Figure 3
Affinity purification of salusin-β-binding proteins/receptor. (a) Truncation of the N-terminal three amino acids of salusin-β after binding to membrane proteins-embedded liposomes. Synthetic salusin-β (500 pmol) was allowed to bind to liposomes embedded with membrane proteins prepared from rat cardiac tissues and subjected to MALDI-MS analysis after elution. (b) MALDI-MS/MS spectra revealing truncation of salusin-β to salusin-β(4-20). MS/MS spectra were assigned theoretical m/z values for a, a-H2O, and b ions. Parent peptide and MS/MS tolerance parameters were set at ± 10 Da and ± 0.03 Da. (c) Retrieval of salusin-β and its truncated forms bound to proteoliposomes prepared from a variety of animal tissues. Membrane proteins-embedded liposomes were prepared from rat, mouse and porcine organs, and binding experiments were performed. The relative amounts of salusin-β (closed column) and its truncated forms (salusin-β(4-20), dark column; salusin-β(5-20), hatched column; salusin-β(6-20), open column) retrieved from the proteoliposomes are expressed in the ordinates. (d) Affinity purification of membrane proteins bound to immobilized salusin-β. Lysates containing membrane proteins prepared from rat hearts were subjected to affinity chromatography using C-terminally (lane 1) and N-terminally (lane 2) immobilized salusin-β, and the eluates were separated by SDS-PAGE. Liposomes embedded with membrane proteins prepared from rat heart were allowed to bind to N-terminally immobilized salusin-β (lane 3). Silver-stained bands (arrows) were dissected, hydrolyzed with trypsin and subjected to LC-MS/MS analyses. The electrophoretic mobilities of low molecular weight standards (lane M) are indicated. From the top of the gel these proteins are phosphorylase B (106.9 kDa), bovine serum albumin (93.6 kDa), ovalbumin (52.3 kDa), carbonic anhydrase (37.2 kDa), and soybean trypsin inhibitor (28.2 kDa).
