QIMRBerghofer - Cytokines in primary cell culture secretome

Antibody-free multiplex measurement of 23 human cytokines in primary cell culture secretome using targeted multiple reaction monitoring mass spectrometry

  • Organism: Homo sapiens
  • Instrument: 6490 Triple Quadrupole LC/MS
  • SpikeIn: Yes
  • Keywords: Cytokine, Cell secretome, Targeted mass spectrometry
  • Lab head: Michelle Hill
Abstract
Cytokines are key mediators of inflammation that commonly serve as biomarkers to monitor immune responses and inflammatory disease. 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.
Created on 9/14/19, 4:29 PM
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20170405_HCy_MRM_AKCMFCS_lvl500_Repl1-4_analysis02_2019-09-12_14-55-48.sky.zip2019-09-14 16:21:42248282351682Primary human keratinocyte conditioned media containing FCS_500 pg/mL cytokines spiked-in_No fractionation_Reproducibility experiment_Day 2_Replicate 1-4_MRM runs
20170405_HCy_MRM_AKCMFCS_analysis01_lvl500_Repl3_2019-09-12_14-54-54.sky.zip2019-09-14 16:21:422482823471082Primary human keratinocyte conditioned media containing FCS_500 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_Reproducibility experiment_Day 2_Replicate 3_MRM runs
20170405_HCy_MRM_AKCMFCS_analysis01_lvl500_Repl2_2019-09-12_14-54-27.sky.zip2019-09-14 16:21:422482823471082Primary human keratinocyte conditioned media containing FCS_500 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_Reproducibility experiment_Day 2_Replicate 2_MRM runs
20170324_HCy_MRM_AKCMFCS_analysis01_lvl500_Repl4_2019-09-12_14-52-12.sky.zip2019-09-14 16:21:422482823471082Primary human keratinocyte conditioned media containing FCS_500 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_Reproducibility experiment_Day 1_Replicate 4_MRM runs
20170405_HCy_MRM_AKCMFCS_analysis01_lvl500_Repl4_2019-09-12_14-55-21.sky.zip2019-09-14 16:21:422482823471082Primary human keratinocyte conditioned media containing FCS_500 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_Reproducibility experiment_Day 2_Replicate 4_MRM runs
20170325_HCy_MRM_AKCMFCS_lvl500_Repl1-4_analysis01_2019-09-12_14-53-38.sky.zip2019-09-14 16:21:42248282351682Primary human keratinocyte conditioned media containing FCS_500 pg/mL cytokines spiked-in_No fractionation_Reproducibility experiment_Day 1_Replicate 1-4_MRM runs
20170324_HCy_MRM_AKCMFCS_analysis01_lvl500_Repl3_2019-09-12_14-51-46.sky.zip2019-09-14 16:21:422482823471082Primary human keratinocyte conditioned media containing FCS_500 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_Reproducibility experiment_Day 1_Replicate 3_MRM runs
20170324_HCy_MRM_AKCMFCS_analysis01_lvl500_Repl2_2019-09-12_14-51-17.sky.zip2019-09-14 16:21:422482823471082Primary human keratinocyte conditioned media containing FCS_500 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_Reproducibility experiment_Day 1_Replicate 2_MRM runs
20170324_HCy_MRM_AKCMFCS_analysis01_lvl500_Repl1_2019-09-12_14-50-52.sky.zip2019-09-14 16:21:412482823471082Primary human keratinocyte conditioned media containing FCS_500 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_Reproducibility experiment_Day 1_Replicate 1_MRM runs
20170210_HCy_MRM_AKCMFCS_analysis01_2019-09-12_14-50-27.sky.zip2019-09-14 16:21:41248282346982Primary human keratinocyte conditioned media containing FCS_0 pg/mL - 1000 pg/mL cytokines spiked-in_No fractionation_MRM runs
20170210_HCy_MRM_AKCM_analysis01_2019-09-12_14-49-54.sky.zip2019-09-14 16:21:41248282351982Primary human keratinocyte conditioned media without FCS_0 pg/mL - 1000 pg/mL cytokines spiked-in_No fractionation_MRM runs
20170206_HCy_MRM_AKCM_analysis01_lvl1000_2019-09-12_14-48-10.sky.zip2019-09-14 16:21:412482823501082Primary human keratinocyte conditioned media without FCS_1000 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_MRM runs
20170206_HCy_MRM_AKCM_analysis01_lvl125_2019-09-12_14-46-44.sky.zip2019-09-14 16:21:412482823501082Primary human keratinocyte conditioned media without FCS_125 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_MRM runs
20170206_HCy_MRM_AKCM_analysis01_lvl500_2019-09-12_14-47-45.sky.zip2019-09-14 16:21:412482823501082Primary human keratinocyte conditioned media without FCS_500 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_MRM runs
20170206_HCy_MRM_AKCM_analysis01_lvl75_2019-09-12_14-46-18.sky.zip2019-09-14 16:21:412482823501082Primary human keratinocyte conditioned media without FCS_75 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_MRM runs
20170201_HCy_MRM_AKCMFCS_analysis01_lvl1000_2019-09-12_14-41-51.sky.zip2019-09-14 16:21:412482823541082Primary human keratinocyte conditioned media containing FCS_1000 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_MRM runs
20170201_HCy_MRM_AKCMFCS_analysis01_lvl500_2019-09-12_14-41-14.sky.zip2019-09-14 16:21:412482823541082Primary human keratinocyte conditioned media containing FCS_500 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_MRM runs
20170405_HCy_MRM_AKCMFCS_analysis01_lvl500_Repl1_2019-09-12_14-54-01.sky.zip2019-09-14 16:21:412482823471082Primary human keratinocyte conditioned media containing FCS_500 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_Reproducibility experiment_Day 2_Replicate 1_MRM runs
20170206_HCy_MRM_AKCM_analysis01_lvl250_2019-09-12_14-47-12.sky.zip2019-09-14 16:21:412482823501082Primary human keratinocyte conditioned media without FCS_250 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_MRM runs
20170206_HCy_MRM_AKCM_analysis01_lvl0_2019-09-12_14-45-15.sky.zip2019-09-14 16:21:412482823511082Primary human keratinocyte conditioned media without FCS_No cytokines spiked-in_High pH reversed-phase peptide fractionation_MRM runs
20170201_HCy_MRM_AKCMFCS_analysis01_lvl250_2019-09-12_14-40-35.sky.zip2019-09-14 16:21:412482823541082Primary human keratinocyte conditioned media containing FCS_250 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_MRM runs
20170206_HCy_MRM_AKCM_analysis01_lvl37-5_2019-09-12_14-45-50.sky.zip2019-09-14 16:21:412482823501082Primary human keratinocyte conditioned media without FCS_37.5 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_MRM runs
20170201_HCy_MRM_AKCMFCS_analysis01_lvl75_2019-09-12_14-38-49.sky.zip2019-09-14 16:21:412482823541082Primary human keratinocyte conditioned media containing FCS_75 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_MRM runs
20170201_HCy_MRM_AKCMFCS_analysis01_lvl37-5_2019-09-12_14-38-01.sky.zip2019-09-14 16:21:402482823541082Primary human keratinocyte conditioned media containing FCS_37.5 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_MRM runs
20170201_HCy_MRM_AKCMFCS_analysis01_lvl0_2019-09-12_14-35-27.sky.zip2019-09-14 16:21:402482823541082Primary human keratinocyte conditioned media containing FCS_No cytokines spiked-in_High pH reversed-phase peptide fractionation_MRM runs
20170201_HCy_MRM_AKCMFCS_analysis01_lvl125_2019-09-12_14-39-48.sky.zip2019-09-14 16:21:402482823541082Primary human keratinocyte conditioned media containing FCS_125 pg/mL cytokines spiked-in_High pH reversed-phase peptide fractionation_MRM runs

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