Contributions of immunoaffinity chromatography to deep proteome profiling of human biofluids

Abstract

Human biofluids, especially blood plasma or serum, hold great potential as the sources of candidate biomarkers for various diseases; however, the enormous dynamic range of protein concentrations in biofluids represents a significant analytical challenge for detecting promising low-abundance proteins. Over the last decade, various immunoaffinity chromatographic methods have been developed and routinely applied for separating low-abundance proteins from the high- and moderate-abundance proteins, thus enabling much more effective detection of low-abundance proteins. Herein, we review the advances of immunoaffinity separation methods and their contributions to the proteomic applications in human biofluids. The limitations and future perspectives of immunoaffinity separation methods are also discussed.

Keywords: Affinity proteomics; Biofluid; Biomarker discovery; Immunoaffinity chromatography; Immunodepletion; Immunoenrichment; Plasma/Serum; Proteomics.

Figure 1

Biofluids in human. Main sources and origins of human biofluids are depicted; the compositions of protein mass for both plasma/serum (data from in-house protein identification result and protein abundances were based on spectral count) and urine (data from Li et al. [162]) are shown in pie charts on the left and right hand side, respectively.

Figure 2

The main workflows of immunodepletion (A) and immunoenrichment (B). For immunodepletion, the Star and Circles represents LAPs and MAPs, respectively. For Immunoenrichment, the Star represent the target molecules and other symbols are sample matrix.

Figure 3

Immunodepletion using the tandem IgY14-SuperMix configuration. (A) IgY14 and SuperMix columns are connected through a 6-port valve. By switching the valve positions, the SuperMix flow-through (SuperMix_FT), IgY14 bound, and SuperMix bound fractions can be collected consecutively. (B) Representative LC chromatograms of 3 replicates of the tandem IgY14-SuperMix separations during the initial, middle and late stage of the column life of a reference plasma sample.

Figure 4

General workflow of applying immunoenrichment and immunodepletion for a biomarker related study. Biofluid samples (e.g., plasma/serum or urine) are subjected to immunodepletion or immunoenrichment to reduce sample complexity. Immunodepletion is used to remove high abundant species, such as HAPs and MAPs, prior to MS-based proteomic studies. Target proteins or peptides can be selectively enriched through immunoenrichment by using MSIA or SISCAPA approaches, which are especially useful for targeted proteomics.