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A comparison of different biotinylation reagents, tryptic digestion procedures, and mass spectrometric techniques for 2‐D peptide mapping of membrane proteins
Author(s) -
Scheurer Simone B.,
Roesli Christoph,
Neri Dario,
Elia Giuliano
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200402069
Subject(s) - biotinylation , chromatography , digestion (alchemy) , chemistry , peptide , tandem mass tag , mass spectrometry , reagent , proteomics , isobaric labeling , biochemistry , protein mass spectrometry , tandem mass spectrometry , quantitative proteomics , gene
2‐D peptide mapping is a novel technique for the relative quantification of membrane proteins (Scheurer S. et al. , Proteomics 2005, in press). Using closely related metastatic and nonmetastatic teratocarcinoma cell lines as a model system, we have performed a comparative analysis of different biotinylation reagents, tryptic digestion procedures, and mass spectrometric techniques, with the aim to increase the number of proteins identified by 2‐D peptide mapping. Our experience indicates that the LC‐MALDI TOF/TOF technique is superior to LC‐ESI MS/MS in terms of the number of proteins identified and confidence in protein identification. Furthermore, the best results were obtained by tryptic digestion of proteins eluted from a streptavidin column using a cleavable biotin derivative.