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

Besnik Muqaku, Astrid Slany, Andrea Bileck, Dominique Kreutz, Christopher Gerner

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 upon inflammatory activation. 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. All experimental data are available via ProteomeXchange, PXD002211/12, and Panorama ( www.panoramaweb.org ). 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 was between 80 and 120 %, and the median coefficient of variation for LC/MS was only 2.2 %. When including the variance of quantification introduced by cell culture and digestion, the coefficient of variation was less than 20 % for most peptides. With appropriate marker molecules, we monitored typical variations introduced by cell culture caused by differences in cell numbers, proliferative states, and 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. Graphical Abstract Work flow diagram: Data processing steps beginning with orbitrap-based shotgun data acquisition and MaxQuant data analysis, followed by peptide and transition selection for MRM analysis using Skyline and experimental validation using triple quadrupole MS.