Evaluation of Multiprotein Immunoaffinity Subtraction for Plasma Proteomics and Candidate Biomarker Discovery Using Mass Spectrometry

Abstract

Strategies for removal of high abundance proteins have been increasingly utilized in proteomic studies of serum/ plasma and other body fluids to enhance the detection of low abundance proteins and achieve broader proteome coverage; however, both the reproducibility and specificity of the high abundance protein depletion process still represent common concerns. Here we report a detailed evaluation of immunoaffinity subtraction performed applying the ProteomeLab IgY-12 system that is commonly used in human serum/plasma proteome characterization in combination with high resolution LC-MS/MS. Plasma samples were repeatedly processed using this approach, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. The removal of target proteins by the immunoaffinity subtraction system and the overall process was highly reproducible. Non-target proteins, including one spiked protein standard (rabbit glyceraldehyde-3-phosphate dehydrogenase), were also observed to bind to the column at different levels but also in a reproducible manner. The results suggest that multiprotein immunoaffinity subtraction systems can be readily integrated into quantitative strategies to enhance detection of low abundance proteins in biomarker discovery studies.

Advances and Challenges in Liquid Chromatography-Mass Spectrometry-based Proteomics Profiling for Clinical Applications

“…As shown in Table II, coupling the immunoaffinity depletion step to LC-MS provides an additional 1-2 orders of magnitude increase in dynamic range, allowing for detection of more low abundance proteins by effectively increasing the sample loading; similar improvements were reported in other studies (40,41). Good reproducibility was demonstrated by performing immunoaffinity depletion with an automated LC system; however, some of the nontarget low abundance proteins have also been observed to bind to the columns but in a reproducible fashion (42). A possible approach to counter this effect is to analyze both the flow-through and bound fractions in more of a "partitioning" method instead of a pure "depletion" approach (39) with the accompanying trade-off of an increased number of required analyses.…”

Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membranes

“…For example, Washburn et al utilized multidimensional protein identification technology (MudPIT) for large scale proteome analysis of Saccharomyces cerevisiae ,32 and, more recently, Liu et al reported more than 300 confidently identified plasma proteins after coupling 2D-LC-MS/MS with immunodepletion. 31 The measurement of HbA1c reflects the level of nonenzymatic glycation inside the erythrocyte; however, it can also reflect differences in blood glucose levels over time, as well as the individual effects of biological factors that influence cellular glucose transport and the nonenzymatic protein glycation/ deglycation cycle. 33 Glycation of the major plasma proteins and erythrocyte membrane proteins more often reflects glycation in the extracellular compartment, which is more directly associated with mean blood glucose over a given time period.…”

“…14,15 Thus, the discovery of very low abundance glycated proteins represents the proverbial "needle in a haystack". Since immunodepletion to remove high-abundance plasma proteins has been reported to greatly improve the dynamic range of proteomic measurements, 15,31 we utilized an IgY-12 immunodepletion column to deplete the 12 most abundant plasma proteins and enhance our ability to identify low level glycated proteins. As a result, we were able to identify many proteins present in the moderate-to low-abundance range in the plasma proteome.…”

Enhanced Interferon Signaling Pathway in Oral Cancer Revealed by Quantitative Proteome Analysis of Microdissected Specimens Using 16O/18O Labeling and Integrated Two-dimensional LC-ESI-MALDI Tandem MS

“…Blood samples are often used in clinical studies because they are less invasive and more convenient than other types of bodily samples and can be analyzed using automatic and high throughput techniques. Unfortunately the extremely dynamic range of protein concentrations in serum and plasma impedes the direct discovery of potential biomarkers (23,24). Proteins can be released into the blood from diseased tissues during cell death or via secretory pathways.…”