Abstract
The platform for precise proteomic profiling of targeted cell populations from heterogeneous tissue sections is developed. We demonstrate a seamless and systematic integration of LCM with an automated cap-IA for the handling of a very small-sized dissected tissues section from the kidney, liver and pancreatic Langerhans islet of rats. Our analysis reveals that the lowest LCM section area ≥ 0.125 mm2 with 10 µm thickness can be optimized for the detection of proteins through LCM-cap-IA integration. We detect signals ranging from a highly-abundant protein, β-actin, to a low-abundance protein, LC-3AB, using 0.125 mm2 LCM section from rat kidney, but, so far, a relatively large section is required for good quality of results. This integration is applicable for a highly-sensitive and accurate assessment of microdissected tissue sections to decipher hidden proteomic information of pure targeted cells. To validate this integration, PCK2 protein expression is studied within Langerhans islets of normal and diabetic rats. Our results show significant overexpression of PCK2 in Langerhans islets of rats with long-term diabetes.
Highlights
The last several years have seen rapid development of technologies and methods that permit a detailed analysis of the genome and proteome
We demonstrate for the first time, a seamless and systematic integration of laser capture microdissection (LCM) with automated cap-IA to study the expression of phosphoenolpyruvate within microdissected sections of Langerhans islets
Multiple antibody-based methods for quantifying proteins in single cells have been developed such as CyTOF [20,21,22], scWestern blots [11,23], and Proseek Multiplex, an immunoassay readout by PCR [24]
Summary
The last several years have seen rapid development of technologies and methods that permit a detailed analysis of the genome and proteome. Even experimental approaches that utilize single-cell analysis are an effective means to understand how cell networks work in concert to coordinate a response at the population level. This interdisciplinary frontier of analytical chemistry, biology, and medicine has provided a unique platform to study a certain type of specific cells in the population to identify heterogeneity between cells. Various molecular biological techniques have been combined with LCM, such as next-generation sequencing [1], or microarray and quantitative RT-PCR [2,3] These advancements in the genomic revolution have increased the interest of researchers to combine LCM with proteomics to discover new information [1,4,5,6,7,8,9]. The major obstacles in proteomic studies are their variability from gene
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