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Immunohistochemical staining, laser capture microdissection, and filter-aided sample preparation-assisted proteomic analysis of target cell populations within tissue samples.

ABSTRACT: An important problem involves isolating subpopulations of cells defined by protein markers in clinical tissue samples for proteomic studies. We describe a method termed Immunohistochemical staining, laser capture microdissection (LCM) and filter-aided sample preparation (FASP)-Assisted Proteomic analysis of Target cell populations within tissue samples (ILFAPT). The principle of ILFAPT is that a target cell population expressing a protein of interest can be lit up by immunohistochemical staining and isolated from tissue sections using LCM for FASP and proteomic analysis. Using this method, we isolated a small population of CD90(+) stem-like cells from glioblastoma multiforme tissue sections and identified 674 high-confidence (false discovery rate < 0.01) proteins from 32 nL of CD90(+) cells by LC-MS/MS using an Orbitrap Elite mass spectrometer. We further quantified the relative abundance of proteins identified from equal volumes of LCM-captured CD90(+) and CD90(-) cells, where 109 differentially expressed proteins were identified. The major group of these differentially expressed proteins was relevant to cell adhesion and cellular movement. This ILFAPT method has demonstrated the ability to provide in-depth proteome analysis of a very small specific cell population within tissues. It can be broadly applied to the study of target cell populations within clinical specimens.

SUBMITTER: He J

PROVIDER: S-EPMC3766629 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Immunohistochemical staining, laser capture microdissection, and filter-aided sample preparation-assisted proteomic analysis of target cell populations within tissue samples.

He Jintang J Zhu Jianhui J Liu Yashu Y Wu Jing J Nie Song S Heth Jason A JA Muraszko Karin M KM Fan Xing X Lubman David M DM

Electrophoresis 20130427 11

An important problem involves isolating subpopulations of cells defined by protein markers in clinical tissue samples for proteomic studies. We describe a method termed Immunohistochemical staining, laser capture microdissection (LCM) and filter-aided sample preparation (FASP)-Assisted Proteomic analysis of Target cell populations within tissue samples (ILFAPT). The principle of ILFAPT is that a target cell population expressing a protein of interest can be lit up by immunohistochemical staining ...[more]

PMID: 23436586

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Project description:Due to its outstanding throughput and analytical resolution, gel-free LC-based shotgun proteomics represents the gold standard of proteome analysis. Thereby, the efficiency of sample preparation dramatically affects the correctness and reliability of protein quantification. Thus, the steps of protein isolation, solubilization, and proteolysis represent the principal bottleneck of shotgun proteomics. The desired performance of the sample preparation protocols can be achieved by the application of detergents. However, these compounds ultimately compromise reverse-phase chromatographic separation and disrupt electrospray ionization. Filter-aided sample preparation (FASP) represents an elegant approach to overcome these limitations. Although this method is comprehensively validated for cell proteomics, its applicability to plants and compatibility with plant-specific protein isolation protocols remain to be confirmed. Thereby, the most important gap is the absence of the data on the linearity of underlying protein quantification methods for plant matrices. To fill this gap, we address here the potential of FASP in combination with two protein isolation protocols for quantitative analysis of pea (Pisum sativum) seed and Arabidopsis thaliana leaf proteomes by the shotgun approach. For this aim, in comprehensive spiking experiments with bovine serum albumin (BSA), we evaluated the linear dynamic range (LDR) of protein quantification in the presence of plant matrices. Furthermore, we addressed the interference of two different plant matrices in quantitative experiments, accomplished with two alternative sample preparation workflows in comparison to conventional FASP-based digestion of cell lysates, considered here as a reference. The spiking experiments revealed high sensitivities (LODs of up to 4 fmol) for spiked BSA and LDRs of at least 0.6 × 102. Thereby, phenol extraction yielded slightly better recoveries, whereas the detergent-based method showed better linearity. Thus, our results indicate the very good applicability of FASP to quantitative plant proteomics with only limited impact of the protein isolation technique on the method's overall performance.

Dataset Information

Immunohistochemical staining, laser capture microdissection, and filter-aided sample preparation-assisted proteomic analysis of target cell populations within tissue samples.

Publications

Immunohistochemical staining, laser capture microdissection, and filter-aided sample preparation-assisted proteomic analysis of target cell populations within tissue samples.

Similar Datasets

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