Comparative proteomic analysis using samples obtained with laser microdissection and saturation dye labelling.
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 mug 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 mug 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.
Wilson
Kate E
KE
MRC Building, Newcastle General Hospital, Newcastle upon Tyne, UK. k.e.wilson@ncl.ac.uk
Marouga
Rita
R
Prime
John E
JE
Pashby
D Paul
DP
Orange
Paul R
PR
Crosier
Steven
S
Keith
Alexander B
AB
Lathe
Richard
R
Mullins
John
J
Estibeiro
Peter
P
Bergling
Helene
H
Hawkins
Edward
E
Morris
Christopher M
CM
D000544:Alzheimer Disease / Q000235:genetics / Q000473:pathology; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D004396:Coloring Agents; D004195:Disease Models, Animal; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006624:Hippocampus / Q000737:chemistry* / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D007834:Lasers; D008297:Male; D042282:Microdissection / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D017966:Pyramidal Cells / Q000737:chemistry; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D016564:Amyloid beta-Protein Precursor; D004396:Coloring Agents; D011506:Proteins