Developments in quantitative mass spectrometry for the analysis of proteome dynamics.
Hughes, C. & Krijgsveld, J.
Trends Biotechnol. 2012 Dec;30(12):668-76. doi: 10.1016/j.tibtech.2012.09.007.Epub 2012 Oct 27.
Two of the primary responses in a cell when perturbed are modulation of the dynamics of its constituent gene expression and protein abundance to restore steady-state homeostasis. To obtain a detailed model of the restoration of this balance, it is necessary to examine the kinetics of transcription and translation, thus going beyond establishing mere abundance levels of transcripts and proteins. In this review we discuss proteomic approaches that utilize genomic tagging and metabolic labeling to reveal turnover kinetics for cellular proteins in a high-throughput manner. Novel metabolic and multiplexed labeling techniques coupled to mass spectrometry, in combination with next-generation sequencing approaches, provide tools for studying the principles of cellular adaptation and dynamics in unprecedented detail.
Selective enrichment of newly synthesized proteins for quantitative secretome analysis.
Eichelbaum, K., Winter, M., Diaz, M.B., Herzig, S. & Krijgsveld, J.
Nat Biotechnol. 2012 Oct;30(10):984-90. doi: 10.1038/nbt.2356. Epub 2012 Sep 23.
Secreted proteins constitute a large and biologically important subset of proteins that are involved in cellular communication, adhesion and migration. Yet secretomes are understudied because of technical limitations in the detection of low-abundance proteins against a background of serum-containing media. Here we introduce a method that combines click chemistry and pulsed stable isotope labeling with amino acids in cell culture to selectively enrich and quantify secreted proteins. The combination of these two labeling approaches allows cells to be studied irrespective of the complexity of the background proteins. We provide an in-depth and differential secretome analysis of various cell lines and primary cells, quantifying secreted factors, including cytokines, chemokines and growth factors. In addition, we reveal that serum starvation has a marked effect on secretome composition. We also analyze the kinetics of protein secretion by macrophages in response to lipopolysaccharides.
Proteomic cornerstones of hematopoietic stem cell differentiation: distinct signatures of multipotent progenitors and myeloid committed cells.
Klimmeck, D., Hansson, J., Raffel, S., Vakhrushev, S.Y., Trumpp, A. & Krijgsveld, J.
Mol Cell Proteomics. 2012 Aug;11(8):286-302. doi: 10.1074/mcp.M111.016790. Epub2012 Mar 27.
Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem/progenitor cells (HSPCs, Lin(neg)Sca-1(+)c-Kit(+)) or myeloid committed precursors (Lin(neg)Sca-1(-)c-Kit(+)). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical evaluation, 893 proteins were found differentially expressed between multipotent and myeloid committed cells. The differential protein content in these cell populations points to a distinct structural organization of the cytoskeleton including remodeling activity. In addition, we found a marked difference in the expression of metabolic enzymes, including a clear shift of specific protein isoforms of the glycolytic pathway. Proteins involved in translation showed a collective higher expression in myeloid progenitors, indicating an increased translational activity. Strikingly, the data uncover a unique signature related to immune defense mechanisms, centering on the RIG-I and type-1 interferon response systems, which are installed in multipotent progenitors but not evident in myeloid committed cells. This suggests that specific, and so far unrecognized, mechanisms protect these immature cells before they mature. In conclusion, this study indicates that the transition of hematopoietic stem/progenitors toward myeloid commitment is accompanied by a profound change in processing of cellular resources, adding novel insights into the molecular mechanisms at the interface between multipotency and lineage commitment.
Highly Coordinated Proteome Dynamics during Reprogramming of Somatic Cells to Pluripotency.
Hansson J., Rafiee M.R., Reiland S., Polo J.M., Gehring J., Okawa S., Huber W., Hochedlinger K., Krijgsveld J.
Cell Rep. 2012 Dec 27;2(6):1579-92. doi: 10.1016/j.celrep.2012.10.014.
Generation of induced pluripotent stem cells (iPSCs) is a process whose mechanistic underpinnings are only beginning to emerge. Here, we applied in-depth quantitative proteomics to monitor proteome changes during the course of reprogramming of fibroblasts to iPSCs. We uncover a two-step resetting of the proteome during the first and last 3 days of reprogramming, with multiple functionally related proteins changing in expression in a highly coordinated fashion. This comprised several biological processes, including changes in the stoichiometry of electron transport-chain complexes, repressed vesicle-mediated transport during the intermediate stage, and an EMT-like process in the late phase. In addition, we demonstrate that the nucleoporin Nup210 is essential for reprogramming by its permitting of rapid cellular proliferation and subsequent progression through MET. Along with the identification of proteins expressed in a stage-specific manner, this study provides a rich resource toward an enhanced mechanistic understanding of cellular reprogramming.
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