Tandem mass spectrometry (MS/MS) is an attractive technique for sequen
cing membrane proteins because it can be applied to peptides in mixtur
es that are difficult to separate chromatographically. To evaluate the
suitability of MS/MS sequencing for membrane proteins and to develop
protocols for the preparation of the cleaved peptides, we employed the
well characterized apoproteins of bacteriorhodopsin and bovine rhodop
sin, ie. bacterioopsin and opsin, respectively. Without separation, ni
ne out of ten peptides resulting from cyanogen bromide cleavage of bac
terioopsin were detected by fast atom bombardment MS, the single undet
ected fragment being a tetrapeptide that was presumably hidden in the
low-m/z matrix background. Furthermore, MS/MS was used to confirm the
sequence of all the peptides detected with m/z values below 3.5 kDa (4
0% of the protein). Bovine opsin was analyzed in a similar fashion. Ta
ndem MS/MS has thus allowed the sequencing of substantial portions of
two integral membrane proteins by the analysis of unseparated peptide
mixtures, demonstrating for the first time that this technique can obv
iate some of the most serious difficulties associated with sequencing
membrane proteins, namely the difficult-to-achieve separation of the '
sticky' peptide fragments.