Selective Labeling and Identification of the Tumor Cell Proteome of Pancreatic Cancer In Vivo
Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers. Dissecting the tumor cell proteome from that of the non-tumor cells in the PDAC tumor bulk is critical for tumorigenesis studies, biomarker discovery, and development of therapeutics. However, investigating the tumor cell proteome has proven evasive due to the tumor's extremely complex cellular composition. To circumvent this technical barrier, we have combined bioorthogonal noncanonical amino acid tagging (BONCAT) and data-independent acquisition mass spectrometry (DIA-MS) in an orthotopic PDAC model to specifically identify the tumor cell proteome in vivo. Utilizing the tumor cell-specific expression of a mutant tRNA synthetase transgene, this approach provides tumor cells with the exclusive ability to incorporate an azide-bearing methionine analogue into newly synthesized proteins. The azide-tagged tumor cell proteome is subsequently enriched and purified via a bioorthogonal reaction and then identified and quantified using DIA-MS. Applying this workflow to the orthotopic PDAC model, we have identified thousands of proteins expressed by the tumor cells. Furthermore, by comparing the tumor cell and tumor bulk proteomes, we showed that the approach can distinctly differentiate proteins produced by tumor cells from those of non-tumor cells within the tumor microenvironment. Our study, for the first time, reveals the tumor cell proteome of PDAC under physiological conditions, providing broad applications for tumorigenesis, therapeutics, and biomarker studies in various human cancers.
© 2020 American Chemical Society. Received: August 27, 2020; Published: December 8, 2020. We greatly appreciate Dr. Marina Pasca di Magliano for generously providing the murine pancreatic cancer cell line (4292). We would also like to thank members of David Tirrell and Erin Schuman labs for sharing protocols on BONCAT procedures. Mass spectrometry analyses were conducted in the Mass Spectrometry Laboratory at the University of Texas Health Science Center at San Antonio. This work was supported in part by NIH K22CA207598 (Y.L.), CPRIT RP200472 (Y.L.), and NIH GM008042 (D.K.S.; UCLA-Caltech Medical Scientist Training Program). Support from the University of Texas System Proteomics Core Network for the purchase of the Lumos mass spectrometer is gratefully acknowledged. Author Contributions: N.G.A., S.T.W., and Y.L. designed the study and developed the approach. N.G.A. carried out all cell culture, animal studies, and click chemistry experiments. S.P. and D.M. conducted the DIA-MS analysis. D.K.S., S.T.W., L.N., N.G.A., and Y.L. analyzed the data. J.C. advised the study. N.G.A. and Y.L. wrote and S.T.W. edited the manuscript. All authors reviewed and approved the manuscript. The authors declare no competing financial interest.
Submitted - 2020.05.25.113670v2.full.pdf
Supplemental Material - pr0c00666_si_001.pdf
Supplemental Material - pr0c00666_si_002.xlsx