Dataset Information

A ‘proteomic ruler’ for protein copy number and concentration estimation without spike-in standards

ABSTRACT: Absolute protein quantification using mass spectrometry (MS)-based proteomics delivers protein concentrations or copy numbers per cell. Existing methodologies typically require a combination of isotope-labeled spike-in references, cell counting and protein concentration measurements. Here we present a novel concept that delivers similar quantitative results directly from deep eukaryotic proteome datasets without any additional experimental steps. We show that the MS-signal of histones can be used as a ‘proteomic ruler’ because is proportional to the amount of DNA in the sample, which in turn depends of the number of cells. As a result, our proteomic ruler approach adds an absolute scale to the MS readout and allows the estimation of the copy numbers of individual proteins per cell. We compare our protein quantifications with values derived from the SILAC-PrEST method, which combines spike-in protein fragment standards with precise isotope label quantification. The proteomic ruler approach yields quantitative readouts that are in remarkably good agreement with the precision method. We attribute this surprising result to the fact that the proteomic ruler approach omits error-prone steps such as cell counting or protein concentration measurements. The proteomic ruler approach is readily applicable to any deep eukaryotic proteome dataset – even in retrospect analysis – and we demonstrate its usefulness on a series of mouse organ proteomes.

INSTRUMENT(S): LTQ Orbitrap, LTQ Orbitrap Velos, Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Mario Oroshi

PROVIDER: PXD000661 | Pride | 2014-09-17

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20110429_Velos4_MaZe_SA_HeLaH-PrESTsL11.RAW	Raw
	20110429_Velos4_MaZe_SA_HeLaH-PrESTsL3.RAW	Raw
	20110429_Velos4_MaZe_SA_HeLaH-PrESTsL4.RAW	Raw
	20110429_Velos4_MaZe_SA_HeLaH-PrESTsL5.RAW	Raw
	20110429_Velos4_MaZe_SA_HeLaH-PrESTsL6.RAW	Raw

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A "proteomic ruler" for protein copy number and concentration estimation without spike-in standards.

Wiśniewski Jacek R JR Hein Marco Y MY Cox Jürgen J Mann Matthias M

Molecular & cellular proteomics : MCP 20140915 12

Absolute protein quantification using mass spectrometry (MS)-based proteomics delivers protein concentrations or copy numbers per cell. Existing methodologies typically require a combination of isotope-labeled spike-in references, cell counting, and protein concentration measurements. Here we present a novel method that delivers similar quantitative results directly from deep eukaryotic proteome datasets without any additional experimental steps. We show that the MS signal of histones can be use ...[more]

PMID: 25225357

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Project description:Aiming at defining the protein content and composition of a single mitochondrion of Arabidopsis thaliana, shotgun analyses were performed on purified mitochondria from cultured heterotrophic Arabidopsis cells. Based on microscopic observations on the size of mitochondria, as well as biochemical properties, such as protein concentration, average molecular mass of proteins, and the average length of the signal peptide, intensity-based absolute quantification (iBAQ) values were applied to calculate the copy number of each identified protein species within a single mitochondrion. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40,000 for Voltage-Dependent Anion Channel 1 (VDAC1) to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat (PPR) proteins that modify mitochondrial transcripts. Aiming at defining the protein content and composition of a single mitochondrion of Arabidopsis thaliana, shotgun analyses were performed on purified mitochondria from cultured heterotrophic Arabidopsis cells. Based on microscopic observations on the size of mitochondria, as well as biochemical properties, such as protein concentration, average molecular mass of proteins, and the average length of the signal peptide, intensity-based absolute quantification (iBAQ) values were applied to calculate the copy number of each identified protein species within a single mitochondrion. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40,000 for Voltage-Dependent Anion Channel 1 (VDAC1) to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat (PPR) proteins that modify mitochondrial transcripts. Aiming at defining the protein content and composition of a single mitochondrion of Arabidopsis thaliana, shotgun analyses were performed on purified mitochondria from cultured heterotrophic Arabidopsis cells. Based on microscopic observations on the size of mitochondria, as well as biochemical properties, such as protein concentration, average molecular mass of proteins, and the average length of the signal peptide, intensity-based absolute quantification (iBAQ) values were applied to calculate the copy number of each identified protein species within a single mitochondrion. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40,000 for Voltage-Dependent Anion Channel 1 (VDAC1) to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat (PPR) proteins that modify mitochondrial transcripts.