Affinity Capture Enrichment versus Affinity Depletion: A Comparison of Strategies for Increasing Coverage of Low-Abundant Human Plasma Proteins

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.

Searching for Novel Candidate Biomarkers of RLS in Blood by Proteomic Analysis

“…The detection of FGA in this study was because the depletion efficiency of FGA was not perfect. 85 Hence, despite we observed a significant change in abundance of FGA among the different groups, this difference might have been caused by the bias introduced by the depletion steps. More investigations need to be performed for the expressions of FGA in RLS in future studies.…”

Clinical Proteomics of Biofluids in Haematological Malignancies

“…Furthermore, combining ammonium sulfate precipitation and protein A affinity chromatography was found to be an affordable and effective method of plasma HAP removal [ 24 ]. A recent comparison of chemi-proteomic affinity-based probes against the popular MARS14 and ProteoMiner techniques revealed the affinity capture enrichment as an effective alternative to current methods with several advantages associated with the use of these probes [ 25 ]. Unfortunately, the discovery of biomarkers in serum/plasma can be obstructed during HAP depletion as protein–protein interactions between LAPs, such as cytokines, and off-target effects, can risk the concomitant removal of less abundant proteins that are of potential interest [ 26 , 27 ].…”

Proteomics and Nucleotide Profiling as Tools for Biomarker and Drug Target Discovery

“…The studies presented in this Special Issue cover a variety of diseases, from brain tumours [1,2] to colorectal cancer (CRC) [3,4], thyroid cancer [5], heart failure [6] and renal failure treated with transplantation [7]. Several different platforms are used, from microarrays [6] and antibody arrays [1] to gel-based proteomics using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with mass spectrometry (MS) protein identification [3,4], strategies using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for protein identification [4] and imaging [5] and liquid chromatography-tandem mass spectrometry (LC-MS/MS) with either data-dependent acquisition (DDA) [1,3,[6][7][8] or data-independent acquisition (DIA) using sequential window acquisition of all theoretical fragment ion spectra (SWATH) technology [2]. Quantification strategies include label-free quantification [2,3,6,8] as well as labelling with tandem mass tags (TMT) [7] and isobaric tags for relative and absolute quantification (iTRAQ) [1].…”

“…Several different platforms are used, from microarrays [6] and antibody arrays [1] to gel-based proteomics using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with mass spectrometry (MS) protein identification [3,4], strategies using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for protein identification [4] and imaging [5] and liquid chromatography-tandem mass spectrometry (LC-MS/MS) with either data-dependent acquisition (DDA) [1,3,[6][7][8] or data-independent acquisition (DIA) using sequential window acquisition of all theoretical fragment ion spectra (SWATH) technology [2]. Quantification strategies include label-free quantification [2,3,6,8] as well as labelling with tandem mass tags (TMT) [7] and isobaric tags for relative and absolute quantification (iTRAQ) [1]. The material analysed varies from cultured cell lines [1,3] to tissue biopsies [3,4], formalin-fixed paraffin-embedded (FFPE) tissue [5,7], extracellular vesicles (EVs) [2] and plasma [4,8].…”

“…Quantification strategies include label-free quantification [2,3,6,8] as well as labelling with tandem mass tags (TMT) [7] and isobaric tags for relative and absolute quantification (iTRAQ) [1]. The material analysed varies from cultured cell lines [1,3] to tissue biopsies [3,4], formalin-fixed paraffin-embedded (FFPE) tissue [5,7], extracellular vesicles (EVs) [2] and plasma [4,8].…”