title: Array-Based Online Two Dimensional Liquid Chromatography System Applied to Effective Depletion of High-Abundance Proteins in Human Plasma.
abstract: In this work, an array-based online two-dimensional liquid chromatography (2D-LC) system was constructed for protein separation and effective depletion of high-abundance proteins in human plasma. This system employed a strong anion exchange column in the first dimension and eight reversed-phase liquid chromatographic columns in the second dimension. All the protein components in the first dimension were enriched on the trapping columns, simultaneously back-flushed and concurrently separated in the second dimension. LC eluents were then collected on 96-well plates for further analysis. Compared with common 2D-LC system, this system showed an 8-fold increase in throughput and convenient utilization of stop-flow mode for sample separation. The RSD of retention time and peak area were separately below 0.51% and 8%. Recovery rates of four standard proteins were all above 95%. This array-based 2D-LC system was subsequently applied to the analysis of proteins in human plasma. The eluents containing high-abundance proteins were rapidly located according to the results of bicinchoninic acid assay. In all, with the effective depletion of 84 high-abundance proteins, a total of 1332 proteins were identified through our system. The dynamic range of the identified protein concentrations covered 9 orders of magnitude, ranging from 41 g/L level for HSA down to 0.01 ng/mL level for the low-abundance proteins.
authors.affiliation: Department of Chemistry, Fudan University , Shanghai 200433, China.|Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Huang
authors.affiliation: Department of Chemistry, Fudan University , Shanghai 200433, China.|Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, China.
authors.forename: Guoquan
authors.initials: G
authors.lastname: Yan
authors.affiliation: Department of Chemistry, Fudan University , Shanghai 200433, China.|Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, China.
authors.forename: Mingxia
authors.initials: M
authors.lastname: Gao
authors.affiliation: Department of Chemistry, Fudan University , Shanghai 200433, China.|Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, China.
authors.forename: Xiangmin
authors.initials: X
authors.lastname: Zhang
mesh_terms: D001798:Blood Proteins / Q000032:analysis / Q000302:isolation & purification*; D056148:Chromatography, Reverse-Phase; D006801:Humans; D009862:Online Systems; D040081:Protein Array Analysis
chemical_list: D001798:Blood Proteins