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-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.
Liang
S
S
College of Life Science, Hunan Normal University, Changsha, China. liangsp@public.cs.hn.cn
Li
X
X
Cao
M
M
Xie
J
J
Chen
P
P
Huang
R
R
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000032:analysis*; D020539:Sequence Analysis, Protein; D016622:Silver Staining; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013111:Spider Venoms / Q000032:analysis* / Q000302:isolation & purification; D013112:Spiders / Q000737:chemistry*
D010455:Peptides; D013111:Spider Venoms