Single Cell Immuno-Laser Microdissection Coupled to Label-Free Proteomics to Reveal the Proteotypes of Human Brain Cells After Ischemia

Abstract

Cerebral ischemia entails rapid tissue damage in the affected brain area causing devastating neurological dysfunction. How each component of the neurovascular unit contributes or responds to the ischemic insult in the context of the human brain has not been solved yet. Thus, the analysis of the proteome is a straightforward approach to unraveling these cell proteotypes. In this study, post-mortem brain slices from ischemic stroke patients were obtained corresponding to infarcted (IC) and contralateral (CL) areas. By means of laser microdissection, neurons and blood brain barrier structures (BBB) were isolated and analyzed using label-free quantification. MS data are available via ProteomeXchange with identifier PXD003519. Ninety proteins were identified only in neurons, 260 proteins only in the BBB and 261 proteins in both cell types. Bioinformatics analyses revealed that repair processes, mainly related to synaptic plasticity, are outlined in microdissected neurons, with nonexclusive important functions found in the BBB. A total of 30 proteins showing < 0.05 and fold-change> 2 between IC and CL areas were considered meaningful in this study: 13 in neurons, 14 in the BBB and 3 in both cell types. Twelve of these proteins were selected as candidates and analyzed by immunohistofluorescence in independent brains. The MS findings were completely verified for neuronal SAHH2 and SRSF1 whereas the presence in both cell types of GABT and EAA2 was only validated in neurons. In addition, SAHH2 showed its potential as a prognostic biomarker of neurological improvement when analyzed early in the plasma of ischemic stroke patients. Therefore, the quantitative proteomes of neurons and the BBB (or proteotypes) after human brain ischemia presented here contribute to increasing the knowledge regarding the molecular mechanisms of ischemic stroke pathology and highlight new proteins that might represent putative biomarkers of brain ischemia or therapeutic targets.

Multi-region proteome analysis quantifies spatial heterogeneity of prostate tissue biomarkers

“…The number of proteins quantified in our study exceeds by 1-2 orders of magnitude the number of proteins typically quantified by tissue staining, which is the current standard method for protein quantification in clinical tissue samples. Our workflow is also compatible with laser capture microdissected samples which can also be analyzed by shotgun proteomics (Buczak et al, 2018, Garcia-Berrocoso, Llombart et al, 2018, Grosserueschkamp, Bracht et al, 2017). Our data did not achieve single cell resolution like the CyTOF technology.…”

Cell-Type-Specific Proteomics: A Neuroscience Perspective

“…Furthermore, mass spectrometry analysis of four different compartments in FFPE fetal human brain tissue identified a total of 3041 proteins [55]. Two recent reports isolated cells from human post-mortem tissue using LCM to identify a small number of potential biomarkers from AD [56] and ischemic stroke [57] patients via mass spectrometry. LCM was also recently used to quantify approximately 1000 proteins from 10–18 cells (100-µm-diameter) isolated from different rat brain regions [26].…”

Proteomic-Based Approaches for the Study of Ischemic Stroke

“…Based on label-free spectral counting, 30 differentially expressed proteins between infarcted and contralateral areas were identified. Among them, SAHH2 was subsequently verified in the plasma of ischemic stroke patients and considered meaningful as a prognostic biomarker of neurological improvement [17].…”