Stabilizing the Proteomes of Acute Myeloid Leukemia Cells: Implications for Cancer Proteomics.

Previous work has shown that inhibition of abundant myeloid azurophil granule-associated serine proteases (ELANE, PRTN3, CTSG, and NSP4) is required to stabilize some proteins in myeloid cells. We therefore hypothesized that effective inhibition of these proteases may be necessary for mass-spectrometry based proteomics of samples containing myeloid cells. To test this hypothesis, we thawed viably preserved acute myeloid leukemia (AML) cells from cryovials in the presence or absence of diisopropyl flurophosphate (DFP), a cell-permeable, irreversible serine protease inhibitor. Global proteomic analysis was performed, using label-free and isobaric peptide-labeling quantitation. The presence of DFP resulted in an increase of identified tryptic peptides (14-57%) and proteins (9-31%). In the absence of DFP, 11-31% of all detected peptide intensity came from non-tryptic peptides; most (52-75%) had cleavage specificity consistent with the activity of ELANE/PRTN3. Treatment with DFP reduced the intensity of non-tryptic peptides to 4-8% of the total peptide intensity. Consistent with this reduction of tryptic peptide generation, ELANE inhibition was 95%, based on diisopropyl phosphate modification of active site serine residue. Overall, the measured abundance of 20% of proteins was significantly altered by DFP treatment. These results suggest that active myeloid serine proteases, released during sample processing, can skew quantitative proteomic measurements through artifactual changes in relative protein abundance. Finally, significant ELANE activity was also detected in Clinical Proteomics Tumor Analysis Consortium (CPTAC) datasets of solid tumors (many of which have known myeloid infiltration). In the pancreatic cancer dataset, the median percentage of non-tryptic intensity detected across patient samples was 34%, with many patient samples having more than half of their detected peptide intensity from non-tryptic cleavage events consistent with ELANE cleavage specificity. This study suggests that in vitro cleavage of proteins by myeloid serine proteases may be relevant for proteomic studies of any tumor that contains infiltrating myeloid cells.