Selective Depletion of High Abundance Proteins from Human Plasma by Imprinting Techniques

In proteome study, it is indispensable to deplete high abundance proteins to identify low abundance ones1. Antibody techniques have been proven the most effective methods. However, with the consideration of stability, robustness and cost, the development of artificial antibody, molecularly imprinted materials, might be a state-of-the-art solution. Here, we developed two molecularly imprinted polymers (MIP) by two different protocols, protein hierarchical imprinting and multi-epitope imprinting. In the hierarchical imprinting, porcine serum albumin (PSA), an analogue of the highest abundance proteins human serum albumin (HSA), was used as the template. PSA was first adsorbed on the pore walls of silica particles which were packed in a SPE cartridge. Methacrylamide and methacrylic acid were used as the functional monomers, while piperazine diacrylamide was applied as the crosslinker. Pre-polymerization solution was pushed inside silica beads by vacuum. After polymerization, silica and PSA were removed by NH4HF2 and acetic acid-SDS solution to gain the recognition sites on the surface of the polymer microspheres which were of the same shape, size, but complementary structure as the silica beads. The saturated binding amounts to MIP and its control (NIP) were 12 mg g-1 and 3.5 mg g-1 respectively. In the protein mixture, the MIP exhibit excellent selectivity for PSA, with imprinting factors as about 3.6, much higher than those for non-template proteins. Then, MIP was applied in the human plasma to remove the HSA. As shown in Table 1, under stringent filtering criteria for plasma analysis, the identified peptides number and ratio of HSA after HSA removal was decreased to 1/3 of the original sample, which increase the identified protein group number from 348 to 422. It indicated that the PSA imprinted microspheres might be promising alternatives of antibodies for the depletion of high abundance proteins, with good stability, high robustness, and low cost. Table 1. Number of identified peptides and protein groups before and after MIP application in the plasma Original serum After MIP application Peptides of HSA 3076 949 Percentage of HSA peptides in total peptides (%) 37.8 13.9 Total protein groups 348 422 * filtering criteria: false positive rate less than 1% Multi-epitope imprinting was developed to overcome the difficulty in obtaining the pure protein as the template, while it could contribute to deplete several corresponding proteins at the same time. Three epitopes, corresponding with top three high abundance proteins in plasma, were identified and synthesized. And, submicrometer core-shell magnetic microspheres were prepared as the matrix. After the matrix was grafted by aldehyde group, the epitopes were immobilized through the coupling between –NH2 and –CHO. Then, surface imprinting was conducted by employing the microspheres in the miniemulsion polymerization. After the formation of the imprinted shell layer, the immobilized templates were removed by hydrolysis to get the imprinted particle finally. The application of these imprinted particles in the plasma was undergoing in our lab. 1. Issaq HJ, Xiao Z, Veenstra TD, Serum and plasma proteomics, Chemical Reviews, 2007, 107, 3601-362