Membrane protein proteomics - Novel method for membrane protein identification and quantification

Sammanfattning: Membrane proteins are fairly refractory to digestion especially by trypsin. Less specific proteases like elastase and pepsin are much more effective. However database searching using non-tryptic peptides is much less effective due to the lack of charge localisation at the N- and C-termini and the absence of sequence specificity. We describe a method for N-terminal specific labelling of peptides from non-tryptic digestions of membrane proteins which facilitates database searching and can be used for relative quantitation. The conditions for digestion using the non-specific enzyme Proteinase K have been optimised to obtain peptides of a suitable length for MS fragmentation. We show the effectiveness of the identification of membrane proteins using a plasma membrane preparation from a leukaemia cell line and demonstrate a large increase in the number of membrane proteins with small extra-membranar domains being identified in comparison to previous published methods. Our method was then further developed for relative quantitation of membrane proteins. We describe this by using membrane preparations from Bacillus subtilis grown with and without the presents of 0.5% glucose and an isotopic labelling strategy. The results show good reproducibility of the calculated fold changes of the identified peptides within the same protein. The method was then applied on plasma membrane proteins from cancer cells grown with and without the presence of oxygen. The aim was to find biomarkers for tissue hypoxia.