Immunoaffinity separation of plasma proteins by IgY microbeads: Meeting the needs of proteomic sample preparation and analysis

Abstract

Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95-99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein-protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics.

Human body fluid proteome analysis

“…Comparing to Cibacron blue dye and Protein G methods, immunoaffinity depletion using multiple affinity removal columns (MARC) is more effective because it can simultaneously remove multiple abundant proteins, with minimal carryover, high longevity, and minimal nonspecific binding [16,17,21,22]. Immunodepletion can be effectively realized with IgY antibody microbeads or peptidebased affinity medium [23,24]. Protein precipitation with TCA/acetone or NaCl/ethanol also appears to be useful for depletion of albumins [25,26].…”

Profiling of vitreous proteomes from proliferative diabetic retinopathy and nondiabetic patients

“…IgY-12 columns have been previously used to deplete 12 highly abundant proteins from human or primate biological fluids [32]. Likewise, PDR vitreous samples were treated using IgY-12 columns, and subsequently the high and low abundance protein fractions obtained were subjected to 2-DE.…”

Variation in protein levels obtained from human blood cells and biofluids for platelet, peripheral blood mononuclear cell, plasma, urine and saliva proteomics

“…These sources of variation can obscure the biological changes under investigation. Recently, we and others determined optimal methods to deplete human plasma of its most abundant proteins using immuno-affinity columns, concentrate protein homogenates, and isolate PBMC with minimal platelet contamination [6,9,20,29,31]. Here we present data on the amount and the variability of proteins available from platelets, PBMC, plasma, urine and saliva from healthy volunteers for proteomics analysis after overnight or 36 h fasting.…”