Analysis of LDL-associated proteins using the method of digitized native protein mapping

The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel-cutting and quantitative LC-MS/MS (in data-independent acquisition mode, or MSE), was improved by using a new MS/MS mode, ion mobility separation enhanced-MSE (HDMSE) and applied to analyze the area of human plasma low-density lipoproteins (LDL). An 18 mm * 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid-cut into 72 square gel pieces and subjected to quantitative LC-MS/MS. Compared with MSE, HDMSE showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. Totally 253 proteins were assigned with LC-HDMSE and the quantity distribution of each was reconstructed as a native protein map. The maps showed Apo B-100 was the most abundant protein in the grid-cut area, concentrated at pI 5.4-6.1 and apparent mass ca. 1000 kDa, which corresponded to four gel pieces, squares 39-42. An Excel macro was prepared to search protein maps which show protein quantity peaks localized within the concentrated region of Apo B-100 (squares 39-42). Twenty-two proteins out of the 252 matched this criteria, in which nineteen proteins have been reported to be associated with LDL. This method require only several microliters of a plasma sample and the principle of the protein separation is totally different from ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL.