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.

Verification of protein biomarker specificity for the identification of biological stains by quadrupole time‐of‐flight mass spectrometry

“…Shortening the assay's 1 h per sample run time, however, would better meet the higher throughput needs of some larger forensic laboratories . Possible avenues for increasing sample throughput could include the use of commercially available automation systems that process higher density microwell plates in parallel ; transferring the chromatographic separation process to an Ultra Performance Liquid Chromatography system (UPLC) and/or; porting the existing assay to a faster mass spectrometry platform. One such option would be the triple quadrupole mass spectrometers performing multiple reaction monitoring (QQQ‐MRM).…”

Developing front‐end devices for improved sample preparation in MS‐based proteome analysis

“…Similarly, sophisticated “online” approaches have been proposed to manipulate protein samples within a fluidic network, which is not only automated but can be directly integrated to MS analysis. Such platforms can incorporate multiple columns including immobilized enzyme microreactors, columns to enrich desired analytes (eg, TiO 2 for phosphopeptides), or deplete abundant interferences (by immunoaffinity), in addition to conventional separation columns, being integrated by way of simple switching valves. Suited to the analysis of smaller cell populations of trace samples, electrophoretically driven microfluidic chips, ( ) or discrete droplet microarrays, are capable of processing samples including isolating digestion, purifying, and separating protein or peptide mixtures ahead of MS.…”

Mass Spectrometry-Based Plasma Proteomics: Considerations from Sample Collection to Achieving Translational Data

“…This characteristic can be utilized to improve detectability of low abundance proteins by systematic depletion of high abundance proteins 58 . For example, immunodepletion spin columns, immuno-depletion-LC and magnetic beads have been commercialized to remove up to the 20 most abundant plasma proteins [59][60][61][62][63][64] . This strategy has also been extended to deplete moderately abundant proteins (e.g., complement proteins, fibronectin, plasminogen) using Affibody molecules, bead-bound peptide hexamers, or antibodies 65 60,66,67 .…”

“…In cases where comprehensive characterization of the entire plasma proteome is not necessary, a strategy around enrichment and concentration might be better suited. In this approach, workflows are used to increase the sensitivity for specific proteins of interest 62 . For example, methods have been developed to focus on specific sub-proteomes (plasma glycoproteome and phosphoproteome) [76][77][78] , proteins that have a specific activity (cytokines for signaling), or those that originate from specific compartments (membrane proteins) 79 .…”