Functional protease profiling with reporter peptides in serum specimens of colorectal cancer patients: demonstration of its routine diagnostic applicability

Abstract

BackgroundThe progression of many solid tumors is characterized by the release of tumor-associated proteases and the detection of tumor specific proteolytic activity in serum specimens is a promising diagnostic tool in oncology. Here we describe a mass spectrometry-based functional proteomic profiling approach that tracks the ex-vivo degradation of a synthetic endoprotease substrate in serum specimens of colorectal tumor patients.MethodsA reporter peptide (RP) with the amino acid sequence WKPYDAAD was synthesized that has a known cleavage site for the cysteine-endopeptidase cancer procoagulant (EC 3.4.22.26). The RP was added to serum specimens from colorectal cancer patients (n = 30), inflammatory controls (n = 30) and healthy controls (n = 30) and incubated under strictly standardized conditions. The proteolytic fragment of the RP was quantified with liquid chromatography / mass spectrometry (LC/MS).ResultsRP-spiking showed good intra- and inter-day reproducibility with coefficients of variation (CVs) that did not exceed a value of 10%. The calibration curve for the anchor peptide was linear in the concentration range of 0.4 – 50 μmol/L. The median concentration of the RP-fragment in serum specimens from tumor patients (TU: 17.6 μmol/L, SD 9.0) was significantly higher when compared to non-malignant inflammatory controls (IC: 11.1 μmol/L, SD 6.1) and healthy controls (HC: 10.3 μmol/L, SD 3.1). Highest area under receiver operating characteristic (AUROC) values were seen for discrimination of TU versus HC (0.89) followed by TU versus IC (0.77). IC and HC could barely be separated indicated by an AUROC value of 0.57. The proteolytic activity towards the RP was conserved in serum specimens that were kept at room temperature for up to 24 hours prior to the analysis.ConclusionThe proteolytic cleavage of reporter peptides is a surrogate marker for tumor associated proteolytic activity in serum specimens of cancer patients. A simple, robust and highly reproducible LC/MS method has been developed that allows the quantification of proteolytic fragments in serum specimens. The preanalytical impact of sample handling is minimal as the tumor-associated proteolytic activity towards the reporter peptide is stable for at least up to 24 h. Taken together, the functional protease profiling shows characteristics that are in line with routinely performed diagnostic assays. Further work will focus on the identification of additional reporter peptides for the construction of a multiplex assay to increase diagnostic accuracy of the functional protease profiling.

Multiplex profiling of tumor‐associated proteolytic activity in serum of colorectal cancer patients

“…The reproducibility of RP spiking was tested with quality control specimens (QCH and QCT) that were randomly interspersed into measurements of serum from tumor patients and controls. The CV was inversely correlated to the concentration of AP [10]. Lowest CV of 5.1% was found in QCT specimens for RP132 with median concentration of 37.0 mol/L (SD 1.9).…”

“…Only five of 144 RPs showed clearly increased cleavage in pooled serum specimens from tumor patients (QCT) when compared to HC (QCH). RP132 has already been validated in previous studies [10,11], whereas RP14, RP46, RP48, and RP83 were newly identified targets for detection of tumor-associated proteolytic activity.…”

“…Recently, we described the use of a synthetic RP for the detection of tumor-associated proteolytic activity in blood of colorectal cancer patients [10][11][12]. These initial studies were performed with only one single RP that has a cleavage site originated form coagulation factor X (P00742) and is a known substrate of the cysteine protease cancer procoagulant.…”

“…4B). This overlap of signal intensities between different groups does result in limited diagnostic power of one single marker [10] and the combination of multiple markers might improve the classification accuracy.…”

“…Most interesting for diagnostic purpose is the fact that some of these tumor‐associated proteases are secreted and released into the bloodstream . By adding appropriate synthetic RPs the tumor‐associated protease activity in specimens of cancer patients can be monitored ex vivo .…”