Development of a selected reaction monitoring mass spectrometry-based assay to detect asparaginyl endopeptidase activity in biological fluids
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Anindita Dutta1,2,*, David N. Potier1,*, Michael J. Walker1, Oliver J. Gray3, Catriona Parker4, Mark Holland4, Andrew J.K. Williamson1, Andrew Pierce1, Richard D. Unwin1,5, Shekhar Krishnan2, Vaskar Saha2,4,*, Anthony D. Whetton1,3,*
1Stem Cell and Leukaemia Proteomics Laboratory, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
2Tata Translational Cancer Research Centre, Kolkata, India
3Stoller Biomarker Discovery Centre, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
4Children’s Cancer Group, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
5Current address: Centre for Advanced Discovery and Experimental Therapeutics, Central Manchester University Hospitals NHS Foundation Trust and Institute of Human Development, University of Manchester, Manchester, UK
*These authors have contributed equally to this work
Anthony D. Whetton, email: [email protected]
Keywords: legumain, AEP, biomarker, SRM, protease
Received: May 16, 2016 Accepted: September 15, 2016 Published: September 23, 2016
Cancer Biomarkers have the capability to improve patient outcomes. They have potential applications in diagnosis, prognosis, monitoring of disease progression and measuring response to treatment. This type of information is particularly useful in the individualisation of treatment regimens. Biomarkers may take many forms but considerable effort has been made to identify and quantify proteins in biological fluids. However, a major challenge in measuring protein in biological fluids, such as plasma, is the sensitivity of the assay and the complex matrix of proteins present. Furthermore, determining the effect of proteases in disease requires measurement of their activity in biological fluids as quantification of the protein itself may not provide sufficient information. To date little progress has been made towards monitoring activity of proteases in plasma. The protease asparaginyl endopeptidase has been implicated in diseases such as breast cancer, leukaemia and dementia. Here we describe a new approach to sensitively and in a targeted fashion quantify asparaginyl endopeptidase activity in plasma using a synthetic substrate peptide protected from nonspecific hydrolysis using D-amino acids within the structure. Our selected reaction monitoring approach enabled asparaginyl endopeptidase activity to be measured in human plasma with both a high dynamic range and sensitivity. This manuscript describes a paradigm for future development of assays to measure protease activities in biological fluids as biomarkers of disease.
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