Identification of protein spots in silver-stained two-dimensional gels by MALDI-TOF mass peptide map analysis

Protein spots in silver-stained two dimensional gels were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including: i) In-gel reduction, alkylation and enzymatic digestion; ii) Extraction and desalting by using a pipette tip containing a small C18 micro-column (ZipTip(TM)); iii) Direct MALDI-TOF mass analysis and protein database searching. The results demonstrated that single silver-stained protein spots in a 2-DE-gel could be identified rapidly by this procedure and the use of the ZipTip(TM) pipette tip could increase evidently the sensitivity of the MALDI-TOF analysis. By using this procedure, 10 protein spots in a silver-stained gel of 2-DE of crude venom of the spider S. Huwena were analyzed and identified.