Selective Labeling and Identification of the Tumor Cell Proteome of Pancreatic Cancer In Vivo

Abstract

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. KEYWORDS: pancreatic ductal adenocarcinoma (PDAC), bioorthogonal noncanonical amino acid tagging (BONCAT), data-independent acquisition mass spectrometry (DIA-MS), methionyl-tRNA synthetase (MetRS), azidonorleucine (ANL), patient-derived xenografts (PDX)

Cell-selective proteomics segregates pancreatic cancer subtypes by extracellular proteins in tumors and circulation

“…Conceptually, methionyl-tRNA synthetase L274G (MetRS*)-based azidonorleucine (Anl) labeling offers unique possibilities for analyzing intercellular interactions in complex heterocellular systems. However, the achieved proteomic depth in our initial experiments and previously published MetRS*-based studies did not exceed 4000 proteins 23 , 28 and was therefore significantly lower than state-of-the-art with modern mass spectrometers and software 29 , limiting the discovery potential. Hence, we set out to identify and overcome technical bottlenecks.…”

“…This effectively avoids cell-damage-related losses, selection bias for more robust cell populations, and potential protein expression or modification state artifacts by stresses and environmental changes during the enzymatic and mechanical treatment necessary to extract cells from tissues 24 – 27 . However, the achieved proteome coverage has generally been low, and even the deepest studies remained under 4000 specifically enriched proteins 23 , 28 , leaving open the feasibility of comprehensive Anl enrichment-based proteomics analysis.…”

Recent advancements in mass spectrometry–based tools to investigate newly synthesized proteins

“…In contrast to AHA or HPG, labeling with ANL provides the opportunity to retain some spatial information in MS-based NSP analysis, as a prerequisite for the metabolic incorporation of ANL is the (cell-specific) expression of a mutant methionine-tRNA ligase that recognizes ANL. Several studies have used this technique to monitor cell types of interest in vivo [ 43 , 44 ]. A tumor-specific proteome was consequently labeled and identified by using ANL in vivo [ 44 ].…”

“…Several studies have used this technique to monitor cell types of interest in vivo [ 43 , 44 ]. A tumor-specific proteome was consequently labeled and identified by using ANL in vivo [ 44 ]. Importantly, with this method, synthesis events in cell types of interest could be studied as well.…”

Characterization of the Secretome of a Specific Cell Expressing Mutant Methionyl-tRNA Synthetase in Co-Culture Using Click Chemistry

“…ANL-tagged proteins can be selectively conjugated and enriched through azide-alkyne cycloaddition. Therefore, if we express MetRS L274G in a specific cell, then we can specifically enrich the newly synthesized proteins, which have ANL residues in their sequences, from that cel [ 17 , 18 , 19 ].…”