Dataset Information

Quantification of cytokines secreted by primary human cells using MRM: evaluation of analytical parameters – shotgun secretome data of untreated HUVECs

ABSTRACT: The determination of secreted proteins may provide highly valuable information about cell functions. While the typical methods for the determination of biologically relevant but low-abundant molecular species still relies on the use of specific antibodies, mass spectrometry-based methods are now gaining sufficient sensitivity to cope with such challenges. In the current study we have identified several cytokines and chemokines which were induced by inflammatory activation of primary human umbilical vein endothelial cells. Based on the high-resolution mass spectrometry data obtained with a Q Exactive orbitrap, we built an MRM method to quantify the most relevant molecules selected from the screening experiment. Using nano-flow Chip-HPLC coupled to a 6490 triple-quadrupole MS for MRM analyses we achieved calibration curves covering a linear range of four orders of magnitude and detection limits in the low attomol per microliter concentration range. Carryover was consistently less than 0.005%, the accuracy between 80% and 120% and the median coefficient of variation for LC/MS was only 2.2%. When including the variance introduced by biological replicates and the digestion procedure, the coefficient of variation was less than 20% for most peptides. Selection of appropriate marker molecules allowed us to monitor typical cell culture variations such as different cell densities, proliferative states and the occurrence of cell death. As a result, here we present a robust and efficient MRM-based assay for the accurate and sensitive determination of cytokines and chemokines representative for functional cell states and including comprehensive quality controls.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Endothelial Cell Of Umbilical Vein, Umbilical Vein

SUBMITTER: Christopher Gerner

LAB HEAD: Christopher Gerner

PROVIDER: PXD002212 | Pride | 2015-06-29

REPOSITORIES: Pride

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Quantification of cytokines secreted by primary human cells using multiple reaction monitoring: evaluation of analytical parameters.

Muqaku Besnik B Slany Astrid A Bileck Andrea A Kreutz Dominique D Gerner Christopher C

Analytical and bioanalytical chemistry 20150620 21

Determination of secreted proteins provides highly valuable information about cell functions. While the typical methods for the determination of biologically relevant but low abundant molecular species still rely on the use of specific antibodies, mass spectrometry-based methods are now gaining sufficient sensitivity to cope with such challenges as well. In the current study, we have identified several cytokines and chemokines which were induced in primary human umbilical vein endothelial cells ...[more]

PMID: 26092402

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	HUVEC_con1a_sn.mzid.gz	Mzid
	HUVEC_con1a_sn.pride.mgf.gz	Mgf
	HUVEC_con1a_sn.pride.mztab.gz	Mztab
	HUVEC_con1a_sn.raw	Raw