database (http://kaiko2ddb.dna.affrc.go.jp/cgi-bin/ search_2DDB.cgi).

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“…Proteome analysis of total FB tissue (V instar, day 3) assigned as many as 380 proteins [ 16 ]. However, major hemolymph proteins such as lipophorin [ 17 ], vitellogenin, storage protein, egg specific protein and 30 kDa proteins, which were also found in FB tissue, drew the most attention in the past as they are needed for silkworm growth, development and reproduction [ 11 , 18 - 20 ].…”

Section: Introductionmentioning

confidence: 99%

“…No comprehensive analysis on the protein level has been carried out so far, apart from first assignments of short terminal stretches of a hemolymph 30 kDa component by Edman sequencing (matching 17 amino acids to LP3 (P09336)/L301 (Q00802) [ 28 ]) and its detection in global proteome analyses of hemolymph [ 46 ] and total FB [ 16 ]. However, RNA and protein levels hardly correlate so that detailed proteomic investigations are adamant for functional studies.…”

Section: Introductionmentioning

confidence: 99%

A proteomic view on the developmental transfer of homologous 30 kDa lipoproteins from peripheral fat body to perivisceral fat body via hemolymph in silkworm, Bombyx mori

et al. 2012

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BackgroundA group of abundant proteins of ~30 kDa is synthesized in silkworm larval peripheral fat body (PPFB) tissues and transported into the open circulatory system (hemolymph) in a time-depended fashion to be eventually stored as granules in the pupal perivisceral fat body (PVFB) tissues for adult development during the non-feeding stage. These proteins have been shown to act anti-apoptotic besides being assigned roles in embryogenesis and defense. However, detailed protein structural information for individual PPFB and PVFB tissues during larval and pupal developmental stages is still missing. Gel electrophoresis and chromatography were used to separate the 30 kDa proteins from both PPFB and PVFB as well as hemolymph total proteomes. Mass spectrometry (MS) was employed to elucidate individual protein sequences. Furthermore, 30 kDa proteins were purified and biochemically characterized.ResultsOne- and two-dimensional gel electrophoresis (1/2D-PAGE) was used to visualize the relative changes of abundance of the 30 kDa proteins in PPFB and PVFB as well as hemolymph from day 1 of V instar larval stage to day 6 of pupal stage. Their concentrations were markedly increased in hemolymph and PVFB up to the first two days of pupal development and these proteins were consumed during development of the adult insect. Typically, three protein bands were observed (~29, 30, 31 kDa) in 1D-PAGE, which were subjected to MS-based protein identification along with spots excised from 2D-gels run for those proteomes. Gas phase fragmentation was used to generate peptide sequence information, which was matched to the available nucleotide data pool of more than ten highly homologous insect 30 kDa lipoproteins. Phylogenetic and similarity analyses of those sequences were performed to assist in the assignment of experimentally identified peptides to known sequences. Lipoproteins LP1 to LP5 and L301/302 could be matched to peptides extracted from all bands suggesting the presence of full length and truncated or modified protein forms in all of them. The individual variants could not be easily separated by classical means of purification such as 2D-PAGE because of their high similarity. They even seemed to aggregate as was indicated by native gel electrophoresis. Multistep chromatographic procedures eventually allowed purification of an LP3-like protein. The protein responded to lipoprotein-specific staining.ConclusionsIn B. mori larvae and pupae, 30 kDa lipoproteins LP1 to LP5 and L301/302 were detected in PPFB and PVFB tissue as well as in hemolymph. The concentration of these proteins changed progressively during development from their synthesis in PPFB, transport in hemolymph to storage in PVFB. While the 30 kDa proteins could be reproducibly separated in three bands electrophoretically, the exact nature of the individual protein forms present in those bands remained partially ambiguous. The amino acid sequences of all known 30 kDa proteins showed very high homology. High-resolution separation techniques will be necessary before MS a...

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Proteome analysis of silkworm 2. Hemolymph

Cited by 7 publications

References 12 publications

Comparative analysis of proteome maps of silkworm hemolymph during different developmental stages

Comparative analysis of proteome maps of silkworm hemolymph during different developmental stages

Proteome analysis of silkworm 3. Malpighian tube

A proteomic view on the developmental transfer of homologous 30 kDa lipoproteins from peripheral fat body to perivisceral fat body via hemolymph in silkworm, Bombyx mori

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