Chapter 3
Utilization of Laser Capture Microdissection Coupled
to Mass Spectrometry to Uncover the Proteome of Cellular
Protrusions
Ana Gordon and Karine Gousset
Abstract
Laser capture microdissection (LCM) provides a fast, specific, and versatile method to isolate and enrich
cells in mixed populations and/or subcellular structures, for further proteomic study. Furthermore, mass
spectrometry (MS) can quickly and accurately generate differential protein expression profiles from small
amounts of samples. Although cellular protrusions—such as tunneling nanotubes, filopodia, growth cones,
invadopodia, etc.—are involved in essential physiological and pathological actions such as phagocytosis or
cancer-cell invasion, the study of their protein composition is progressing slowly due to their fragility and
transient nature. The method described herein, combining LCM and MS, has been designed to identify the
proteome of different cellular protrusions. First, cells are fixed with a novel fixative method to preserve the
cellular protrusions, which are isolated by LCM. Next, the extraction of proteins from the enriched sample
is optimized to de-crosslink the fixative agent to improve the identification of proteins by MS. The efficient
protein recovery and high sample quality of this method enable the protein profiling of these small and
diverse subcellular structures.
Key words Cellular protrusion, Laser capture microdissection, Proteomics, Fixation, DTBP, Mass
spectrometry
1 Introduction
Laser capture microdissection (LCM) is a tool that facilitates, via
direct visualization of cells or tissues, the enrichment and isolation
of cells of interest in a heterogeneous sample [1]. There are different classes of LCM depending on the type of laser and capture
method. Overall, laser capture microdissection is an optical microscope, which can be coupled to fluorescence, with a finely focused
laser and a system for capturing dissected samples. The cells of
interest can be selected from the heterogeneous sample through a
touch screen, enabling the selection of regions of interest (ROI)
using an interactive pen or the mouse. Each ROI can be drawn
freehand or using predefined geometrical shapes. This technique
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
25
Utilization of Laser Capture Microdissection Coupled
to Mass Spectrometry to Uncover the Proteome of Cellular
Protrusions
Ana Gordon and Karine Gousset
Abstract
Laser capture microdissection (LCM) provides a fast, specific, and versatile method to isolate and enrich
cells in mixed populations and/or subcellular structures, for further proteomic study. Furthermore, mass
spectrometry (MS) can quickly and accurately generate differential protein expression profiles from small
amounts of samples. Although cellular protrusions—such as tunneling nanotubes, filopodia, growth cones,
invadopodia, etc.—are involved in essential physiological and pathological actions such as phagocytosis or
cancer-cell invasion, the study of their protein composition is progressing slowly due to their fragility and
transient nature. The method described herein, combining LCM and MS, has been designed to identify the
proteome of different cellular protrusions. First, cells are fixed with a novel fixative method to preserve the
cellular protrusions, which are isolated by LCM. Next, the extraction of proteins from the enriched sample
is optimized to de-crosslink the fixative agent to improve the identification of proteins by MS. The efficient
protein recovery and high sample quality of this method enable the protein profiling of these small and
diverse subcellular structures.
Key words Cellular protrusion, Laser capture microdissection, Proteomics, Fixation, DTBP, Mass
spectrometry
1 Introduction
Laser capture microdissection (LCM) is a tool that facilitates, via
direct visualization of cells or tissues, the enrichment and isolation
of cells of interest in a heterogeneous sample [1]. There are different classes of LCM depending on the type of laser and capture
method. Overall, laser capture microdissection is an optical microscope, which can be coupled to fluorescence, with a finely focused
laser and a system for capturing dissected samples. The cells of
interest can be selected from the heterogeneous sample through a
touch screen, enabling the selection of regions of interest (ROI)
using an interactive pen or the mouse. Each ROI can be drawn
freehand or using predefined geometrical shapes. This technique
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
25