Analysis of PEG‐fractionated high‐molecular‐mass proteins in human plasma by non‐denaturing micro 2‐DE and MALDI‐MS PMF

Abstract

The process of 0-4% PEG precipitation of high-molecular-mass proteins (around and above 1x10(3) kDa) in human plasma was analyzed by non-denaturing micro 2-DE employing agarose IEF gels in the first dimension, and by MALDI-MS PMF. The PEG 0-2% precipitate could be mostly re-dissolved (fraction PS2%), but some part remained as precipitate (fraction PP2%). Non-denaturing 2-DE followed by MALDI-MS PMF showed that PS2% contained fibrinogen (Fb), fibronectin (FN), and their oligomers as the major components, and von Willebrand factor and coagulation factor XIII as the minor components. PP2% was directly subjected to MALDI-MS PMF and FN and Fb were assigned, suggesting it was formed by co-precipitation of the two proteins. The PEG 2-4% precipitate could be totally dissolved, but after freezing and thawing the solution, a small amount of precipitate appeared. MALDI-MS PMF analysis of the precipitate (PP4%) suggested that its major constituent is complement C1. The soluble fraction of the PEG 2-4% precipitate (PS4%), analyzed by non-denaturing 2-DE and MALDI-MS PMF, contained C4b-binding protein and its complex with complement C4, low-density lipoproteins, IgM, and complement C1 subcomponent q, together with Fb, FN, and their oligomers. When the PS4% fraction was analyzed by a Type-II 2-DE technique (non-denaturing IEF followed by SDS-PAGE, no reducing agent being used), several proteins in molecular mass range from 7x10(1) to 2x10(2) kDa appeared, suggesting that these proteins interacted with the high-molecular-mass proteins, then dissociated in the presence of SDS.