Optimising the use of TRIzol-extracted proteins in surface enhanced laser desorption/ ionization (SELDI) analysis.
BACKGROUND
Research with clinical specimens is always hampered by the limited availability of relevant samples, necessitating the use of a single sample for multiple assays. TRIzol is a common reagent for RNA extraction, but DNA and protein fractions can also be used for other studies. However, little is known about using TRIzol-extracted proteins in proteomic research, partly because proteins extracted from TRIzol are very resistant to solubilization.


RESULTS
To facilitate the use of TRIzol-extracted proteins, we first compared the ability of four different common solubilizing reagents to solubilize the TRIzol-extracted proteins from an osteosarcoma cell line, U2-OS. Then we analyzed the solubilized proteins by Surface Enhanced Laser Desorption/Ionization technique (SELDI). The results showed that solubilization of TRIzol-extracted proteins with 9.5 M Urea and 2% CHAPS ([3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate]) (UREA-CHAPS) was significantly better than the standard 1% SDS in terms of solubilization efficiency and the number of detectable ion peaks. Using three different types of SELDI arrays (CM10, H50, and IMAC-Cu), we demonstrated that peak detection with proteins solubilized by UREA-CHAPS was reproducible (r > 0.9). Further SELDI analysis indicated that the number of ion peaks detected in TRIzol-extracted proteins was comparable to a direct extraction method, suggesting many proteins still remain in the TRIzol protein fraction.


CONCLUSIONS
Our results suggest that UREA-CHAPS performed very well in solubilizing TRIzol-extracted proteins for SELDI applications. Protein fractions left over after TRIzol RNA extraction could be a valuable but neglected source for proteomic or biochemical analysis when additional samples are not available.
Texas Children's Cancer Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA. tman@bcm.tmc.edu
Tsz-Kwong
TK
Man
Yiting
Y
Li
Tu Anh
TA
Dang
Jianhe
J
Shen
Laszlo
L
Perlaky
Ching C
CC
Lau