Structural and functional studies with native mass spectrometry : insights into membranes and glycoproteins

The membrane is a boundary between extracellular and intracellular environments or separates intracellular spaces. It also plays a vital and irreplaceable role in hosting membrane protein and is a matrix or media to exchange material and information in and out of the cell. Hence understanding the protein structure, tracing the signalling and investigating active ligands are essential. This thesis studies the structure details of glycoproteins, molecular events of rhodopsin in disc membrane, and the endogenous ligands of voltage-dependent potassium ion channel beta subunits. Native mass spectrometry (native MS) monitors the photoactivation and regeneration of rhodopsin in membrane in Chapter 3. The retinal isomerisation and hydrolysis process are slower than in the detergent environment. It is also discovered that the membrane component, N-ret-PE is a source of cis-retinal for rhodopsin regeneration. This pathway is independent of other cells and does not require conversion between retinal and retinol, which may improve the efficiency of vision persistence. Later in Chapter 4, the phototransduction of rhodopsin in membrane is further studied with native MS. The GDP release from the transducin is directly observed on transducin when it couples with rhodopsin. Hence the activated Gtα-PDE6 hydrolyses and releases cGMP. This detection of photoactivation and transduction in the membrane can be used for rhodopsin-targeting compounds. These provide insights into the regulation of rhodopsin-base active ligands for rhodopsin signalling. Apart from the static observation of protein structure, employment of native MS reveals occupancy of N- and O-glycans, LacNAcs, GlcNAc branches on N-glycans and linkage of glycans applied in transferrin, fetuin, epoetin and alpha 1-acid glycoprotein in Chapter 5. In Chapter 6, native MS detects cofactor and active ligands that monitor Kvβ2, both aldo-keto reductase and potassium ion channel beta subunit. An endogenous inhibitory ligand is likely to be an inhibitor of Kvβ2, maintaining an increasing redox state in the ratio of NADPH/NADP+. The finding of a small ligand may result in a possible drug prototype for sleep rhythm. This thesis shows that the new application of native MS providing detailed information in structural, functional and metabolomic biology studies. Together these studies by the means of native MS advance the understanding in protein structure and functions.