ABSTRACT: we prepared synthetic proteins corresponding to 80 of the ribosome subunits by stable isotope labeling and then performed absolute quantification.
Project description:we prepared synthetic proteins corresponding to 30 of the proteasome subunits by stable isotope labeling and then performed absolute quantification.
Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. Total liver RNA was mixed with 2.5 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled by 3â ligation. Total RNA mix was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool (n = 6). The synthetic miRNA pool consisted of 2.5 fmol of each of 891 non redundant miRNAs sequences and miRControl 3 sequences. The array data was normalized by calculating the median of the miRControl 3 present in the liver and UR sample. The miRNA amount was calculated with respect to the corresponding miRNA in the UR.
Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. The RNA extracted from 7 x 10^ 5 to 1 x 10^ 6 CD34(+)/CD133(-) cells of three different donors was analyzed. 1 µg of respective total RNA was mixed with 2.5 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled by 3’ ligation. Total RNA mix was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 2.5 fmol of each of 954 non redundant miRNAs sequences and miRControl 3 sequences. The array data was normalized by calculating the median of the miRControl 3 present in the CD34(+)/CD133(-) and UR sample. The miRNA amount was calculated with respect to the corresponding miRNA in the UR.
Project description:The aim of this study was to determine the fate of ribosomes and r-proteins. In this respect, SILAC (Stable Isotope Labeled Amino acids in cell Culture) based experimental approach was used (Fig. 1). E.coli cells were grown in MOPS medium supplemented with “heavy” labeled arginine (Arg10) and lysine (Lys8). At the mid-log phase, the culture was further supplemented with a 20-fold molar excess of “light” unlabeled arginine (Arg0) and lysine (Lys0), divided into 8 aliquots, and grown for 14 days. Cell samples were collected at day one (24h), day two (48h), and subsequently in 48h intervals over the following 12 days. The ribosome particles were isolated using sucrose gradient centrifugation. the quantities of r-proteins in the 70S ribosome fraction were determined using SILAC based LC-MS/MS and normalized to the corresponding values of day one.
Project description:Absolute quantification of proteome is one of the most important tasks in proteomic research. The aim in this analysis is providing proof-of-principle of our originally developed strategy for absolute quantification of multiple proteins present at various concentrations in a cell, using stable isotope-labeled peptide concatenated standard (PCS). This study also provides insights into structures of regulation of metabolic pathway and into biological mechanisms for regulation of expressed protein abundance.
Project description:Major histocompatibility complex (MHC) class I peptides play a critical role in immune cell recognition. Cancer cells modulate surface MHC levels in response to therapy, thereby affecting antitumor immunity. However, understanding the peptide repertoire response to treatment remains challenging and is limited by quantitative mass spectrometry-based strategies lacking robust normalization controls. We describe a novel approach that leverages recombinant heavy isotope-coded peptide MHCs (hipMHCs) and multiplex isotope tagging for quantitation of peptide repertoires using low sample input. HipMHCs improve quantitative accuracy by normalizing for variation across analyses, and enable absolute quantification using internal calibrants to determine copies per cell of MHC antigens. Application of this platform to profile the immunopeptidome response to CDK4/6 inhibition and Interferon gamma, known modulators of antigen presentation, uncovered treatment-specific alterations that connect the intracellular response to extracellular immune presentation. This method quantifies repertoire changes that can inform targeted and combination immunotherapy design.
Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells.
Project description:A quantitative view of cellular functions requires precise measures of the rates of biomolecule production, especially proteins-the direct effectors of biological processes. Here we present a genome-wide approach, based on ribosome profiling, for measuring absolute protein synthesis rates. The resultant E. coli dataset transforms our understanding of the extent to which protein synthesis is precisely controlled to optimize function and efficiency. For example, members of multi-protein complexes are made in precise proportion to their stoichiometry, whereas components of functional modules are produced differentially according to their hierarchical role. Estimates of absolute protein abundance also reveal principles used to optimize design. These include how the level of different types of transcription factors is optimized for rapid response, and how a metabolic pathway (methionine biosynthesis) balances production cost with activity requirements. More broadly, our studies reveal how general principles, important both for understanding natural systems and for synthesizing new ones, emerge from global quantitative analyses of protein synthesis. 4 samples of E. coli ribosome profiling and mRNA-seq, including biological replicates