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Immunohistochemical staining, laser capture microdissection, and filter-aided sample preparation-assisted proteomic
Jintang He1, Jianhui Zhu, Yashu Liu
1Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI 48109-0656, USA.
Insights
A new method, ILFAPT, isolates specific cell populations from tissue for proteomic analysis. This technique successfully identified proteins in rare glioblastoma stem-like cells, advancing cell-specific research.
Area of Science:
- Proteomics
- Cell Biology
- Cancer Research
Background:
- Isolating specific cell subpopulations from clinical tissues for proteomic study is challenging.
- Existing methods often lack the sensitivity to analyze small cell quantities.
- Understanding cell-specific protein expression is crucial for disease research.
Purpose of the Study:
- To develop and validate a novel method for isolating and analyzing proteomes of specific target cell populations within tissue samples.
- To demonstrate the application of this method for analyzing rare stem-like cells in glioblastoma multiforme.
Main Methods:
- Immunohistochemical staining to highlight target cells.
- Laser capture microdissection (LCM) to isolate cells.
- Filter-aided sample preparation (FASP) for proteomic analysis.
- Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for protein identification and quantification.
Main Results:
- The ILFAPT method successfully isolated CD90(+) stem-like cells from glioblastoma multiforme tissue.
- 674 high-confidence proteins were identified from a small volume (32 nL) of isolated cells.
- 109 differentially expressed proteins were quantified between CD90(+) and CD90(-) cells, primarily related to cell adhesion and movement.
Conclusions:
- The ILFAPT method enables in-depth proteome analysis of very small, specific cell populations within complex tissue specimens.
- This technique has broad applicability for studying target cell populations in clinical samples.
- ILFAPT advances the capability for cell-specific proteomic research in oncology and beyond.
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.
