Analyses of disulfide-mediated protein conjugates in human plasma using various proteomics technologies

• Main Authors: Tsung-Han Hsieh, 謝宗翰
• Other Authors: Yeou-Guang Tsay
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/36446345373964094278

碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 96 === Plasma is the part of blood devoid of the cellular elements, containing many proteins with critical roles in various biological processes. Since the functional entities of many intracellular proteins are the multiprotein complexed structures, we are particularly interested in how the extracellular protein complexes, like those in plasma proteome, are assembled. In order to investigate whether interchain disulfide bonds may mediate the complex formation of these proteins, we prepared the plasma with gel filtration column in HPLC to reduce the interference by albumins, and the plasma has been examined in a two-dimensional gel electrophoresis system, namely diagonal gel electrophoresis, which consists of non-reducing SDS-polyacrylamide gel electrophoresis (PAGE) as the abscissa and reducing SDS-PAGE as the ordinate. We observed that proteins without any interchain disulfide bonds were aligned on a virtual diagonal line as the electrophoretic migration along two axes should be similar. On the contrary, proteins with interchain disulfide bonds were positioned in the area beneath the diagonal line since the mobility should increase significantly under the reducing condition. In order to circumvent the interference by highly abundant albumin, gel filtration chromatography was first applied to process the sample such that the albumin content was largely deprived (>75%). We found that more than 35 different plasma proteins do not seem to contain interchain disulfide bridges. On the contrary, more than 21 different plasma protein subunits comprised the disulfide-mediated protein conjugates in plasma. Based on our observations, the subunit structures of haptoglobin protein conjugates are largely consistent with the known model. It is quite intriguing to find that there existed several subunit structures of disulfide-linked plasma proteins. For example, α2-macroglobulin appears to have an alternative structure that consists of a tetrameric subunit linked via disulfide bridges. Complement component 4 is another plasma protein that seems to have an α1/α2 subunit structure in addition to the original α peptide chain. The other plasma proteins, like complement component 3, need to be further investigated for their subunit structure. In order to isolate these protein conjugates at higher amounts as well as to detect scarce species, we were prompted to use several analytical methods to better resolve disulfide-containing plasma protein conjugates. Preparative SDS-PAGE was the first one we tested, with which we separated proteins based on their molecular sizes. Nevertheless, the use of this system was compromised by the poor resolution and interference by abundant proteins like albumin. In contrast, macroporous reverse phase chromatography seemed to have the prowess to effectively separate plasma proteins based on overall protein hydrophobicity. We estimated that the plasma proteome could be resolved into 60~70 groups of protein bands under non-reducing conditions. We will take advantage of this system to characterize the details of subunit structures for these disulfide-mediated protein conjugates, for identifying the novel cleavage sites as well as the locations of involved disulfide bridges.