Dataset Information

Mass spectrometry-based quantitative proteomics of whole cell lysate from breast cancer cell line MCF7 spiked with 57 protein standards

ABSTRACT: Here we present quantitative proteomics data used in the evaluation of quantitative accuracy. A human cell line, MCF7 was split into 9 aliquotes that were spiked with a dilution series of 57 protein standards of known amounts spanning 5 orders of magnitude. The protein extracts were trypsinized, and the peptides were analysed by LC-MS using either a label-free or a label-based (TMT 6-plex and iTRAQ 8-plex) quantification approach. The iTRAQ- and TMT-labelled samples were co-analysed and separated by HiRIEF (high resolution isoelectric focusing) prior LC-MS. Raw MS data was identified and quantified under the software platform Proteome Discoverer 1.3.0.339 (Thermo Fisher Scientific Inc.) or MaxQuant software (version 1.2.0.18) (label-free data). For both protein identification and quantification at least 1 unique (i.e. a peptide that occurs in not more than one database entry) peptide was required. The false discovery rate (FDR) for peptide identification was set to 5% in all analyses. For the iTRAQ and TMT labeled samples, all MS/MS spectra were searched by SEQUEST combined with the Percolator algorithm (version 2.0) for PSM search optimization. Searches were performed against a custom made database consisting of SwissProt human sequences(uniprot.org 2012-01-17, 20242 entries), and the spiked in protein standards (57 protein sequences). Peptide FDR was calculated by a target – decoy approach.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: AnnSofi Sandberg

LAB HEAD: AnnSofi Sandberg

PROVIDER: PXD000578 | Pride | 2013-12-03

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	AnnSofi_Rui_spike-in_iTRAQ-TMT-labelfree_420ugIPG37-49_90mingrad_fr31.raw	Raw
	AnnSofi_Rui_spike-in_iTRAQ-TMT-labelfree_420ugIPG37-49_90mingrad_fr32.raw	Raw
	AnnSofi_Rui_spike-in_iTRAQ-TMT-labelfree_420ugIPG37-49_90mingrad_fr33.raw	Raw
	AnnSofi_Rui_spike-in_iTRAQ-TMT-labelfree_420ugIPG37-49_90mingrad_fr34.raw	Raw
	AnnSofi_Rui_spike-in_iTRAQ-TMT-labelfree_420ugIPG37-49_90mingrad_fr35.raw	Raw

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Publications

Quantitative accuracy in mass spectrometry based proteomics of complex samples: the impact of labeling and precursor interference.

Sandberg AnnSofi A Branca Rui M M RM Lehtiö Janne J Forshed Jenny J

Journal of proteomics 20131105

Knowing the limit of quantification is important to accurately judge the results from proteomics studies. In order to investigate isobaric labels in combination with peptide pre-fractionation by high resolution isoelectric focusing in terms of limit of detection, quantitative accuracy and how to improve it, we used a human cell lysate spiked with 57 protein standards providing reference points across a wide concentration range. Specifically, the impact of precursor mixing (isolation interference ...[more]

PMID: 24211767

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