PXD015407

PXD015407 is an original dataset announced via ProteomeXchange.

Title	Antibody-free multiplex measurement of 23 human cytokines in primary cell culture secretome using targeted multiple reaction monitoring mass spectrometry
Description	Cytokines are commonly measured by immunoassays, which have limited multiplexing capacity, are costly and can exhibit cross-reactivity. Although multiple reaction monitoring (MRM) mass spectrometry is a cost-effective method for multiplex measurement of peptides, the ability to detect the relatively low level of secreted cytokines in a complex biological matrix without extensive processing remains to be fully evaluated. We used human keratinocyte conditioned media as an experimental system to evaluate the assay linearity, reproducibility and limit of detection of a MRM assay targeting 23 human cytokines with and without prior 2D chromatographic separation. Without antibody enrichment or immunodepletion of high abundant proteins, the MRM assay could detect 21 cytokines by two or more unique peptides, and two cytokines by a single unique peptide. In a serum-free matrix, the limit of detection and quantification for the 79 monitored cytokine peptides ranged from 7 to 507pg/mL and 24 to 1689 pg/mL, respectively. The presence of serum reduced assay sensitivity however this could be compensated for by high-pH reversed-phase fractionation. Furthermore, the assay shows good replicate consistency with 8% intra- and 12% inter-day coefficient of variations, with increases in assay variability by circa 10% when a pre-fractionation step is included. In summary, our results show potential utility of a multiplex cytokine MRM for routine measurement of secreted cytokines in cellular experiments. Additional enrichment steps will be required in high complexity matrices such as serum.
HostingRepository	PRIDE
AnnounceDate	2020-02-19
AnnouncementXML	Submission_2020-02-24_04:34:26.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD015407
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Alok Shah
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	Deamidated; Oxidation; Acetyl; Carbamidomethyl
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2019-09-11 01:17:19	ID requested
1	2020-02-19 04:14:11	announced
⏵ 2	2020-02-24 04:34:28	announced	2020-02-24: Updated publication reference for PubMed record(s): 32069036.

Krueger A, Stoll T, Shah AK, Sinha R, Frazer IH, Hill MM, Antibody-Free Multiplex Measurement of 23 Human Cytokines in Primary Cell Culture Secretome Using Targeted Mass Spectrometry. Anal Chem, 92(5):3742-3750(2020) [pubmed]

submitter keyword: Human, Cytokine, Secretome

Michelle M. Hill
contact affiliation	The University of Queensland Diamantina Institute, University of Queensland, Translational Research Institute, Brisbane QLD 4102, Australia and QIMR Berghofer Medical Research Institute, Brisbane QLD 4006, Australia
contact email	Michelle.Hill@qimrberghofer.edu.au
lab head
Alok Shah
contact affiliation	QIMR Berghofer Medical Research Institute
contact email	alok.shah@qimrberghofer.edu.au
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/02/PXD015407

PRIDE project URI

• PRIDE
  1. PXD015407
     1. Label: PRIDE project
     2. Name: Antibody-free multiplex measurement of 23 human cytokines in primary cell culture secretome using targeted multiple reaction monitoring mass spectrometry