Indentification of venom proteins of spider S-huwena on two-dimensional electrophoresis gel by N-terminal microsequencing and mass spectrometric peptide mapping

Venom proteins of the spider Selenocosmia huwena were separated by two-dime nsional gel electrophoresis, with the separation in the first dimension on a wide range of immobilized pH (3-10) gradients. Over 300 protein spots wer e presented on a silver-stained 2D gel. The protein spots with molecular we ight >10 kDa were analyzed, after electrotransferring to polyvinyldene difl uoride (PVDF) membrane, by N-terminal microseqencing. Some of the silver-st ained protein spots with molecular weight over 10 kDa were analyzed and ide ntified by employing an improved procedure of mass spectrometric peptide ma pping, including (1) in-gel reduction, alkylation, and enzymatic digestion; (2) extraction and desalting by using the pipette tip containing a small C 18 microcolumn (Ziptip(TM)); and (3) direct MAIDI-TOF mass analysis and pro tein 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 the se protein spots.