Abstract
The search to date for accurate protein biomarkers in acute ischemic stroke has taken into consideration the stage and/or the size of infarction, but has not accounted for the site of stroke. In the present study, multiple reaction monitoring using labeled reference peptide (LRP) following laser capture microdissection (LCM) is used to identify site-specific protein biomarker candidates. In middle cerebral artery occlusion (MCAO) rat models, both intact and infarcted brain tissue was collected by LCM, followed by on-film digestion and semi-quantification using triple-quadrupole mass spectrometry. Thirty-four unique peptides were detected for the verification of 12 proteins in both tissue homogenates and LCM-captured samples. Six insoluble proteins, including neurofilament light polypeptide (NEFL), alpha-internexin (INA), microtubule-associated protein 2 (MAP2), myelin basic protein (MBP), myelin proteolipid protein (PLP) and 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP), were found to be site-specific. Soluble proteins, such as neuron-specific enolase (NSE) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), and some insoluble proteins, including neurofilament heavy polypeptide (NEFH), glial fibrillary acidic protein (GFAP), microtubule-associated protein tau (MAPT) and tubulin β-3 chain (TUBB3), were found to be evenly distributed in the brain. Therefore, we conclude that some insoluble protein biomarkers for stroke are site-specific, and would make excellent candidates for the design and analysis of relevant clinical studies in the future.
Highlights
Numerous studies have focused on blood biomarker discovery for ischemic stroke and a variety of protein biomarker candidates have been identified [1]
In 2014, genetically labeled neurons from the drosophila melanogaster brain were isolated by laser capture microdissection (LCM), and the catapulted tissue patches were collected on a reversed phase column, and analyzed using an on-column extraction (OCE) that was directly coupled with liquid chromatography–multistage mass spectrometry (LC-MS(n)) [9]
The area of infarction after occlusion of the middle cerebral artery (MCA) is identified by 2,3,5-triphenyltetrazolium chloride (TTC) staining, which makes infarcted regions white (Figure 1B)
Summary
Numerous studies have focused on blood biomarker discovery for ischemic stroke and a variety of protein biomarker candidates have been identified [1]. Laser capture microdissection (LCM) provides an approach to make normalization possible based on the area of tissue sample, and it could become a new assay to assess protein distribution and stability in infarcted brain tissues. LCM followed by MRM using LRP (LCM–LRP) is an established tool for screening site-specific protein biomarkers in both intact and infarcted tissue. By using LCM–LRP, we can normalize the level of targeted protein by the capture area of LCM that remains the same for infarcted tissue and intact tissue. We procured brain tissue samples from middle cerebral artery occlusion (MCAO) rat model brain by LCM and measured proteins of interest by triple-quadrupole mass spectrometry using the LRP method.
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