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.