Kinase-loaded magnetic beads for sequentialin vitrophosphorylation of peptides and proteins

Abstract

Post-translational modifications, including phosphorylation, greatly impact the physiological function of proteins, especially those that are natively unfolded and implicated in many neurodegenerative diseases. However, structural and functional studies of such proteins require fully defined phosphorylation, including those that are not physiological. Thus, the kinases ERK2 and GSK-3β were immobilized to various superparamagnetic beads with carboxylic, aldehyde, Ni, or Co functional groups, with a view to efficiently phosphorylate peptides and proteins in vitro. Full phosphorylation of specific synthetic peptides confirmed that beads were successfully loaded with kinases. Remarkably, enzymes covalently immobilized on carboxylated SeraMag beads remained active upon reuse, with residual activity after 10 uses 99.5 ± 0.34% for GSK-3β and 36.2 ± 2.01% for ERK2. The beads were also used to sequentially phosphorylate recombinant tau, which in vivo is a biomarker of Alzheimer's disease. Thus, a system consisting of two fully active kinases immobilized to magnetic beads is demonstrated for the first time. In comparison to soluble enzymes, the beads are easier to handle, reusable, and thus low-cost. Importantly, these beads are also convenient to remove from reactions to minimize contamination of phosphorylated products or to exchange with other kinases.

Amorphous TiO2 Nanotubes as a Platform for Highly Selective Phosphopeptide Enrichment

“…This might be important, for example, in studies focused on the analysis of hyperphosphorylated proteins related to certain pathology states. 7,8…”

“…5 One of the major goals in phosphoproteomics, the field that studies the dynamic changes of phosphoproteins over time, is to identify and describe the relationship of the phosphorylation regulation with the mechanism and pathogenesis of many serious diseases. 6−8…”

Automated Synthesis of Heavily Phosphorylated Peptides

“…[8] Elucidating the effect of Thr/ Ser phosphorylation pattern on the activity using a full-length protein is very difficult since most biochemical strategies produce either mutated analogs or mixtures of proteins with heterogeneous phosphorylation patterns. [9][10][11][12][13] Synthesizing peptides, each with a specific phosphorylated pattern, is thus pivotal in studying the effect of phosphorylation patterns on the biological activity of proteins.…”

Repetitive Synthesis of High‐Molecular‐Weight Hyaluronic Acid with Immobilized Enzyme Cascades

“…Magnetic carriers offer a time‐saving inexpensive way to separate enzymes from the reaction just by applying a magnetic field [33] . Therefore, easy reusability of enzymes is enabled [35,36] …”