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Comparative proteomic analysis using samples obtained with laser microdissection and saturation dye labelling
Author(s) -
Wilson Kate E.,
Marouga Rita,
Prime John E.,
Pashby D. Paul,
Orange Paul R.,
Crosier Steven,
Keith Alexander B.,
Lathe Richard,
Mullins John,
Estibeiro Peter,
Bergling Helene,
Hawkins Edward,
Morris Christopher M.
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401255
Subject(s) - laser capture microdissection , labelling , proteomics , biology , transgene , microdissection , microbiology and biotechnology , chemistry , biochemistry , gene expression , gene
Comparative proteomic methods are rapidly being applied to many different biological systems including complex tissues. One pitfall of these methods is that in some cases, such as oncology and neuroscience, tissue complexity requires isolation of specific cell types and sample is limited. Laser microdissection (LMD) is commonly used for obtaining such samples for proteomic studies. We have combined LMD with sensitive thiol‐reactive saturation dye labelling of protein samples and 2‐D DIGE to identify protein changes in a test system, the isolated CA1 pyramidal neurone layer of a transgenic (Tg) rat carrying a human amyloid precursor protein transgene. Saturation dye labelling proved to be extremely sensitive with a spot map of over 5,000 proteins being readily produced from 5 μg total protein, with over 100 proteins being significantly altered at p < 0.0005. Of the proteins identified, all showed coherent changes associated with transgene expression. It was, however, difficult to identify significantly different proteins using PMF and MALDI‐TOF on gels containing less than 500 μg total protein. The use of saturation dye labelling of limiting samples will therefore require the use of highly sensitive MS techniques to identify the significantly altered proteins isolated using methods such as LMD.