PXD002212

PXD002212 is an original dataset announced via ProteomeXchange.

Title	Quantification of cytokines secreted by primary human cells using MRM: evaluation of analytical parameters – shotgun secretome data of untreated HUVECs
Description	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.
HostingRepository	PRIDE
AnnounceDate	2015-06-29
AnnouncementXML	Submission_2015-06-29_04:41:17.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD002212
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Christopher Gerner
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	Oxidation; Acetyl; Carbamidomethyl
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2015-05-20 03:50:19	ID requested
⏵ 1	2015-06-29 04:41:19	announced

Muqaku B, Slany A, Bileck A, Kreutz D, Gerner C, Quantification of cytokines secreted by primary human cells using multiple reaction monitoring: evaluation of analytical parameters. Anal Bioanal Chem, 407(21):6525-36(2015) [pubmed]

curator keyword: Biomedical, Technical

submitter keyword: Analytical assay, cytokines, inflammation, primary human cells, proteome profiling, targeted proteomics

Christopher Gerner
contact affiliation	University of Vienna, Faculty of Chemistry, Department of Analytical Chemistry
contact email	christopher.gerner@univie.ac.at
lab head
Christopher Gerner
contact affiliation	University of Vienna
contact email	christopher.gerner@univie.ac.at
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD002212
     1. Label: PRIDE project
     2. Name: Quantification of cytokines secreted by primary human cells using MRM: evaluation of analytical parameters – shotgun secretome data of untreated HUVECs