SEQUENCING MEMBRANE-PROTEINS BY TANDEM MASS-SPECTROMETRY

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.