Abstract
In the present study, we evaluated four small molecule affinity-based probes based on agarose-immobilized benzamidine (ABA), O-Phospho-L-Tyrosine (pTYR), 8-Amino-hexyl-cAMP (cAMP), or 8-Amino-hexyl-ATP (ATP) for their ability to remove high-abundant proteins such as serum albumin from plasma samples thereby enabling the detection of medium-to-low abundant proteins in plasma samples by mass spectrometry-based proteomics. We compared their performance with the most commonly used immunodepletion method, the Multi Affinity Removal System Human 14 (MARS14) targeting the top 14 most abundant plasma proteins and also the ProteoMiner protein equalization method by label-free quantitative liquid chromatography tandem mass spectrometry (LC-MSMS) analysis. The affinity-based probes demonstrated a high reproducibility for low-abundant plasma proteins, down to picomol per mL levels, compared to the Multi Affinity Removal System (MARS) 14 and the Proteominer methods, and also demonstrated superior removal of the majority of the high-abundant plasma proteins. The ABA-based affinity probe and the Proteominer protein equalization method performed better compared to all other methods in terms of the number of analyzed proteins. All the tested methods were highly reproducible for both high-abundant plasma proteins and low-abundant proteins as measured by correlation analyses of six replicate experiments. In conclusion, our results demonstrated that small-molecule based affinity-based probes are excellent alternatives to the commonly used immune-depletion methods for proteomic biomarker discovery studies in plasma. Data are available via ProteomeXchange with identifier PXD020727.
Highlights
Human body fluids like serum, plasma, or cerebrospinal fluids are accessible in a clinical context and carry a large number of proteins that are regarded as highly informative about physiological and pathological states
These include depletion of high-abundant proteins using immune affinity approaches, selective capturing of sub-proteomes [3,4,5], protein corona formation on nanoparticles [6] or enrichment techniques alone [7,8] and/or in combinations with extensive fractionation techniques such as sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) [9], strong cation exchange (SCX) [10], isoelectric focusing (IEF) [11], hydrophilic interaction chromatrography (HILIC), and high-pH separation [9]. Despite these efforts to reduce sample complexity, plasma biomarker studies generally identify a significantly lower number of proteins compared to proteomic analysis of protein extracts from tissue biopsies or isolated from cell cultures, despite the fact that more than 10,000 proteins to date have been identified in plasma, according to the Plasma Proteome Database [12,13]
We head-to-head compared used methods for the depletion of abundant proteins in plasma samples, the Multi Affinity Removal System (MARS) 14 cartridge and the Proteominer immobilized peptide library with four affinity-based approaches, based on approaches based on cAMP, ATP, pTYR, or agarose-immobilized benzamidine (ABA) linked to agarose beads
Summary
Human body fluids like serum, plasma, or cerebrospinal fluids are accessible in a clinical context and carry a large number of proteins that are regarded as highly informative about physiological and pathological states. A great effort is made to reduce sample complexity in biomarker discovery research employing a variety of methods These include depletion of high-abundant proteins using immune affinity approaches, selective capturing of sub-proteomes [3,4,5], protein corona formation on nanoparticles [6] or enrichment techniques alone [7,8] and/or in combinations with extensive fractionation techniques such as sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) [9], strong cation exchange (SCX) [10], isoelectric focusing (IEF) [11], hydrophilic interaction chromatrography (HILIC), and high-pH separation [9]. Many of these techniques for sample complexity reduction are laborious and expensive, thereby hampering their application in a clinical context that most often requires the analysis of hundreds of samples
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