Proteomic analysis of cell fate decision.
Hansson, J. & Krijgsveld, J.
Curr Opin Genet Dev. 2013 Oct;23(5):540-7. doi: 10.1016/j.gde.2013.06.004. Epub2013 Aug 10.
The field of proteomics is progressing at a rapid pace, developing from primarily a specialist technology to a valuable tool in biological research. Importantly, the establishment of mass spectrometry as a quantitative method, miniaturisation of liquid chromatography techniques, and improved sensitivity of mass-spectrometric instrumentation now enable near-complete monitoring of cellular proteome dynamics. An increasing number of studies are therefore now applying quantitative proteomics to study proteins and posttranslational modifications in stem cells, to reveal molecular mechanisms and pathways underlying pluripotency, differentiation and reprogramming.
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.
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.