Česká společnost pro hmotnostní spektrometrii

(Česká konference hmotnostní spektrometrie 2018 - ThS-017)
Triple approach to membrane proteome profiling of human pheochromocytoma and paraganglioma

Ondřej Vít ^1 *, Karel Pacák ², Jiří Petrák ¹

1. 1. lékařská fakulta UK, BIOCEV
2. Eunice Kennedy Shriver National Institute of Child Health and Human Development - NIH

Abstrakt

About a quarter of human genes encode integral membrane proteins (IMPs), which perform various key biological functions, such as receptor signaling, transport of molecules, cell-cell interactions, etc. Proteomic studies of IMPs are, however, made difficult by their amphipathy, the lack of trypsin cleavage sites and low levels of their expression.
Among the most effective approaches for identification of low-abundant IMPs seem to be those that aim to enrich either soluble (extramembrane) or hydrophobic (transmembrane) parts of IMPs. Our method named hpTC (high pH, trypsin, CNBr) allows identification of IMPs based on their hydrophobic alpha-helices protected from trypsin by the phospholipid bilayer after re-cleavage with CNBr. To maximize the membrane proteome coverage, we combine this approach with standard tryptic digest and two methods based on glycopeptide enrichment, one that uses lectin entrapment on ultrafilters (lectin-FASP) and the second using hydrazide chemistry with solid phase extraction (SPEG).
The combination of these parallel approaches targeting both hydrophilic and hydrophobic segments of IMPs allows us to identify a broader range of IMPs than the classical proteomic strategies. We apply our strategy to membrane proteome profiling of human pheochromocytoma and paraganglioma (PHEO & PGL). These neuroendocrine tumors are extremely rare, and there are no known effective therapeutic approaches. Our goal is to gather data for a detailed description of membrane proteome of human PHEO & PGL that could be used for the search of new drug targets and diagnostic markers. So far we were able to identify over 1200 IMPs in each patient sample, nearly 2000 IMPs in total. In each sample, we identified over 20 so-called missing proteins.

* Korespondující autor: ondrvit@gmail.com