Quantitative Proteomics in Laser Capture Microdissected Sleep Nuclei From Rat Brain

Abstract

The combination of stable isotope labeling of amino acids in mammals (SILAM) and laser capture microdissection (LCM) for selective proteomic analysis of the targeted tissues holds tremendous potential for refined characterization of proteome changes within complex tissues such as the brain. The authors have applied this approach to measure changes in relative protein abundance in ventral tegmental area (VTA) of the rat brain that correlate to pharmacological perturbations. Enriched 13C6 15N2-lysine was introduced in vivo via diet. These animals were sacrificed during the middle of the 12-hour light period to extract isotopically “heavy” proteins, which were then used as a reference for extracts from dosed, unlabeled rats. Animals were administered an orexin peptide (Ox-B), an orexin receptor antagonist (ORA), or a mixture of both (Ox-B + ORA). All samples were obtained at same phase of the sleep cycle. Labeled-pair identification and differential quantitation provided protein identification and expression ratio data. Five proteins were found to exhibit decreased relative abundance after administration of an ORA, including α-synuclein and rat myelin basic protein. Conversely, six proteins showed increased relative abundance upon antagonist treatment, including 2’,3’-cyclic nucleotide 3’-phosphodiesterase.

Human blood serum proteome changes after 6 hours of sleep deprivation at night

“…& Tononi, 2011;Davies et al, 2014;Hinard et al, 2012;Jones et al, 2008;Kim et al, 2014;Mackiewicz et al, 2007;Maret et al, 2007;Miller et al, 2014;Moller-Levet et al, 2013;Pellegrino et al, 2012;Porter et al, 2012;Thompson et al, 2010;Vazquez et al, 2009;Vazquez et al, 2008;Vecsey et al, 2012;Wang et al, 2010;Zimmerman et al, 2006;Aho et al, 2016;Gehrman et al, 2018;Honma et al, 2020;Laing et al, 2017;Nollet et al, 2019;Sengupta et al, 2017;Weljie et al, 2015;Yoon …”

Serum Apolipoprotein A-1 Quantification by LC–MS with A Silac Internal Standard Reveals Reduced Levels in Smokers

“…Furthermore, the risk of coronary heart disease is strongly associated with increased adiposity, which can be further increased by smoking behavior [ 3 , 4 ]. Advances in MS, proteomics and protein bioanalytical approaches led to a rapid evolution and expansion in the area of MS-based biomarker discovery and development [ 5 , 6 ]. Once a biomarker has been identified as a clinically relevant target, quantitative assays must be developed for diagnosis and prognosis of disease.…”

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

“…LCM is a powerful technique for the dissection of discrete populations of cells or regions of tissue from tissue sections on a microscope slide and the subsequent isolation of RNA, microRNA, DNA and proteins. The use of LCM in combination with analysis using high-throughput technologies such as microarray, next generation RNA sequencing, epigenetic analysis of DNA and proteomics is becoming more and more common as the sensitivity of these techniques improves and the amount of starting material needed is reduced [8][9][10][11][12] . With LCM, a better anatomical resolution is achieved for a sample, and the understanding from downstream measures of these samples is greatly enhanced.…”