Dataset Information

Absolute Quantification of Endogenous Ras Isoform Abundance.

ABSTRACT: Ras proteins are important signalling hubs situated near the top of networks controlling cell proliferation, differentiation and survival. Three almost identical isoforms, HRAS, KRAS and NRAS, are ubiquitously expressed yet have differing biological and oncogenic properties. In order to help understand the relative biological contributions of each isoform we have optimised a quantitative proteomics method for accurately measuring Ras isoform protein copy number per cell. The use of isotopic protein standards together with selected reaction monitoring for diagnostic peptides is sensitive, robust and suitable for application to sub-milligram quantities of lysates. We find that in a panel of isogenic SW48 colorectal cancer cells, endogenous Ras proteins are highly abundant with ?260,000 total Ras protein copies per cell and the rank order of isoform abundance is KRAS>NRAS?HRAS. A subset of oncogenic KRAS mutants exhibit increased total cellular Ras abundance and altered the ratio of mutant versus wild type KRAS protein. These data and methodology are significant because Ras protein copy number is required to parameterise models of signalling networks and informs interpretation of isoform-specific Ras functional data.

SUBMITTER: Mageean CJ

PROVIDER: S-EPMC4641634 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Absolute Quantification of Endogenous Ras Isoform Abundance.

Mageean Craig J CJ Griffiths John R JR Smith Duncan L DL Clague Michael J MJ Prior Ian A IA

PloS one 20151111 11

Ras proteins are important signalling hubs situated near the top of networks controlling cell proliferation, differentiation and survival. Three almost identical isoforms, HRAS, KRAS and NRAS, are ubiquitously expressed yet have differing biological and oncogenic properties. In order to help understand the relative biological contributions of each isoform we have optimised a quantitative proteomics method for accurately measuring Ras isoform protein copy number per cell. The use of isotopic prot ...[more]

PMID: 26560143

Similar Datasets

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.

Dataset Information

Absolute Quantification of Endogenous Ras Isoform Abundance.

Publications

Absolute Quantification of Endogenous Ras Isoform Abundance.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure