Introduction

The ability to remove highly abundant proteins specifically and with high selectivity is increasingly important in proteomic studies, and success in this procedure is leading to an ever-increasing list of lower abundant proteins being identified in biological fluids.^1–3 Several immunoaffinity columns are commercially available for the purpose of the removal of multiple high-abundant proteins from human plasma.^4,5 These columns have been used by various laboratories for the successful removal of targeted proteins in high throughput proteomic analysis. Beckman Coulter is marketing a series of new products (ProteomeLab IgY-12 proteome partitioning systems) for proteomic sample preparation using polyclonal IgY antibodies immobilized to microbeads packed in spin columns or liquid chromatography (LC) columns to partition (deplete) 12 of the most highly abundant proteins from plasma that collectively constitute up to 96% of the total protein mass in plasma.

Recently, a study reported consistent performance of the mentioned column over a span of 115 sample runs.⁶ Efficient removal of targeted proteins and binding of non-targeted proteins to the column has also been reported.⁷ The current study validated the column performance on the consistent recovery of the low abundant proteins at various concentrations of plasma along with five radiolabelled non-targeted proteins. The low-abundant proteins recovered at various stages were further validated by proteomic analysis for their relative abundance.

We used Beckman Coulter ProteomeLab IgY-12 LC2 (10 × 64 mm) proteome partitioning system with the included buffers and spin filters. The reagents and gels for SDS-PAGE and protein estimation kits were purchased from Bio-Rad Laboratories (Hercules, California, USA). Radiolabelled (¹⁴C) proteins were obtained from Amersham Biochemicals (GE Healthcare, Chicago, Illinois, USA). Spin concentrators of 5K MWCO were purchased from Agilent Technologies (Wilmington, Delaware, USA). All other materials were obtained from Sigma Chemicals (St Louis, Missouri, USA) unless otherwise mentioned.

Table 1: Validation of ProteomeLab IgY-12 chromatography based on variable starting protein concentrations. A, B, C, D and E are the plasma sample preparations for the column. 125 μL of each sample were subjected ProteomeLab IgY-12 chromatography starting from A to E. The protein content of samples and their respective flow through (FT) were measured. The values are expressed as mean ± standard deviation.