Label-free, mass spectrometric (MS) deciphering of enzymatic reactions by direct analysis of substrate-to-product conversion provides the next step toward more physiological relevant assays within drug discovery campaigns. Reduced risk of suffering from compound interference combined with diminished necessity for tailored signal mediators emphasizes the valuable role of label-free readouts. However, MS-based detection has not hitherto met high-throughput screening (HTS) requirements because of the lack of HTS-compatible sample introduction. In the present study, we report on a fully automated liquid-handling concept built in-house to concatenate biochemical assays with matrix-assisted laser desorption/ionization time-of-flight closing this technological gap. The integrated reformatting from 384- to 1536-well format enables cycle times of 0.6 s/sample for automated spotting and 0.4 s/sample for MS analysis, matching the requirements of HTS compatibility. In-depth examination of spotting quality, quantification accuracy, and instrument robustness together with the implementation of a protein tyrosine phosphatase 1B (PTP1B) inhibitor screening (4896 compounds) demonstrate the potential of the heavily inquired HTS integration of the label-free MS readout. Overall, the presented data demonstrate that the introduced automation concept makes label-free MS-based readouts accessible for HTS within drug discovery campaigns but also in other research areas requiring ultrafast MS-based detection.

Cathepsin K is a potent extracellular matrix-degrading protease that requires interactions with soluble glycosaminolycans for its collagenolytic activity in bone and cartilage. The major sources of glycosaminoglycans in cartilage are aggrecan aggregates. Therefore, we investigated whether cathepsin K activity is capable to hydrolyze aggrecan into fragments allowing the formation of glycosaminoglycan-cathepsin K complexes and determined the cleavage site specificity of cathepsin K toward the cartilage-resident link protein and aggrecan. The cleavage site specificity was compared with those of cathepsins S and L. All three cathepsins released glycosaminoglycans from native bovine cartilage at lysosomal pH and to a lesser degree at neutral extracellular pH. Cathepsin-predigested aggrecan complexes and cartilage provided suitable glycosaminoglycan fragments that allowed the formation of collagenolytically active cathepsin K complexes. A detailed analysis of the degradation of aggrecan aggregates revealed two cathepsin K cleavage sites in the link protein and several sites in aggrecan, including one site within the interglobular domain E1. In summary, these results demonstrate that cathepsin K is capable to degrade aggrecan complexes at specific cleavage sites and that cathepsin K activity alone is sufficient to self-provide the glycosaminoglycan fragments required for the formation of its collagenolytically active complex.

The release of aggrecan catabolites from cartilage is an early event in the pathogenesis of degenerative joint diseases. The enzymes involved in this process are unknown, controversial, and the subject of intense investigation. In this paper we have utilized a recombinant substrate containing the interglobular domain (IGD) of aggrecan to study specifically aggrecanase versus matrix metalloproteinase (

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