A homogeneous mass-encoded strategy for mass spectrometric biosensing of multiplex proteins

Protein kinases play important roles in fundamental biological processes. Aberrant activities may result in many diseases, thus the detection of multiplex kinase activities is important in clinical diagnosis. Recent progress has focused on the mass spectrometric biosensing technique to enable highly sensitive detection of multiplex targets. However, the application of the developed methods in multiplex enzyme analysis is greatly challenged due to the substrate immobilization on solid interfaces, which affect the contact between substrates and enzymes to reduce enzyme reaction efficiency.

This work developed a homogeneous mass-encoded method for biosensing of multiplex proteins, which was performed using designed peptides containing the coding sequences and substrate regions. With the assistance of titanium dioxide coated magnetic beads (TiO₂-MBs) to capture the phosphopeptide products, followed by trypsin to cleave the products for releasing the coding sequences, the kinase assays were achieved by submitting the supernatant for ultrahigh performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) analysis. Using protein kinase A (PKA) and human epidermal growth factor receptor 2 (HER2) as model targets, the peak area ratios of the coding sequences to the internal standards showed linear relations of 1.0-100 ng mL^-1 and 0.2-20 U mL^-1, with the detection limits of 0.46 ng mL^-1 and 0.033 U mL^-1 for HER2 and PKA, respectively. The proposed strategy also demonstrated great practicability in inhibition analysis and kinase activity assays in cell lysates.

A homogeneous mass-encoded method for multiplex detection of kinase activities could simplify the assay procedure and reveal the enzyme activities with free substrates. The strategy enabled multiplex kinase activity assays with convenience, high sensitivity, and high specificity, demonstrating promising applications in clinical fields.