Dataset Information

Quantification of cytokines secreted by primary human cells using MRM: evaluation of analytical parameters – shotgun secretome data of inflammatory stimulated 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: PXD002211 | Pride | 2015-06-29

REPOSITORIES: Pride

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Publications

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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Project description:Fibroblasts have only recently been identified as important effector cells in inflammation. In this study, human dermal fibroblasts were inflammatory stimulated with interleukin-1beta and comprehensively analysed with respect to proteins, eicosanoids and metabolites. For eicosadomics, we have established a data-dependent shotgun analysis method capable of identifying inflammation-regulated lipids of yet unknown function. Several classical inflammatory agonists were found induced, including PGA2, PGB2, PGE2 and TXB2, but also modulators such as PGA3 and PGE3, while 8-HETE and several HODE family members remained unaffected. Using targeted metabolomics, several acylcarnithins, phosphatitylcholins and sphingomyelins were found significantly downregulated. Proteome profiling with orbitrap-MS demonstrated the strong induction of several chemokines, metalloproteinases and other effector molecules. Treatment of stimulated fibroblasts with dexamethasone almost completely abrogated the formation of all inflammation-induced eicosanoids and restored levels of acylcarnithins back to normal. As expected, the secretion of IL-6, MMP1, MMP3, CXCL2 and CXCL3 was strongly down-regulated. However, instead of counter-regulating, dexamethasone further enhanced consequences of inflammatory stimulation with respect to CXCL1, CXCL6, complement C3 as well as sphingomyelins. Shotgun secretome data were confirmed by targeted analysis with triple-quadrupol-MS. These molecules have been described to be involved in chronic inflammation. In peripheral blood mononuclear cells, actually dexamethasone successfully downregulated the formation of all detectable inflammation mediators. The present data suggest that successful pharmacological abrogation of the formation of lipid inflammatory mediators in fibroblasts may not suffice to suppress the release of several other powerful inflammatory mediators which we thus understand to be capable of establishing chronic inflammation states.

Dataset Information

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

Publications

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

Similar Datasets

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