Efficient prefractionation of low‐abundance proteins in human plasma and construction of a two‐dimensional map

Human plasma is the most clinically valuable specimen, containing not only a dynamic concentration range of protein components, but also several groups of high‐abundance proteins that seriously interfere with the detection of low‐abundance potential biomarker proteins. To establish a high‐throughput method for efficient depletion of high‐abundance proteins and subsequent fractionation, prior to molecular analysis of proteins, we explored how coupled immunoaffinity columns, commercially available as multiple affinity removal columns (MARC) and free flow electrophoresis (FFE), could apply to the HUPO plasma proteome project. Here we report identification of proteins and construction of a human plasma 2‐DE map devoid of six major abundance proteins (albumin, transferrin, IgG, IgA, haptoglobin, and antitrypsin) using MARC. The proteins were identified by PMF, matching with various internal 2‐DE maps, resulting in a total of 144 nonredundant proteins that were identified from 398 spots. Tissue plasminogen activator, usually present at 10–60 ng/mL plasma, was also identified, indicative of a potentially low‐abundance biomarker. Comparison of representative 2‐D gel images of three ethnic groups (Caucasian, Asian‐American, African‐American) plasma exhibited minor differences in certain proteins between races and sample pretreatment. To establish a throughput fractionation of plasma samples by FFE, either MARC flow‐through fractions or untreated samples of Korean serum were subjected to FFE. After separation of samples on FFE, an aliquot of each fraction was analyzed by 1‐D gel, in which MARC separation was a prerequisite for FFE work. Thus, a working scheme of MARC → FFE → 1‐D PAGE → 2‐D‐nanoLC‐MS/MS may be considered as a widely applicable standard platform technology for fractionation of complex samples like plasma.