Analysis of mouse brain microvascular endothelium using immuno-laser capture microdissection coupled to a hybrid linear ion trap with Fourier transform-mass spectrometry proteomics platform.
The purpose of this study was to identify the protein profile of the mouse brain microvascular endothelium in situ. This involved coupling of a double-label, immuno-laser capture microdissection (LCM) process with LTQ-FT mass spectrometry to perform the in situ proteomic analysis. LCM was utilized to isolate cells from frozen mouse brain tissue sections. Following cell capture, samples were solubilized and proteins separated by gel electrophoresis in preparation for enzymatic digestion and LC-MS analysis. Processed samples were subsequently analyzed using a linear IT coupled with a Fourier transform mass spectrometer (LTQ-FT MS). Overall, in this study, 881 proteins were identified from a specific cell category using immuno-guided LCM to probe these cell types along the entirety of the cerebral microvascular tree. The identification of sufficient numbers of proteins with high biological interest should allow us to study protein expression by specific cell types - as defined by certain cell markers - in complex tissues.
Lu
Qiaozhen
Q
Barnett Institute and Department of Chemistry, Northeastern University, Boston, MA, USA.
Murugesan
Nivetha
N
Macdonald
Jennifer A
JA
Wu
Shiaw-Lin
SL
Pachter
Joel S
JS
Hancock
William S
WS
D000469:Alkaline Phosphatase; D000818:Animals; D001921:Brain / Q000098:blood supply*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism*; D005583:Fourier Analysis; D007124:Immunoenzyme Techniques; D007834:Lasers; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D008833:Microcirculation / Q000378:metabolism; D042282:Microdissection; D020543:Proteome / Q000378:metabolism*
D020543:Proteome; D000469:Alkaline Phosphatase