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Abstract Background:
Transcriptomic analysis alone of steroids and neuropeptides in the human brain is insufficient in allowing us to recapitulate the biological basis for how stress contributes to the development of brain disorders including schizophrenia, major depression and bipolar disorder. A 2023 Nature Biotechnology study by Vicari and colleagues reported a multimodal approach to tissue analysis which combines histology, mass spectrometry and spatial transcriptomics to give a holistic overview of mRNA transcripts, proteins, lipids and low molecular weight metabolites within a single tissue sample (1). If this pipeline could be adapted to detect neurosteroids and neuropeptides, this new workflow could provide critical information about the underlying biological processes which modulate the status of upstream regulation of gene and protein expression by neuropeptides/steroids in pathological conditions, including psychopathology.
Problem:
Limiting factors of steroid and neuropeptide analysis include their low in vivo concentrations (especially when compared to endogenous lipid species), high spatial heterogeneity of expression, and dynamic rates of metabolism/stability in postmortem tissue.
Method Information:
Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is an established technique for spatially resolved molecular analysis which we are establishing to examine neuropeptide/neurosteroid levels in the human brain with spatial resolution. Briefly, sequential 10-12μm sections of fresh frozen human brain cortex tissue are sectioned using a cryostat, then mounted onto a 10x Genomics Visium barcoded gene expression array glass microscope slide. Slides are stored at -80oC, then thawed in a desiccator for an hour prior to use. To promote ion formation and protect the tissue from degradation, a MALDI matrix whose composition depends on the target molecular species (e.g., 2,5-dihydroxybenzoic acid for neuropeptides), is applied to the tissue using an automated spraying system. Following matrix application, samples are analysed on a Thermo Fisher Scientific Orbitrap Fusion. Following MALDI-MSI, samples are washed thrice in pre-chilled methanol to remove residual matrix and can then be stored at -80oC until Visium processing.
Troubleshooting Steps:
So far, we have focused our optimisation on neuropeptides, with the primary troubleshooting steps being:
- Tissue preparation. Thickness of sample, storage conditions, thawing methods.
- Pre-matrix washes to reduce the lipid content of the tissue and limit the interference of comparatively strong lipid signal peaks. Currently using 30s in 100% chloroform.
- Concentrations of matrix and its delivery solvent. Currently using 50% acetonitrile, 50% water and 0.2% trifluoracetic acid with between 25-35mg/mL DHB at between 4-6 passes.
- Instrument settings for increased signal strength in the larger m/z peptide ranges (i.e., >1000m/z), currently working through trialling conditions of 400oC tube temperature, 60-120% RF lens, 500-2000 m/z scan rang and 7-12% laser power in positive mode.
Outcome:
While still in the early stages of establishing methods for the analysis of steroids and neuropeptides in the human brain using our Orbitrap Fusion, I am looking forward to feedback on our intended next steps (especially analysis of lowly concentrated neurosteroids) and potentially identifying additional components for optimisation.
References:
(1) Vicari M, Mirzazadeh R, Nilsson A, Shariatgorji R, Bjärterot P, Larsson L, et al. Spatial multimodal analysis of transcriptomes and metabolomes in tissues. Nature Biotechnology. 2023. |
= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
