Native protein mapping and visualization of protein interactions in human plasma high-density lipoprotein by 2-DE and LC-MS/MS

A human plasma sample was subjected to non-denaturing micro 2-DE and a gel area (5 mm X 18 mm) which includes high-density lipoprotein (HDL) was cut into 1 mm X 1 mm squares, then the proteins in the 90 gel pieces were analyzed by in-gel trypsin digestion and nano-LC-MS/MS. Grid-cutting of the gel was employed to; 1) ensure the total analysis of the proteins in the area, 2) standardize the conditions of analysis by LC-MS/MS, 3) reconstruct the protein distribution patterns from the label-free quantification data. Totally 154 proteins (excluding keratins) were assigned in the 90 gel pieces and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The map of apolipoprotein (Apo) A-I showed a wide apparent mass distribution characteristic to HDL and was compared with the maps of the other 153 proteins. Eleven proteins showed maps of wide distribution that overlapped with the map of Apo A-I, and all have been reported to be the components of HDL. Further, seven minor proteins associated with HDL were detected at the gel positions of high Apo A-I quantity. These results for the first time visualized the localization of HDL apolipoproteins on a non-denaturing 2-DE gel and strongly suggested their interactions.