Functional protease profiling with reporter peptides in serum specimens of colorectal cancer patients: demonstration of its routine diagnostic applicability
© Findeisen et al.; licensee BioMed Central Ltd. 2012
Received: 17 January 2012
Accepted: 4 April 2012
Published: 8 June 2012
The 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.
A reporter peptide (RP) with the amino acid sequence WKPYDAAD was synthesized that has a known cleavage site for the cysteine-endopeptidase cancer procoagulant (EC 22.214.171.124). 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).
RP-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.
The 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.
KeywordsFunctional protease profiling Serum Colorectal cancer Cancer procoagulant diagnosis Reporter peptide Mass spectrometry
Proteases play an important role in different biological processes including cell differentiation, inflammation and tissue remodelling, haemostasis, immunity, angiogenesis, apoptosis and malignant disease . Specifically, proteases are well known factors to promote local progression and distant metastasis of colorectal cancer and many other solid tumors [2, 3]. Furthermore, there is increasing evidence that proteases also have key functions in early stages of tumor development . The tumor-associated proteases are either secreted directly by the tumor or originate from surrounding connective tissue and infiltrating leucocytes as a result of tumor-stroma interaction . Some tumor-associated proteases like cathepsins, matrix-metalloproteases, kallikreins and cancer procoagulant (CP) are released into the bloodstream and can be used for diagnostic and prognostic purposes [6–10]. Tumor-associated protease activity in serum specimens of cancer patients can be monitored using synthetic substrates that are selectively cleaved by the protease of interest [6–9]. With the use of appropriate synthetic reporter-peptides (RPs) for spiking of serum specimens, the reaction conditions that comprise substrate concentration, incubation time and buffer composition can be optimized and standardized accordingly . Furthermore, the proteolytic fragments accumulate to the level that they become readily detectable by mass spectrometry . This approach is similar to established diagnostic assays measuring the proteolytic activity of distinct enzymes, e.g., coagulation factors .
Recently, we have described a functional protease profiling approach using a reporter peptide that is cleaved by the tumor associated protease cancer procoagulant (EC 126.96.36.199) . However, the analysis of proteolytic fragments was performed with MALDI-TOF mass spectrometry that is only a semi-quantitative method  with limited inter-day reproducibility . Furthermore, proteolytic fragments had to be extracted from serum specimens with serial affinity purification that is a rather laborious method with limited throughput and reproducibility. To alleviate these restrictions, we have developed a robust and highly reproducible liquid chromatography-mass spectrometry (LC-MS) assay for the absolute quantification of a targeted proteolytic fragment.
Peptide sequences of reporter peptide, anchor peptide and internal standard
[M + H]2+observed
[M + H]1+theoretical (monoisotopic)
The sufficient preanalytical stability of biomarkers is a prerequisite for routine diagnostic use and we could demonstrate that the tumor-associated proteolytic activity towards the reporter peptide is preserved for up to 24 h. Furthermore a small proof-of-concept experiment (n = 90) was performed to demonstrate the diagnostic power of functional protease profiling with reporter peptide spiking. Systemic inflammation has been recognized as serious threat for cancer biomarker discovery  and we selected the collective of control individuals accordingly. The concentrations of proteolytic fragments were significantly higher in serum specimens from tumor patients (TU) when compared to serum from inflammatory controls (IC) and healthy controls (HC). This indicates the presence of the tumor-associated protease cancer procoagulant that is associated with an increased cleavage of the reporter peptide in serum specimens of tumor patients.
Here we present a method to monitor controlled, ex-vivo peptide breakdown in serum samples using LC/MS with absolute quantification of the respective fragment that might lead to an activity based approach for biomarker discovery and validation.
LC-MS analysis and absolute quantification of the anchor peptide
Optimization of incubation time and reproducibility of RP-spiking
Inhibition of proteolytic reaction with iodoacetamide
The cysteine-endoprotease cancer procoagulant can specifically be inhibited by iodoacetamide  and different concentrations of protease inhibitor were added to spiked serum specimens of a tumor patient. As expected, the concentration of CP-AP is inversely proportional to the amount of iodoacetamide concentrations of serum specimens that were spiked with CP-RP. After 22 h of incubation the amount of CP-AP that accumulated in the serum specimen was taken as 100%. In the presence of 5, 25 and 100 mmol/L jodoacetamide, the CP-AP concentration was reduced down to 88%, 63% and 25% respectively (Additional file 2: Figure S2).
Preanalytical stability of cancer procoagulant activity
Functional protease profiling
Finally, we set up a proof-of-concept experiment to elucidate the applicability of protease profiling for diagnostic purposes. However, the implementation of MS as a routine diagnostic tool clearly depends on good inter-day reproducibility of the method. Three aliquots of a serum specimen from one tumor patient were randomly integrated into small series of serum specimens from patients and control individuals on four consecutive days. The median concentration of CP-AP was 31.9 μmol/L with SD of 3.3 μmol/L and CV of 10.2% (Additional file 3: Figure S3). As expected, the inter-day reproducibility is not as good as the intra-day reproducibility (see Figure 3B). However, CVs of 10% or even more are acceptable for many routine laboratory assays .
The dysregulation of protease activity plays an important role for the initiation and progression of malignant disease [1, 4]. Tumor-associated proteases like matrix metalloproteases, cathepsins, kallikrein related peptidases and members of the plasminogen activator system are secreted into the bloodstream and might be candidates for functional protease profiling (for review see ). Specifically, the tumor-associated protease cancer procoagulant is secreted from numerous malignancies including colorectal cancer into the bloodstream . Under in vivo conditions this can cause paraneoplastic coagulopathy throughout cleavage and activation of the coagulation factor X heavy chain (P00742) . The reporter peptide CP-RP comprises the cleavage site WKPYDAAD that is part of the coagulation factor X and is preferably cleaved in serum specimens of tumor patients . Adding reporter peptides to serum specimens enables the monitoring of tumor-related proteolytic activity for diagnostic use [7–9, 23, 24]. Furthermore, reporter peptide spiking offers major advantages over native MS-based peptide profiling concerning the standardization of preanalytical variabilities [6, 11]. The main focus of our present work was to optimize functional protease profiling with respect to simplified sample preparation and increased inter-day reproducibility to make it amenable as a laboratory assay for routine diagnostic use.
Recently, a sample clean-up with trichloroacetic acid (TCA) has been described that showed a sufficient recovery for peptides with a molecular weight of less than 3000 Da . Furthermore, the LC-MS technique is the method of choice for the reproducible quantification of small molecules like peptides in clinical specimens , and accordingly this technology was selected for assay development. Even at low CP-AP concentrations of 0.4 μmol/L the extracted ion chromatogram of CP-AP with m/z 515.795 shows only one single peak (see Figure 1) and this excellent signal to noise ratio makes quantitative LC/MS analyses amenable [27, 28]. Recently, criticism has been raised against functional protease profiling and it has been suggested to characterize the proteolytic activity in more detail . Here, we demonstrate that the proteolytic processing of CP-RP and thus the accumulation of CP-AP can be inhibited by the addition of a protease inhibitor. Iodoacetamide is a known cysteine protease inhibitor and reacts readily with the free thiol of cysteine residues required for the hydrolyzing proteases such as cancer procoagulant [18, 30]. The amount of CP-AP that is generated in the serum of cancer patients is inversely proportional to the concentration of iodoacetamide added (Additional file 2: Figure S2). This demonstrates that the cleavage of CP-RP and the accumulation of CP-AP is a specific reaction that is related to cysteinprotease activity.
Most interestingly, the proteolytic activity of serum specimens towards CP-RP is conserved for up to 24 h indicating a good preanalytical stability making it useful for diagnostic application (Figure 4).
Patient demographics and clinical characteristics
Here we present an optimized LC/MS assay for the quantification of a reporter peptide fragment that correlates with tumor-associated proteolytic activity in serum specimens of colorectal cancer patients. With this improved method three major observations could be made: First, the reproducibility of the assay is excellent with coefficients of variation that did not exceed 10%. Second, the tumor-associated proteolytic activity towards the reporter peptide is stable in serum specimens for up to 24 hours. Specifically, good reproducibility and sufficient preanalytical stability are major prerequisites of laboratory diagnostic assays. Third, inflammatory controls (IC) could fairly be separated from tumorpatients (TP) and this is most important as inflammation is an inherent component of cancer and many studies have identified biomarkers that are associated with inflammation rather than malignancy . However, there is a considerable overlap concerning the concentration of CP-AP in serum specimens from controls and tumorpatients. The combination of multiple reporter peptides that are processed by different tumor-associated proteases will be necessary to increase diagnostic accuracy of functional protease profiling. However, if suitable reporter peptides are available, the simultaneous quantification of multiple anchorpeptides could easily be adopted for the presented LC/MS method.
Materials and methods
Materials and chemicals
The reporter peptide (CP-RP), the anchor peptide (CP-AP) and the internal standard (IS) (Table 1) were synthesized in the functional genome analysis laboratory of the German Cancer Research Centre (Heidelberg, Germany). HPLC-grade acetonitrile was purchased from Fisher Chemicals (Germany). Formic acid was purchased from Sigma (Germany). Phosphate buffered saline pH 7.4 (PBS) was purchased from PAA Laboratories. Protease buffer: 200 mol/L TrisHCl, 20 mmol/L CaCl2, pH 7.8. Iodoacetamide and trichloroacetic acid were purchased from Sigma and Fluka respectively. All reagents and chemicals were at least of analytical grade.
Whole blood specimens were acquired from patients with metastatic colorectal tumors (n = 30) and patients without malignant disease but elevated acute phase protein CRP (n = 30) at the University Hospital Mannheim. Blood from healthy control individuals (n = 30) was taken from employees of the University Hospital Mannheim during routine laboratory testing at the works doctor’s office. Patient characteristics are summarized in Table 2. Blood collection was performed after we obtained institutional review board approval and patients’ written informed consent. After a 30 min clotting time at room temperature the specimens were centrifuged at 20°C for 10 min at 3000 x g. The serum was aliquoted and stored at −80°C until further use. All serum specimens were refrigerated within 6 hours after blood withdrawal. Any handling and processing of serum specimens from tumor patients and controls was performed in a strictly randomized and blinded manner. Measurements of C-reactive protein (CRP) and carcinoembryonic antigene (CEA) were performed on the Dimension VistaTM System (Siemens).
Serum specimens were diluted in the ratio of 1:3 with PBS to a final volume of 100 μL. The reporter peptide (CP-RP) and the internal standard (IS) were dissolved in protease buffer to a concentration of 100 μmol/L for CP-RP and 20 μmol/L for the IS. The diluted serum (50 μL) and the mix of RP and IS (50 μL) were incubated at 37°C for 3 h, 6 h or 22 h as depicted in results. The incubation was terminated by adding 100 μL of 10% (v/v) trichloroacetic acid (TCA) and the resulting mixture was kept at 4°C for 30 min prior to centrifugation for 15 min. at 4°C and 12.000 rpm in a microcentrifuge (Eppendorf). The supernatant was again centrifuged for 5 min. at 4°C and 12.000 rpm and 2 μL of the supernatant were injected onto the HPLC-column.
Liquid chromatography – mass spectrometry (LC-MS) analysis
LC-MS was performed using a nano HPLC system (UltiMate3000, Dionex) coupled to a linear ion trap Fourier Transform Ion Cyclotron Resonance mass spectrometer (LTQ-FTICR, Thermo Fisher Scientific) with a chip interface (TriVersa NanoMate, Advion). Analytical chromatography of CP-AP and IS (see Table 1) was performed on a 75 μm ID C-18 column (Dionex) with a flow of 300 nL/min and a gradient from 20-35% of buffer B in 23 min. The composition of buffer A was water with 0.1% formic acid and buffer B was 80% acetonitrile with 0,08% formic acid. Each LC run was preceded by a blank run ensuring lack of carryover of the material from the previous run. MS analysis was performed in positive ion mode, with a mass range of 250–600 m/z. MS/MS analyses were performed on the reporter peptide fragment CP-AP for sequence confirmation.
Reproducibility of reporter peptide spiking
To monitor the reproducibility of reporter peptide spiking, two distinct quality control samples were generated comprising serum specimens from five colorectal tumor patients (QCT) and five healthy control individuals (QCH), respectively. Both samples were aliquoted and stored at −80°C until further use. The QCT and QCH-samples were spiked with the reporter peptide and internal standard and incubated for 3 h, 6 h and 22 h at 37°C as described above. The proteolytic processing of the reporter peptide CP-RP resulted in the accumulation of CP-AP and the respective peak areas were used for quantification using LCQuan that is part of the Xcalibur software package (Thermo Fisher Scientific). Each QC-specimen was processed 5 times and median, standard deviation (SD) and coefficient of variation (CV) of the m/z 515.795 peak was calculated with Microsoft Excel software.
The D’Agostino-Pearson test, Mann–Whitney test and the receiver operating characteristics (ROC) calculations were performed with MedCalc (MedCalc Software). Results for continuous variables were expressed with the medians and standard deviations. Calculated P values of less than 0.05 were considered to indicate statistical significance. Correlation analyses were performed with Microsoft Excel 2002 SP-2 using the ‘add trendline’ functionality.
Liquid chromatography mass spectrometry
Coefficient of variation
Matrix-assisted laser desorption/ionization–time of flight mass spectrometry
High pressure liquid chromatography
Mass to charge ratio
Pooled serum specimens from tumor patients designated as ‘quality control tumor’
Pooled serum specimens from healthy controls designated as ‘quality control healthy’
Receiver operating characteristic
Area under the curve
Extracted ion chromatogram
We gratefully acknowledge that the costs of publication were supported by the LESSER-LOEWE Foundation e.V.
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