This method has been successfully used to visualize mRNA translation in situ however, it requires predepletion of intracellular endogenous methionine followed by supplementation of the amino acid analog, both of which can result in cellular stress and potential alteration of translation patterns ( Vaughan et al. 2009).Īnother proteomic approach is based on incorporation of a modified methionine analog called azidohomoalanine (AHA), which is subsequently derivatized to tagged reporter molecules or an affinity purification matrix through click chemistry. This method, termed pulsed SILAC (pSILAC), is successfully used to detect long-lasting changes in protein production and degradation but is incompatible for studying rapid fluctuations because accurate quantification of SILAC pairs requires relatively long pulses ( Schwanhausser et al. To distinguish newly synthesized from pre-existing proteins, cells are pulse-labeled with SILAC amino acids, allowing only proteins produced during the pulse to incorporate the label. For high-resolution identification and quantification of proteins, metabolic labeling was combined with mass spectrometric (MS) analysis, and the radioactive amino acids were replaced by stable isotope-labeled (SILAC ) amino acids. Traditionally, translation rates have been monitored by metabolic labeling of cells with radioactive amino acids. The ability to identify and quantify the proteins produced in a population of cells under various conditions is therefore essential to our understanding of the processes underlying gene expression. This provides cells with the plasticity needed to rapidly modulate gene expression in response to changes in environmental conditions (e.g., cellular stress) and also for fine-tuning of protein levels during cell cycle progression, proliferation, and differentiation ( Calkhoven et al. Although mRNA levels are commonly used as a proxy of protein amounts, comparative genomic and proteomic analyses of different species and cell types have shown that mRNA and protein levels do not correlate perfectly, thereby stressing the important contribution of translation control and protein stability to gene expression ( Vogel and Marcotte 2012). In past decades, major efforts were invested in studying transcription regulation, while research focusing on post-transcriptional control has lagged behind. MRNA translation is a key step in gene expression that attracts increasing attention at the systems biology level. This simple and economical technique is broadly applicable to any cell type and tissue, enabling the identification and quantification of rapid proteome responses under various biological conditions. Using PUNCH-P, we measured cell cycle-specific fluctuations in synthesis for >5000 proteins in mammalian cells, identified proteins not previously implicated in cell cycle processes, and generated the first translational profile of a whole mouse brain. In this study, we describe a novel system-wide proteomic approach for direct monitoring of translation, termed puromycin-associated nascent chain proteomics (PUNCH-P), which is based on incorporation of biotinylated puromycin into newly synthesized proteins under cell-free conditions followed by streptavidin affinity purification and liquid chromatography-tandem mass spectrometry analysis. Mass-spectrometric and RNA sequencing methods have been recently developed for investigating mRNA translation at a global level, but these still involve technical limitations and are not widely applicable. Of course, new languages are being added all the time, so if you don't see the language or dialect you need in our list of supported languages, keep checking back.Monitoring protein synthesis is essential to our understanding of gene expression regulation, as protein abundance is thought to be predominantly controlled at the level of translation. Īnd our text translator is available in more than 60 languages for clear, seamless instant messaging. Our voice translator can currently translate conversations from following languages, including Arabic, Bulgarian, Catalan, Chinese (Simplified), Chinese (Traditional), Croatian, Czech, Danish, Dutch, German, Greek, English (UK), English (US), Spanish (Spain), Spanish (Mexico), Estonian, Finnish, French (Canada), French (France), Hindi, Hungarian, Indonesian, Italian, Japanese, Korean, Lithuanian, Latvian, Malay, Norwegian Bokmål, Polish, Portuguese (Portugal), Portuguese (Brazil), Romanian, Russian, Slovak, Slovenian, Swedish, Thai, Turkish, Vietnamese. Whether you need to translate English to Spanish, English to French, or communicate in voice or text in dozens of languages, Skype can help you do it all in real time – and break down language barriers with your friends, family, clients and colleagues.
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