Clinical Chemistry 56: 272-280, 2010; 10.1373/clinchem.2009.133462
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(Clinical Chemistry. 2010;56:272-280.)
© 2010 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

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

Teresa Peccerella1, Nadine Lukan2, Ralf Hofheinz2, Dirk Schadendorf3, Markus Kostrezewa4, Michael Neumaier1 and Peter Findeisen1,a

1 Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany; 2 III. Medical Clinic, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany; 3 Skin Cancer Unit, German Cancer Research Center Heidelberg, and Department of Dermatology, University Hospital of Mannheim, Mannheim, Germany; 4 Bruker Daltonik, Bremen, Germany.

aAddress correspondence to this author at: Klinikum Mannheim, Institut für Klinische Chemie, Mannheim 68167, Germany. Fax +49-(0)-621-383-3819; e-mail peter.findeisen{at}umm.de.

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.