A comparison of different biotinylation reagents, tryptic digestion procedures, and mass spectrometric techniques for 2-D peptide mapping of membrane proteins.
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.
Scheurer
Simone B
SB
Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology-Zurich, Wolfgang-Pauli-Str. 10, CH-8093 Zurich, Switzerland.
Roesli
Christoph
C
Neri
Dario
D
Elia
Giuliano
G
D000818:Animals; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism*; D030562:Databases, Protein; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry; D008954:Models, Biological; D008956:Models, Chemical; D015335:Molecular Probes / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D019809:Streptavidin; D014357:Trypsin / Q000737:chemistry*
D008565:Membrane Proteins; D015335:Molecular Probes; D010455:Peptides; D011506:Proteins; D001710:Biotin; D019809:Streptavidin; D014357:Trypsin