Quantification of beta-catenin signaling components in colon cancer cell lines, tissue sections, and microdissected tumor cells using reaction monitoring mass spectrometry.
Reaction monitoring mass spectrometry has emerged as a powerful tool for targeted detection and quantification of proteins in clinical samples. Here, we report the use of gel electrophoresis for protein fractionation and liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM) screening for quantitative analysis of components from the Wnt/beta-catenin signaling pathway, which contributes to colon tumor formation and progression. In silico tools are used to design LC-MRM screens for each target protein. Following successful peptide detection, stable isotope labeled peptides are synthesized and developed as internal standards. Then, the assays are implemented in colon cancer cell lines to achieve detection in minimal amounts of cells, compatible with direct translation to clinical specimens. Selected assays are compared with qualitative results from immunoblotting (Westerns) and translated to individual frozen colon tissue sections and laser capture microdissected tumor cells. This LC-MRM platform has been translated from in vitro models to clinical specimens, forming the basis for future experiments in patient assessment.
Chen
Yi
Y
H. Lee Moffitt Cancer Center & Research Institute at the University of South Florida, Tampa, FL 33612, USA.
Gruidl
Mike
M
Remily-Wood
Elizabeth
E
Liu
Richard Z
RZ
Eschrich
Steven
S
Lloyd
Mark
M
Nasir
Aejaz
A
Bui
Marilyn M
MM
Huang
Emina
E
Shibata
David
D
Yeatman
Timothy
T
Koomen
John M
JM
D015153:Blotting, Western; D045744:Cell Line, Tumor; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D003110:Colonic Neoplasms / Q000378:metabolism / Q000503:physiopathology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D015398:Signal Transduction / Q000502:physiology*; D051176:beta Catenin / Q000378:metabolism*
D051176:beta Catenin