Novel mass spectrometry-based proteomic approaches to understand how the composition and dynamics of the proteome underlies key properties of cancer and stem cells
Proteins are often referred to as the work-horses of the cell, meant to indicate that they execute the biochemical processes that are essential for cellular life. In addition, they give cells their identity, endowing specialized functions to the hundreds of different cell types in the human body. What is more, protein expression even in individual cell types is highly dynamic, yet strictly controlled, to ensure that proteins of specific functionality are available in the correct amount and at the right time, allowing cells to respond to changes in their environment. Much like proteins drive normal physiological processes, they are also at the basis of many diseases, e.g. in cancer where genetic mutations result in altered protein expression to promote cell growth.
Because of the tight connection between protein expression and cellular function and phenotype, the core interest of our group is to investigate proteome composition and dynamics to reveal mechanisms that underlie cellular adaptation and that drive disease. In particular, we are interested in regulatory pathways and protein networks that are central in cancer biology, and in the processes that regulate differentiation and reprogramming in stem cells. To this end, we use state-of-the-art mass spectrometric technologies, and we develop dedicated proteomic approaches to understand the regulatory layers in the proteome, with a particular focus on secreted proteins, chromatin- and RNA-associated proteins, and nascent proteome analysis to understand wiring of signaling networks. In addition, we have a strong focus on miniaturization and automation of proteomic workflows to make proteomics a more sensitive and robust technology, especially for clinical applications.
Our research focuses on three main themes:
1. Protein expression profiling in stem cells and cancer
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Characterizing the composition of proteomes in cells and tissues is at the center of our expertise, where deep and quantitative proteome profiling is key to understand the functional differences between cell populations or to determine biological processes that underlie cell plasticity. For instance, using high-resolution mass spectrometry, this has allowed us to characterize stem and progenitor cells that shape the top of the hierarchy in the hematopoietic system (Cabezas-Wallscheid et al, Cell Stem Cell 2014), and to show that cells follow a distinct molecular trajectory during reprogramming from fibroblasts to induced pluripotent stem cells (Hansson et al, Cell reports 2012). Over the years we have implemented various improvements in the proteomic workflow, e.g. developing SP3 to streamline sample preparation especially for low-input applications (Hughes et al, Mol Syst Biol 2014; Hughes et al, Nat Protocols 2019), as shown for the characterization of human oocytes at the single-cell level (Virant-Klun et al, Mol Cell Proteomics 2016). We have integrated SP3 with ultrasonication to establish an automated proteomic pipeline for tissue proteomics, performing all steps including tissue lysis, protein extraction, clean-up, and digestion, producing peptides for direct analysis by mass spectrometry (Müller et al, Mol Syst Biol 2020). The ability to process minute sample amounts from a range of difference sources in a standardized and multiplexed manner is the basis for current and future work in various clinical proteomic applications. A major focus will be to understand tumor relapse in various cancer entities.
2. Nascent proteome and secretome analysis to understand cellular response and signaling networks
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Biological systems are highly dynamic, continuously adapting to their environment, or responding to specific extracellular signals. For instance, cancer cells shape their proteome as a consequence of genetic mutations or exposure to growth factors, with profound implications for disease progression. To understand the impact of such events at the proteome level, we have developed a nascent proteomics approach that combines pulsed-SILAC labeling, click-chemistry and mass spectrometry, and that specifically captures the immediate changes in the proteome elicited by a perturbation (Eichelbaum et al, Nat Biotechnol 2012). This can be similarly applied to intracellular proteomes and secretomes, providing a powerful means to study proteome response in a wide range of scenarios as we have shown by studying the spatio-temporal host response to bacterial infection (Selkrig et al, Nat Microbiol 2020), and by identifying secretory factors that play a crucial role in tissue remodeling upon cardiac infarction (Kuhn et al, Circulation 2020). Using nascent proteome analysis, we currently focus our efforts on the characterization of the proteome effects of oncogenic mutations and cancer drugs, aiming to understand the function and crosstalk of signaling pathways, and to reconstruct the composition of signaling networks. The long-term aim is to identify novel drug targets, and to determine drug mechanism of action.
3. Protein interaction networks with RNA and chromatin
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Among the many functions of proteins as the downstream effectors of gene expression is the regulation of their own genesis during transcription and translation. Specifically, highly elaborate protein machineries interact with DNA in chromatin to regulate transcription of genes, often in a gene-specific manner. Subsequently, proteins accompany RNA throughout their lifetime to regulate RNA transport, translation, and decay. Given this role of proteins at the core of cellular regulation, we are interested to characterize chromatin- and RNA-bound proteomes to gain a mechanistic understanding of genome regulation. We have developed a method that combines chromatin immuno-precipitation with mass spectrometry for the selective isolation of chromatin-associated proteins (ChIP-SICAP), enabling the characterization of protein networks that accompany a chromatin-bound protein of interest. When applied to the pluripotency factors Sox2, Oct4 and Nanog in stem cells, this has allowed us to identify Trim28 as a novel protein that boosts the efficiency of reprogramming (Rafiee et al, Mol Cell 2016; Rafiee et al, Mol Syst Biol 2020). In addition, we have shown that the chromatin-bound interaction network of Sox2 is highly dynamic, changing partners during differentiation to neurons to drive expression of neuron-specific genes (Bunina et al, Cell Syst 2020). The characterization of mRNA-bound proteomes is based on the capture of RNA-protein conjugates from crosslinked cells using poly-dT beads (Castello et al, Cell 2012). We have recently extended this to a more generic approach that is not restricted to mRNA, isolating protein-crosslinked RNA by organic phase extraction (Trendel et al, Cell 2019). Quantitative profiling of these interactions has revealed drastic remodeling of the RNA-bound proteome during arsenite-induced stress, pointing to adaptive processes in the translational machinery, concomitant with a rapid and previously unrecognized form of ribophagy (Trendel et al, Cell 2019). Future work will use these chromatin- and RNA-directed approaches in the context of cell signaling, to identify mechanisms that relay upstream signaling events to a downstream proteomic response.
Key publications
1. Protein expression profiling in stem cells and cancer
Cabezas-Wallscheid N, Klimmeck D, Hansson J, Lipka DB, Reyes A, Wang Q, Weichenhan D, Lier A, von Paleske L, Renders S, Wünsche P, Zeisberger P, Brocks D, Gu L, Herrmann C, Haas S, Essers MA, Brors B, Eils R, Huber W*, Milsom MD*, Plass C*, Krijgsveld J*, Trumpp A*. Identification of Regulatory Networks in HSCs and Their Immediate Progeny via Integrated Proteome, Transcriptome, and DNA Methylome Analysis. Cell Stem Cell (2014), 15(4):507-22. PMID: 25158935
Hansson J, Rafiee MR, Reiland S, Polo JM, Gehring J, Okawa S, Huber W, Hochedlinger K, Krijgsveld J. Highly coordinated proteome dynamics during reprogramming of somatic cells to pluripotency. Cell Reports (2012), 2(6):1579-92. PMID: 23260666
Hughes C, Foehr S, Garfield DA, Furlong EE, Steinmetz LM, Krijgsveld J. Ultrasensitive proteome analysis using paramagnetic bead technology. Mol. Syst. Biol. (2014); 10: 757. PMID: 25358341
Hughes C, Moggridge S, Müller T, Sorensen P, Morin G, and Krijgsveld J. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat Protocols (2019), 14(1):68-85. PMID: 30464214
Müller T, Kalxdorf M, Longuespée R, Kazdal DN, Stenzinger A, Krijgsveld J. Automated sample preparation with SP3 for low-input clinical proteomics. Mol Syst Biol. (2020), 16(1):e9111. PMID: 32129943.
Virant-Klun I, Leicht S, Hughes C, Krijgsveld J. Identification of maturation-specific proteins by single-cell proteomics of human oocytes. Mol Cell Proteomics. (2016), 15(8):2616-27. PMID: 27215607
2. Nascent proteome and secretome analysis to understand cellular response and signaling networks
Eichelbaum, K., M. Winter, M. Berriel Diaz, S. Herzig, and J. Krijgsveld. Selective enrichment of newly synthesized proteins for quantitative secretome analysis. Nat Biotechnol (2012), 30:984-990. PMID: 23000932
Kuhn TC, Knobel J, Burkert-Rettenmaier S, Li X, Meyer IS, Jungmann A, Sicklinger F, Backs J, Lasitschka F, Müller OJ, Katus HA, Krijgsveld J*, Leuschner F*. Secretome analysis of cardiomyocytes identifies PCSK6 as a novel player in cardiac remodeling after myocardial infarction. *Joint senior authors. Circulation (2020), 141:1628–1644. PMID: 32100557.
Selkrig J, Li N, Hausmann A, ManganM, Zietek M, Mateus A, Bobonis J, Sueki A, Imamura H, El Debs B, Sigismondo G, Florea BI, Overkleeft HS, Kopitar-Jerala N, Turk B, Beltrao P, Savitski M, Latz E, Hardt WD, Krijgsveld J*, Typas A*. Spatiotemporal proteomics uncovers cathepsin-dependent host cell death during bacterial infection. Nat Microbiol (2020), 5(9):1119-1133. PMID 32514074
3. Protein interaction networks with RNA and chromatin
Bunina D, Abazova N, Diaz N, Noh K-M*, Krijgsveld J*, Zaugg JB*. Genomic rewiring of SOX2 chromatin interaction network during differentiation of ESCs to postmitotic neurons. Cell Syst (2020), 10(6):480-494.e8. PMID: 32553182
Castello A, Fischer B, Schuschke K, Horos R, Beckmann B, Strein C, Humphreys DT, Preiss T, Steinmetz L, Krijgsveld J* and Hentze MW*. Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell (2020), 149(6):1393-406. PMID: 22658674
Rafiee MR, Sigismondo G, Kalxdorf M, Förster L, Brügger B, Béthune J, Krijgsveld J. Protease-resistant streptavidin for interaction proteomics. Mol Syst Biol (2020), 16(5):e9370. PMID: 32400114
Rafiee M.-R., C. Girardot, G. Sigismondo, J. Krijgsveld. Expanding the circuitry of pluripotency by selective isolation of chromatin-associated proteins. Mol Cell (2016), 64(3):624-635. PMID: 27773674
Trendel J, Schwarzl T, Horos R, Prakash A, Bateman A, Hentze MW, Krijgsveld J. 2019. The human RNA-binding proteome and its dynamics during arsenite-induced translational arrest. Cell (2019), 176(1-2):391-403. PMID: 30528433