Endoprotease Profiling with Double-Tagged Peptide Substrates: A New Diagnostic Approach in Oncology

Abstract

Background: The measurement of disease-related proteolytic activity in complex biological matrices like serum is of emerging interest to improve the diagnosis of malignant diseases. We developed a mass spectrometry (MS)-based functional proteomic profiling approach that tracks degradation of artificial endoprotease substrates in serum specimens. Methods: The synthetic reporter peptides that are cleaved by tumor-associated endopeptidases were systematically optimized with regard to flanking affinity tags, linkers, and stabilizing elements. Serum specimens were incubated with reporter peptides under standardized conditions and the peptides subsequently extracted with affinity chromatography before MS. In a pilot study an optimized reporter peptide with the cleavage motif WKPYDAADL was added to serum specimens from colorectal tumor patients (n = 50) and healthy controls (n = 50). This reporter peptide comprised a known cleavage site for the cysteine-endopeptidase “cancer procoagulant.” Results: Serial affinity chromatography using biotin- and 6xHis tags was superior to the single affinity enrichment using only 6xHis tags. Furthermore, protease-resistant stop elements ensured signal accumulation after prolonged incubation. In contrast, signals from reporter peptides without stop elements vanished completely after prolonged incubation owing to their total degradation. Reporter-peptide spiking showed good reproducibility, and the difference in proteolytic activity between serum specimens from cancer patients and controls was highly significant (P < 0.001). Conclusions: The introduction of a few structural key elements (affinity tags, linkers, d-amino acids) into synthetic reporter peptides increases the diagnostic sensitivity for MS-based protease profiling of serum specimens. This new approach might lead to functional MS-based protease profiling for improved disease classification.

Substrate optimization and clinical validation of reporter peptides for MS-based protease profiling in serum specimens: A new approach for diagnosis of malignant disease

“…With the use of appropriate synthetic reporter peptides (RPs) for the spiking of serum specimens, the reaction conditions that comprise of substrate concentration, incubation time and buffer composition, can be optimized and standardized accordingly. Furthermore, the proteolytic fragments accumulate to a level where they become readily detectable with mass spectrometry (6). This approach is similar to established diagnostic assays measuring the proteolytic activity of distinct enzymes, e.g., coagulation factors (11).…”

“…Nevertheless, tumor-associated protease activity in serum specimens of cancer patients can be monitored by using specific synthetic substrates that are selectively cleaved by the protease of interest (4)(5)(6)(7). With the use of appropriate synthetic reporter peptides (RPs) for the spiking of serum specimens, the reaction conditions that comprise of substrate concentration, incubation time and buffer composition, can be optimized and standardized accordingly.…”

“…The success of mass spectrometry-based functional endoprotease profiling with RP spiking has two essential prerequisites (6). Firstly, the RPs must be protected from unwanted proteolytic processing by genuine serum proteases.…”

“…Pooled serum specimens from tumor patients and healthy controls were diluted 1:3 with PbS pH 7.4 (PAA Laboratories). Subsequently, 50 µl from 100 µM/ml substrate solutions were mixed with 50 µl 1:3 diluted serum sample and incubated at 37˚C for 2 h. Proteolytic fragments of RPs were extracted from serum specimens as previously described (6). Briefly, streptavidin sepharose high-performance slurry (gE Healthcare) was washed twice and resuspended in PbS pH 7.4 to yield 50% slurry.…”

“…Some tumor-associated proteases, such as matrix metalloproteinases (MMPs), cathepsines or cancer procoagulant (cP) are released into the bloodstream and can be used for diagnostic (4)(5)(6)(7) and prognostic purposes (8). However, due to their low concentration, the detection of tumor-associated proteolytic activity in serum specimens is very challenging.…”