Robust Substrate Profiling Method Reveals Striking Differences in Specificities of Serum and Lung Fluid Proteases

Watson, Douglas S.; Jambunathan, Kalyani; Askew, David; Kodukula, Krishna; Galande, Amit K.

doi:10.2144/000113717

Cited by 10 publications

(29 citation statements)

References 32 publications

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“…This correlates to earlier studies profiling proteolytic activities using internally quenched fluorogenic probes [32,33]. Different metabolic markers resulting from thrombin assisted protein degradation, e.g., FPA and its metabolites, provide some information about the coagulation progress and the proteolytic activity in serum and plasma [34,35].…”

Section: Discussionsupporting

confidence: 81%

Differential stability of therapeutic peptides with different proteolytic cleavage sites in blood, plasma and serum

2017

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Proteolytic degradation of peptide-based drugs is often considered as major weakness limiting systemic therapeutic applications. Therefore, huge efforts are typically devoted to stabilize sequences against proteases present in serum or plasma, obtained as supernatants after complete blood coagulation or centrifugation of blood supplemented with anticoagulants, respectively. Plasma and serum are reproducibly obtained from animals and humans allowing consistent for clinical analyses and research applications. However, the spectrum of active or activated proteases appears to vary depending on the activation of proteases and cofactors during coagulation (serum) or inhibition of such enzymes by anticoagulants (plasma), such as EDTA (metallo- and Ca2+-dependent proteases) and heparin (e.g. thrombin, factor Xa). Here, we studied the presumed effects on peptide degradation by taking blood via cardiac puncture of CD-1 mice using a syringe containing a peptide solution. Due to absence of coagulation activators (e.g. glass surfaces and damaged cells), visible blood clotting was prevented allowing to study peptide degradation for one hour. The remaining peptide was quantified and the degradation products were identified using mass spectrometry. When the degradation rates (half-life times) were compared to serum derived freshly from the same animal and commercial serum and plasma samples, peptides of three different families showed indeed considerably different stabilities. Generally, peptides were faster degraded in serum than in plasma, but surprisingly all peptides were more stable in fresh blood and the order of degradation rates among the peptides varied among the six different incubation experiments. This indicates, that proteolytic degradation of peptide-based therapeutics may often be misleading stimulating efforts to stabilize peptides at degradation sites relevant only in vitro, i.e., for serum or plasma stability assays, but of lower importance in vivo.

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Section: Discussionsupporting

confidence: 81%

Differential stability of therapeutic peptides with different proteolytic cleavage sites in blood, plasma and serum

2017

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“…To achieve this goal, it will be necessary to systematically identify reporter peptide sequences that are most efficiently cleaved by disease-specific proteases. However, any multiplex assay for functional protease profiling might implement the development of kinetic measurements and the need for chromogenic protease substrates [36]. Further work will focus on the identification of additional reporter peptides that are cleaved by other tumor-associated proteases e.g.…”

Section: Discussionmentioning

confidence: 99%

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

Findeisen

Costina

Yepes

et al. 2012

J Exp Clin Cancer Res

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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.

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“…These probes remain optically silent in the uncleaved state, but when recognized and cleaved by an appropriate protease they emit a fluorescent signal with the signal intensity proportional to the extent of cleavage. This technique was initially used to determine substrate specificities of purified recombinant proteases (Backes et al, 2000; Thomas et al, 2006) and we have extended it to map the proteolytic signatures of serum, BAL and other complex biological fluids (Watson et al, 2011a; Watson et al, 2011b). …”

Section: Introductionmentioning

confidence: 99%

Proteolytic Fingerprinting of Complex Biological Samples Using Combinatorial Libraries of Fluorogenic Probes

et al. 2012

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Proteases have garnered interest as candidate biomarkers and therapeutic targets for many human diseases. A key challenge is the identification and characterization of disease-relevant proteases in the complex milieu of biological fluids such as serum, plasma and bronchoalveolar lavage, in which a multitude of hydrolases act in concert. We have successfully utilized a concise combinatorial library of internally quenched fluorogenic peptide probes (IQFPs) to map the global proteolytic substrate specificities of complex biological samples. The substrate profiling approach provides a global and a quantitative comparison of protease specificities between different biological samples. Such a comparative analysis can lead to the identification of disease-specific ‘fingerprints’ of proteolytic activities with potential utility in diagnosis and therapy.

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Robust Substrate Profiling Method Reveals Striking Differences in Specificities of Serum and Lung Fluid Proteases

Cited by 10 publications

References 32 publications

Differential stability of therapeutic peptides with different proteolytic cleavage sites in blood, plasma and serum

Differential stability of therapeutic peptides with different proteolytic cleavage sites in blood, plasma and serum

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

Proteolytic Fingerprinting of Complex Biological Samples Using Combinatorial Libraries of Fluorogenic Probes

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