Researchers from Washington University School of Medicine have made a significant breakthrough in the field of cancer proteomics, particularly in the understanding of acute myeloid leukemia (AML) cells. Published in the highly-regarded journal Molecular & Cellular Proteomics, their study could change the way scientists examine the proteomes of AML and other cancers infiltrated by myeloid cells. The discoveries discussed in the study are now available online with DOI: 10.1016/j.mcpro.2024.100716, offering new insights into the stabilization of proteomes within AML cells and the implications for broader cancer proteomics.
The study was led by a team of experts including Robert W. Sprung, Qiang Zhang, Michael H. Kramer, Matthew C. Christopher, Petra Erdmann-Gilmore, Yiling Mi, James P. Malone, Timothy J. Ley, and R. Reid Townsend. The cornerstone of the investigation lies in the manipulation of serine proteases, which researchers previously identified as disruptive to proteomic analyses due to their abundant presence in myeloid azurophil granules and their effect on protein stability in myeloid cells.
The researchers hypothesized that the inhibition of these serine proteases – specifically ELANE, PRTN3, CTSG, and NSP4 – is crucial for accurate mass-spectrometry-based proteomics in samples containing myeloid cells. To test their theory, they thawed viably preserved AML cells from cryovials with and without the application of diisopropyl fluorophosphate (DFP), a cell-permeable, irreversible inhibitor of serine proteases.
The proteomic analysis revealed staggering results. The presence of DFP led to a notable increase in the quantity of identified tryptic peptides, ranging from 14% to 57%, and proteins, by 9% to 31%. Without DFP, non-tryptic peptides accounted for a significant 11%-31% of all detected peptide intensity. A majority of the cleavages producing non-tryptic peptides (52%-75%) were consistent with the actions of ELANE/PRTN3. Once DFP was added, it drastically cut the intensity of non-tryptic peptides to just 4%-8% of the total.
Their findings didn’t stop there. The inhibition of ELANE reached an astounding 95%, observable through the diisopropyl phosphate modification of the active site serine residue. By reducing the generation of non-tryptic peptides, the researchers observed that the measured abundance of approximately 20% of proteins was significantly affected by DFP treatment. This compelling evidence demonstrates the overarching impact that the active serine proteases can have when released during sample processing, potentially skewing the quantitative outcomes of proteomic measurements by artificially altering the relative abundance of proteins.
Adding another layer to their research, the team also delved into the Clinical Proteomics Tumor Analysis Consortium (CPTAC) datasets, specifically those involving solid tumors with known myeloid infiltration. In the pancreatic cancer dataset, they found that the median percentage of non-tryptic intensity detected across patient samples was a concerning 34%. Some patients even exhibited over half of their detected peptide intensity arising from non-tryptic cleavages in line with ELANE’s cleavage specificity.
The significance of their research is profound. It implies that the in vitro processing of proteins by myeloid serine proteases could have far-reaching implications for proteomic studies of any cancer type that possesses myeloid cell infiltration.
The findings of Sprung et al. have bolstered the notion that precision in proteomic measurements is not just a matter of sophisticated equipment and methodology but also hinges on thorough preparatory handling of samples to eliminate external variables such as enzymatic degradation.
The implications of this research are far-reaching, setting a precedent for future studies in cancer proteomics. The addition of DFP or similar serine protease inhibitors during the preparation of AML samples and potentially other cancers with myeloid infiltration could revolutionize the accuracy and reliability of proteomic data, leading to a better understanding of the molecular underpinnings of cancer and enhancing the development of targeted treatments.
For those involved in the research and study of cancer proteomics, the article’s full text is a must-read discussion of this ground-breaking work. The researchers’ dedication to improving our fundamental understanding is a testament to the importance of quality control measures in scientific research, particularly within the realms of oncology and proteomics. The scientific community and future cancer research will undoubtedly benefit from the implications of this study.
References
1. Sprung, R. W., Zhang, Q., Kramer, M. H., Christopher, M. C., Erdmann-Gilmore, P., Mi, Y., … & Townsend, R. R. (2024). Stabilizing the Proteomes of Acute Myeloid Leukemia Cells: Implications for Cancer Proteomics. Molecular & Cellular Proteomics, 100716. https://doi.org/10.1016/j.mcpro.2024.100716
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Keywords
1. Acute Myeloid Leukemia Proteomics
2. Serine Protease Inhibition Cancer
3. Mass-Spectrometry Proteome Analysis
4. Myeloid Cell Proteome Stability
5. Proteomic Sample Preparation