Endoprotease profiling with double-tagged peptide substrates: a new diagnostic approach in oncology

Clinical Chemistry
Teresa PeccerellaPeter Findeisen

Abstract

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. 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." 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 pep...Continue Reading

References

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Citations

May 9, 2015·International Journal of Oncology·Bassel SabbaghPeter Findeisen
Mar 20, 2015·Scientific Reports·Zaian DengTony Y Hu
Jan 4, 2012·Proteomics. Clinical Applications·Peter Findeisen, Michael Neumaier
Mar 13, 2014·Proteomics. Clinical Applications·Diego YepesPeter Findeisen
Apr 12, 2016·Proteomics. Clinical Applications·Bassel SabbaghPeter Findeisen
Aug 21, 2013·American Journal of Clinical Pathology·Peter FindeisenMichael Neumaier
Jul 30, 2015·The Cancer Journal·Jiang YangMarsha A Moses

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