Fig 1.
Schematic representation of the combination of differential PEG precipitation and immunoaffinity chromatography.
Diluted human plasma was fractionated by PEG precipitation followed by IAD of seven major high-abundant proteins. The obtained flow-through fractions were subjected to in-solution tryptic digestion, and the resultant peptides were analyzed by LC-MS/MS. The obtained data was analyzed using MaxQuant software.
Fig 2.
One-dimensional non-denaturing (A) and denaturing (B) electrophoretic analysis of plasma proteins following differential PEG fractionation steps, or no PEG fractionation step.
The PEG concentrations used for precipitation are indicated above each lane, lane “S” represents the final supernatant from the 30% PEG precipitation that was collected following further treatment with cold acetone, and lane “P” shows results with a reference plasma sample. The molecular weight markers (M1, M2) are shown on the left of the images. Prior to electrophoresis analysis, all resulting pellets were re-dissolved with non-denaturing or denaturing buffer, as described in the Methods section. a: Fn; b: α, β, and γ-chain of Fb; c: IgG heavy chain; d: IgG light chain; e: transferrin; f: albumin.Unlike the majority of protein-fractionation agents, PEG separates protein components from natural mixtures by an exclusion mechanism [36]. Atha et al. analyzed the mechanism of PEG precipitated proteins and concluded that PEG, regard as an inert solvent sponge, can discretionarily raise the effective concentration of all proteins, and the proteins of larger size are more sensitive than the smaller proteins [37]. As expected, our non-denaturing electrophoresis results showed that some relatively high-MW proteins were first precipitated at a low PEG concentration (Fig 2A). Because the precipitation procedures were performed at low temperature, the component types in the obtained fractions might represent native protein complexes. To some extent, such a phenomenon has been observed and supported by the initial analysis with non-denaturing electrophoresis. For example, a 4% PEG precipitate was reported to contain fibronectin (Fn)-fibrinogen (Fb) complexes [38]. This phenomenon implied that attributable to the non-ionic and non-denaturing nature of PEG, the proteins fractionated by PEG could recover its native form, and could be used in subsequent non-denaturing immune affinity chromatography.
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.
Fig 4.
Plots of MW and pI versus iBAQ values of the plasma proteins identified from the BF and CF.
In the two fractions, values for each replicate are plotted separately to illustrate consistency in the overall trends.
Fig 5.
Evaluation of effect of combining the PEGF procedure with IAD-based characterization of the human plasma proteome in three independent experiments.
In panels A, B and C, the values for each replicate are plotted separately to illustrate consistency in the overall trends. NF, non-fractionation.
Fig 6.
Comparison and distribution of the identified proteins including the known LAPs (A, C) and their matching unique peptides (B, D) using the conventional IAD method and the novel PEGF-IAD method developed in this study.
In panels A–D, the replicate values are plotted separately to illustrate consistency in the overall trends.
Fig 7.
Comparison of MAPs (A, B, and C) and moderate-intensity peptides (D, E, F) identified from the three indicated fractions in replicate experiments, using the PEGF-IAD method.
In panels A–F, the values for each replicate are plotted separately to illustrate consistency in the overall trends.