Enhanced Detection of Low-Abundance Human Plasma Proteins by Integrating Polyethylene Glycol Fractionation and Immunoaffinity Depletion

Abstract

The enormous depth complexity of the human plasma proteome poses a significant challenge for current mass spectrometry-based proteomic technologies in terms of detecting low-level proteins in plasma, which is essential for successful biomarker discovery efforts. Typically, a single-step analytical approach cannot reduce this intrinsic complexity. Current simplex immunodepletion techniques offer limited capacity for detecting low-abundance proteins, and integrated strategies are thus desirable. In this respect, we developed an improved strategy for analyzing the human plasma proteome by integrating polyethylene glycol (PEG) fractionation with immunoaffinity depletion. PEG fractionation of plasma proteins is simple, rapid, efficient, and compatible with a downstream immunodepletion step. Compared with immunodepletion alone, our integrated strategy substantially improved the proteome coverage afforded by PEG fractionation. Coupling this new protocol with liquid chromatography-tandem mass spectrometry, 135 proteins with reported normal concentrations below 100 ng/mL were confidently identified as common low-abundance proteins. A side-by-side comparison indicated that our integrated strategy was increased by average 43.0% in the identification rate of low-abundance proteins, relying on an average 65.8% increase of the corresponding unique peptides. Further investigation demonstrated that this combined strategy could effectively alleviate the signal-suppressive effects of the major high-abundance proteins by affinity depletion, especially with moderate-abundance proteins after incorporating PEG fractionation, thereby greatly enhancing the detection of low-abundance proteins. In sum, the newly developed strategy of incorporating PEG fractionation to immunodepletion methods can potentially aid in the discovery of plasma biomarkers of therapeutic and clinical interest.

Artificial intelligence identified peptides modulate inflammation in healthy adults

“…While the determination of the LOD is not possible for an untargeted approach, the sensitivity of the analytical method was confirmed by the detection of peptide fragments of several lowabundancy human proteins (UniProt accession, typical concentrations from literature references): prothrombin, P00734, 1.2 ng mL −1 ; multimerin-1, Q13201, 7.2 ng mL −1 ; osteocalcin, P02818, 2-15 ng mL −1 ; insulin C-peptide, P01308, 0.8-3.9 ng mL −1 ; neurosecretory protein VGF, O15240, 3 ng mL −1 ; neurogranin, Q92686, 1.4 ng mL −1 . [23][24][25][26] Taken together, the selected peptides were absent from blood samples at physiologically relevant concentrations down to the lower picomolar range.…”

Proteomic and network analysis of human serum albuminome by integrated use of quick crosslinking and two-step precipitation

“…In view of the aforementioned issue, herein we attempted to utilize the relatively inexpensive PEG. In our previous study, PEG precipitation was integrated into immunoaffinity depletion to enhance the detection of low-abundance human plasma proteins 41 . Unlike the majority of other agents, PEG precipitates protein components from natural mixtures by an exclusion mechanism 42 .…”

Plasma prefractionation methods for proteomic analysis and perspectives in clinical applications

“…In organic solvent precipitation (such as acetone and ACN), medium and large proteins which have poor solubility in nonpolar solvents are precipitated out, whereas low molecular weight proteins in the supernatant can be concentrated by evaporation . Other common precipitants such as ammonium sulfate , polyethylene glycol , and trichloroacetic acid can also be used. This simple precipitation has been used for biomarkers with clinical relevance , such as hepcidin‐25 and insulin‐like growth factor .…”