Stable isotope-labeled paper spray mass spectrometry enables high-sensitivity profiling of protease activity

Proteases play critical roles in disease pathogenesis, and their activity detection is essential in the diagnostic processes for inflammatory disorders, cancers, and neurodegenerative diseases. Conventional approaches for protease detection, such as traditional mass spectrometry detection methods, are susceptible to interference from complex matrices. In recent decades, stable isotope-labeled internal standards (IS) have been widely employed as an effective strategy to mitigate matrix effects. However, existing stable isotope IS face challenges, including difficulty in finding suitable compounds, complex synthesis processes, and high costs. In this study, we demonstrated a novel class of stable isotope labeling reagents and applied them to construct stable isotope-labeled nanoprobes (SIL-nanoprobes). These nanoprobes allow direct, sensitive and specific quantitation of calpain-2 (CAPN2) protease activity in serum using paper spray mass spectrometry (PS-MS). The CAPN2-specific recognition peptide was covalently conjugated to AuNPs, followed by labeling with stable isotope reagents via click chemistry. Upon CAPN2 cleavage, these SIL-nanoprobes generated characteristic MS signal pairs from light and heavy isotope-labeled fragments, enabling precise MS detection. Matrix effects were significantly reduced and the detection limit was achieved to 0.3807 ng/mL. By using an internal isotope-labeled standardization approach, our present strategy enables direct and accurate quantification of CAPN2 activity using PS-MS platform, without external procurement. This strategy holds great promise for other types of protease detection systems by substituting the protease-specific peptide substrates.