posted on 2021-10-20, 19:18authored byFrank Klont, Marcel Kwiatkowski, Alen Faiz, Thea van den Bosch, Simon D. Pouwels, Frank J. Dekker, Nick H.T. ten Hacken, Péter Horvatovich, Rainer Bischoff
Affinity ligands
such as antibodies are widely used in (bio)medical
research for purifying proteins from complex biological samples. These
ligands are generally immobilized onto solid supports which facilitate
the separation of a captured protein from the sample matrix. Adsorptive
microtiter plates are commonly used as solid supports prior to immunochemical
detection (e.g., immunoassays) but hardly ever prior to liquid chromatography–mass
spectrometry (LC-MS-)-based detection. Here, we describe the use of
adsorptive microtiter plates for protein enrichment prior to LC-MS
detection, and we discuss opportunities and challenges of corresponding
workflows, based on examples of targeted (i.e., soluble receptor for
advanced glycation end-products (sRAGE) in human serum) and discovery-based
workflows (i.e., transcription factor p65 (NF-κB) in lysed murine
RAW 264.7 macrophages and peptidyl-prolyl cis–trans isomerase
FKBP5 (FKBP5) in lysed human A549 alveolar basal epithelial cells).
Thereby, we aim to highlight the potential usefulness of adsorptive
microtiter plates in affinity purification workflows prior to LC-MS
detection, which could increase their usage in mass spectrometry-based
protein research.