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

Abstract

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 codigested within undiluted and undigested sera decreasing the ability to detect and quantify cytokines by 10,000-fold compared with pure standards and predigested 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.

Patterns of serum immune biomarkers during elephant endotheliotropic herpesvirus viremia in Asian and African elephants

“…We cannot yet rule out such pathological involvement, but our inability to detect cytokine responses in several of the fatal cases of EEHV HD suggests that aberrant over-expression of cytokines, at least systemically, may not be involved in progression of EEHV HD. Indeed, it remains a possibility that an inadequate immune response may be involved in more severe cases of EEHV HD, so more sensitive tools for quantifying cytokines, such as mass spectrometry techniques [ 87 ], and comparison of tissue-specific and circulating concentrations would be beneficial to investigating differential responses between cases. Further investigation into changes in these biomarkers throughout additional cases of EEHV viremia, including between African and Asian elephants, would be beneficial to understand differential responses and increase our knowledge of disease pathogenesis before immune moderating treatments can be explored.…”

Unrevealing the connection between real sample analysis and analytical method. The case of cytokines

“…Mendoza‐Porras et al elaborated a hyphenated detection method for the simultaneous analysis of IL‐1β, IL‐6, IFN‐γ, and TNF‐α, that combines liquid chromatography with detection by multiple reaction monitoring MS (LC/MRM‐MS). This method permits the detection with LOD of fmol/μL in complex samples such as bovine sera 92 …”

CSF neopterin and quinolinic acid are biomarkers of neuroinflammation and neurotoxicity in FIRES and other infection‐triggered encephalopathy syndromes

“…The first limitation is that most cytokine approaches use research kits that are not licenced for use in clinical care, and the second limitation is the need for batching in some ELISA or multiplex platforms. However, there is potential that key cytokines can be detected and quantified using CSF proteomic approaches using mass spectrometry in the future, 35 and this is particularly relevant for cytokines that are targetable with therapy, such as interleukin-1, interleukin-6, tumour necrosis factor-alpha and interferon-alpha. The severe phenotypes in our cohort were treated empirically with biologics and monoclonal antibodies targeting immune molecules, including anakinra and tocilizumab.…”