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Proc Natl Acad Sci U S A. 2014 May 27;111(21):7594-9. doi: 10.1073/pnas.1405987111. Epub 2014 May 12.

Circulating proteolytic signatures of chemotherapy-induced cell death in humans discovered by N-terminal labeling.

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Departments of Laboratory Medicine,Pharmaceutical Chemistry, and.
Division of Hematology and Medical Oncology and.
Departments of Laboratory Medicine,Laboratory Medicine Service, Veterans Affairs Medical Center, San Francisco, CA 94121.
Departments of Laboratory Medicine.
Pharmaceutical Chemistry, andCellular and Molecular Pharmacology, University of California, San Francisco, CA 94158; and


It is known that many chemotherapeutics induce cellular apoptosis over hours to days. During apoptosis, numerous cellular proteases are activated, most canonically the caspases. We speculated that detection of proteolytic fragments released from apoptotic cells into the peripheral blood may serve as a unique indicator of chemotherapy-induced cell death. Here we used an enzymatic labeling process to positively enrich free peptide α-amines in the plasma of hematologic malignancy patients soon after beginning treatment. This N-terminomic approach largely avoids interference by high-abundance proteins that complicate traditional plasma proteomic analyses. Significantly, by mass spectrometry methods, we found strong biological signatures of apoptosis directly in the postchemotherapy plasma, including numerous caspase-cleaved peptides as well as relevant peptides from apoptotic and cell-stress proteins second mitochondria-derived activator of caspases, HtrA serine peptidase 2, and activating transcription factor 6. We also treated hematologic cancer cell lines with clinically relevant chemotherapeutics and monitored proteolytic fragments released into the media. Remarkably, many of these peptides coincided with those found in patient samples. Overall, we identified 153 proteolytic peptides in postchemotherapy patient plasma as potential indicators of cellular apoptosis. Through targeted quantitative proteomics, we verified that many of these peptides were indeed increased post- vs. prechemotherapy in additional patients. Our findings reveal that numerous proteolytic fragments are released from dying tumor cells. Monitoring posttreatment proteolysis may lead to a novel class of inexpensive, rapid biomarkers of cell death.

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