Seperation And Identification Of The Basic Proteins In The Hemolymph Of The Larval Silkworm

Bombyx mori is an important biological reactor, it goes through 4 stages in its life. Hemolymph plays a vital role in these stages, because of supplying nutrients for other organs. The hemolymph has a variety of proteins relate to storage, immunity, nutrition, conveyance and so on. So, it is significant for us to research the proteins in the hemolymph of Bombyx mori, especially those few basic protein.Iron exchange chromatography and linear ion trap quadrupole(LTQ) mass spectrometry were used to separate and analyse the hemolymph proteins of Bombyx mori. The hemolymph of p50 was collected in the 3rd day of fifth instar, then it was separated by DEAE Sepharose anion exchange column and MonoS cation exchange column at pH7.5. Afterwards, the samples collected from chromatography were analyzed by SDS-PAGE and LTQ mass spectrometry. SDS-PAGE showed main proteins in samples, their molecular weight were 45 kDa, 80 kDa and bigger than 116 kDa. LTQ mass spectrometry identified 233 proteins, 55 proteins have been reported in 73 characterized proteins, other 18 unreported proteins are related to metamorphosis(Death-related protein, Ubiquitin-conjugating enzyme E2 isoform 2, DNA cytosine-5 methyltransferase, Nuclear excision repair protein Rad23, Eukaryotic translation initiation factor 3 subunit E, Eukaryotic translation initiation factor 3, subunit C and 3 Cytochrome P450 proteins), enzyme and enzyme inhibitors(Chymotrypsin inhibitor SCI-II, S-phase kinase-associated protein, CP8), signal transmission(Gustatory receptor 9, Antennal-specific protein 3c), cell structure(Actin-4), heat shock protein(Hsp90-related protein TRAP1) and other proteins(Bm122, Kiser). 160 proteins of identified proteins have unique peptides but not confirmed names, 123 proteins are unreported.5 proteins among the 18 unreported protein were chosen to detect mRNA expression in several organs of p50 and Qiufeng, the results shows that The relative expression of Actin4 is far more than other genes in fat body, midgut, posterior silk gland, malpighian tubule and has no difference between p50 and Qiufeng. The relative expression of Kiser is higher in midgut of Qiufeng and posterior silk gland of p50 than other tissues. S-phase kinase-associated protein is similar to Kiser. Ubiquitin-conjugating enzyme E2 isoform 2 has poor expressions in all tissues especially malpighian tubule of p50 and Qiufeng. Hsp90-related protein TRAP1 also has poor expressions in all tissues.The experiment shows that Iron Exchange Chromatography combined with Linear Ion Trap Quadrupole(LTQ) mass spectrometry are significant to analyze proteins in the hemolymph of silkworm.