Enhanced Detection of Low-Abundance Human Plasma Proteins by Integrating Polyethylene Glycol Fractionation and Immunoaffinity Depletion
Fig 3
Two-dimensional gel images of protein mixtures from the BF (A, C) and CF (B, D) obtained following the PEGF-IAD method.
Analysis was performed on pH 3–10 NL (A, B), pH 5–8 L (C), and pH 4–7 L (D) IPG strips in the first dimension and 12% SDS gels in the second dimension.To gain comprehensive insight into the bias characteristics of proteins from the BF and CF samples, we further employed an iBAQ-based proteomics approach. In the present study, three independent experiments were performed for technical replicates of the overall PEGF-IAD operational process. To rank the absolute abundance of different proteins within a single sample, we used the iBAQ algorithm. Although this algorithm is not quantitative because the two fractionated samples were markedly different in terms of protein compositions, this estimation still facilitated the identification of relative protein abundances. The computed iBAQ values were plotted as a function of either the theoretical MW or pI values for each identified protein. As shown in Fig 4, the proteins with high iBAQ values tended to show high MWs in the BF and moderate MWs in the CF. In terms of pI, the proteins with high iBAQ values mainly distributed in a pI range of 6–8 in the BF and 5–7 in the CF. We also employed the corresponding reciprocals of the iBAQ values to probe the distributions of LAPs in the BF and CF. As shown in S1 Fig, it was observed that more LAPs with high MW values were identified in the BF than in the CF.