Cytokines in the grass, a lesson learnt: Measuring cytokines in plasma using multiple reaction monitoring mass spectrometry.

Rationale: Cytokines are cell regulatory molecules of high importance as indicators for homeostasis and pathology in many species. The current method to measure cytokines in body fluids is reagent dependent, requiring highly specific paired antibodies.

Methods: A liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS)-based approach was developed to simultaneously establish the limits of detection (LODs) and quantification (LOQs) for recombinant cytokines IL-1β, IL-6, IFNγ and TNFα as pure standards and in bovine sera. All experimental LC/MRM-MS data are available at CSIRO Data Access Portal repository under identifier doi.org/10.25919/5de8a0232a862.

Results: The present method enabled LODs and LOQs as low as 1.05 and 1.12 fmol/μL in the experiment comprised of pure standards. Comparable results were obtained in the experiment where digested cytokines were mixed with pre-digested sera proteins. The intrinsic matrix effects were evident when intact cytokines were co-digested within undiluted and undigested sera decreasing the ability to detect and quantify cytokines by 10,000-fold compared with pure standards and pre-digested sera.

Conclusions: The developed LC/MRM-MS method provided insights into the difficulties in detecting the target peptides when embedded in complex matrices. Nonetheless, the method may potentially be readily applied in biomarker-focused research interrogating fluids of lesser complexity such as synovial fluid, cerebrospinal fluid and tissue culture media.