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An optimum method designed for 2‐D DIGE analysis of human arterial intima and media layers isolated by laser microdissection
Author(s) -
de la Cuesta Fernando,
AlvarezLlamas Gloria,
Maroto Aroa S.,
Donado Alicia,
JuarezTosina Rocio,
RodriguezPadial Luis,
Pinto Angel G.,
Barderas Maria G.,
Vivanco Fernando
Publication year - 2009
Publication title -
proteomics – clinical applications
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.948
H-Index - 54
eISSN - 1862-8354
pISSN - 1862-8346
DOI - 10.1002/prca.200900053
Subject(s) - laser capture microdissection , laser , microdissection , materials science , pathology , biomedical engineering , chemistry , optics , medicine , physics , biochemistry , gene , gene expression
The formation and progression of atherosclerotic lesions involve complex mechanisms which are still not fully understood. A variety of cell types from the distinct arterial layers are implicated in the whole process from lipid accumulation within the vascular wall to plaque development and final rupture. In the present work, we employ the combination of laser microdissection and pressure catapulting and 2‐D DIGE saturation labeling to investigate the human intima and media sub‐proteomes isolated from atherosclerotic (coronary and aorta) or non‐atherosclerotic vessels (preatherosclerotic coronary arteries). Laser microdissection and pressure catapulting allows the specific isolation of regions of interest. In turn, DIGE saturation labeling overcomes the limitation of extensive microdissection times to recover the protein amount required to perform comparative 2‐DE, particularly when dealing with tissue regions rich in myofilament proteins, which result in low protein recovery. The compatibility and optimum performance of both techniques were investigated in detail, paying special attention to tissue staining and protein solubilization. Since scarce amount of protein obtained from microdissected tissue made it impossible to directly perform protein identification from 2‐DE spots by MS, we performed in‐solution digestion followed by LC‐MS/MS analysis of total protein extracts from intima and media in order to get an overall picture of protein composition. Proteins so identified confirm the nature of the isolated regions. Finally, similar spot resolution on 2‐D DIGE gels was obtained for the different human artery types (coronary, aorta) and studied layers (intima, media), setting the basis for future clinical comparative studies.