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FastCAT accelerates absolute quantification of proteins by using multiple short non-purified chimeric standards

ABSTRACT: Absolute (molar) quantification of proteins provides the analytical rationale for system-level modelling of diverse molecular mechanisms. FastCAT method employs multiple short (<50 kDa) stable-isotope labeled chimeric proteins (CPs) composed of concatenated quantotypic (Q-) peptides representing the quantified proteins. Each CP also comprises scrambled sequences of reference (R-) peptides that relate its abundance to a single protein standard (BSA). FastCAT not only alleviates the need in purifying CP or using SDS-PAGE, but also improves the accuracy, precision and dynamic range of the absolute quantifications by grouping Q-peptides according to the expected abundance of target proteins. We benchmarked FastCAT against the reference method of MS Western and tested it in the direct molar quantifications of neurological markers in human cerebrospinal fluid at the low ng/mL level.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cerebrospinal Fluid

SUBMITTER: Ignacy Rzagalinski

LAB HEAD: Andrej Shevchenko

PROVIDER: PXD030728 | Pride | 2022-04-01

REPOSITORIES: Pride

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			Action	DRS
	CSF_pool_HpH_F1.raw	Raw
	CSF_pool_HpH_F1_F058188.xml	Xml
	CSF_pool_HpH_F2.raw	Raw
	CSF_pool_HpH_F2_F058190.xml	Xml
	CSF_pool_HpH_F3.raw	Raw

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FastCAT accelerates absolute quantification of proteins by using multiple short non-purified chimeric standards

Project description:Absolute (molar) quantification of proteins provides the analytical rationale for system-level modelling of diverse molecular mechanisms. FastCAT method employs multiple short (<50 kDa) stable-isotope labeled chimeric proteins (CPs) composed of concatenated quantotypic (Q-)peptides representing the quantified proteins. Each CP also comprises scrambled sequences of reference (R-)peptides that relate its abundance to a single protein standard (BSA). FastCAT not only alleviates the need in purifying CP or using SDS-PAGE, but also improves the accuracy, precision and dynamic range of the absolute quantifications by grouping Q-peptides according to the expected abundance of target proteins. We benchmarked FastCAT against the reference method of MS Western and tested it in the direct molar quantifications of neurological markers in human cerebrospinal fluid at the low ng/mL level.

2022-04-04 | PXD030762 | panorama

Drosophila melanogaster eye Proteomics

Project description:Absolute (molar) quantification determines proteins stoichiometry in complexes, networks and metabolic pathways. We employed MS Western workflow to determine molar abundances of proteins critical for morphogenesis and phototransduction (PT) in eyes of Drosophila melanogaster using a single chimeric 264 kDa protein standard that covers, in total, 197 peptides from 43 proteins. Each protein was independently quantified with 2 to 4 proteotypic peptides with the coefficient of variation of less than 15 %, better than 1000-fold dynamic range and sub-femtomole sensitivity. We determined molar abundances and stoichiometric ratios of the components of the PT machinery and the rhabdomere, and how they are changing when rhabdomere morphogenesis is perturbed by genetic manipulation of the evolutionary conserved gene crumbs (crb).

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The hematopoietic stem cell in chronic phase CML is characterized by a transcriptional profile resembling normal myeloid progenitor cells and reflecting loss of quiescence

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