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Absolute quantification of mTOR related signaling molecules

ABSTRACT: To determine protein concentration in cells, we performed spike-in DIA experiments. Isotopically labeled concatemers (for mTOR-related signaling proteins) were spiked into lysates from p18Rev cells (each 20 mg) with four three-fold serial dilutions from 5,000 fmol. The lysates were subjected to digestion with trypsin. For absolute quantification of proteins, the digests containing isotopically labeled recombinant proteins were separated on a column (EV1106, Evosep) using a pre-programmed gradient (15 samples per day) at a flow rate of 200 nL/min using the Evosep One system (Evosep).

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Masaki Matsumoto

PROVIDER: PXD042413 | JPOST Repository | Thu May 23 00:00:00 BST 2024

REPOSITORIES: jPOST

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			Action	DRS
	20211023_EVO15SPD_FAIMS-45_8uDIA_mTORconcat-250fmol.raw	Raw
	20211023_EVO15SPD_FAIMS-45_8uDIA_mTORconcat-27fmol.raw	Raw
	20211023_EVO15SPD_FAIMS-45_8uDIA_mTORconcat-83fmol.raw	Raw
	20211023_EVO15SPD_FAIMS-45_8uDIA_mTORconcat-9fmol.raw	Raw
	report.tsv	Tabular

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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.

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Absolute quantification of mTOR related signaling molecules

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