Indentification of venom proteins of spider S. huwena on two-dimensional electrophoresis gel by N-terminal microsequencing and mass spectrometric peptide mapping |
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Authors: | Liang S Li X Cao M Xie J Chen P Huang R |
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Institution: | (1) College of Life Science, Hunan Normal University, Changsha, 410081, China;(2) College of Life Science, Hunan Normal University, Changsha, 410081, China |
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Abstract: | Venom proteins of the spider Selenocosmia huwena were separated by two-dimensional gel electrophoresis, with the separation in the first dimension on a wide range of immobilized pH (3–10) gradients. Over 300 protein spots were presented on a silver-stained 2D gel. The protein spots with molecular weight > 10 kDa were analyzed, after electrotransferring to polyvinyldene difluoride (PVDF) membrane, by N-terminal microseqencing. Some of the silver-stained protein spots with molecular weight over 10 kDa were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including (1) in-gel reduction, alkylation, and enzymatic digestion; (2) extraction and desalting by using the pipette tip containing a small C18 microcolumn (Ziptip ); and (3) direct MAIDI-TOF mass analysis and protein database searching. Several known toxins such as HWTX-I, HWTX-II, HWTX-IV, and SHL-I were identified and some new components were found among these protein spots. |
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Keywords: | MALDI-TOF mass proteome spider venom 2D-PAGE |
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