Interrogating the Membrane Proteome
As quantitative investigations flesh out the texture of the proteome, it has become evident that around one-third is composed of membrane proteins. However, membrane proteins tend be to be large and unwieldy, difficult to purify while still retaining functionality, and hard to crystallize.
For these reasons they are underrepresented in proteomics studies, and investigators have often ignored them despite their pivotal importance in cell function.
This has changed in recent years, explained Kathryn Lilley, Ph.D., assistant director of research at the Cambridge Centre for Proteomics and the department of biochemistry at the University of Cambridge. She noted that membrane proteins are of special interest to the pharma industry, given the importance of receptors as targets of specific drugs.
There are still challenges to be considered, however. “Questions raised when targeting membrane proteins include abundance, solubility, and suitability for standard proteomics workflow operations,” stated Dr. Lilley.
She and her co-workers addressed these issues through the development of proteomics tools to define the expression of resistance nodulation division efflux pumps—which are low abundance, membrane-bound proteins—in Pseudomonas aeruginosa. Mutations in these proteins are a frequent cause of multiple drug resistance.
Absolute quantification of the amount of protein requires the use of a marker peptide as a standard. Dr. Lilley chose enolase and calibrated the absolute abundance based on the performance of its top three scoring peptides. This approach is extremely flexible, she said, and applies over several orders of magnitude.
Dr. Lilley discussed a second example of her approach to membrane protein characterization known as selected reaction monitoring, a complementary proteomic procedure based on the targeted analysis of a set of predetermined proteins and peptides.
Selected peptides, based on their mass to charge ratio, are fragmented in the collision cell of a triple quadrupole mass spectrometer. The detected fragment ions, referred to as transitions, are used to construct a specific and highly sensitive assay for the detection of a particular peptide in a sample.
This approach was also combined with global mass spectrometry-based protein localization studies in experiments aimed at localizing particular membrane proteins to the plant Golgi apparatus.
“We found that a global membrane proteome analysis is more achievable by a combination of a variety of different mass spectrometry approaches,” Dr. Lilley said. “We have employed quantification of the membrane protein targets, identification of their binding partners, and a focus on the subcellular location of possible candidates for development of workable hypotheses.”