[Removal of high-abundance proteins in plasma of the obese by improved TCA/acetone precipitation method].
OBJECTIVE
To develop an improved trichloroacetic acid (TCA)/acetone precipitation method for removal of high-abundance proteins in plasma of the obese.


METHODS
Volumes of TCA/acetone solution (1, 3, 4, 5, 6, 8, 10 and 20 times of the sample) and concentrations of TCA (10%, 30%, 50%, 60%, 70% TCA/acetone solution) have been investigated to optimize the conditions of sample preparation. SDS-PAGE were used to separate and tested proteins in the supernatant and sediment. The best concentration of the TCA/acetone solution was first determined by SDS-PAGE. The protein in precipitation from 10% TCA/acetone solution processing and the new determined concentration TCA/acetone solution processing were verified by 2-D-SDS-PAGE. And then the digested products of the protein in precipitation and supernatant by trypsin were analyzed by nano HPLC-Chip-MS/MS to verify which is the best concentration to process the plasma.


RESULTS
The best volume of TCA/acetone is four times to sample, which less or more TCA/acetone would reduce the removal efficiency of high-abundance proteins. The concentration of TCA in acetone solution should be 60%, which may remove more high-abundance proteins in plasma than 10%, 30%, 50% TCA in acetone solution. If the TCA concentration is more than 60%, the reproducibility will be much poorer due to fast precipitation of proteins. The results of mass identification showed that human plasma prepared with 60% TCA/acetone (4 times sample volume) could be verified more low-abundance proteins than 10%.


CONCLUSIONS
The most desirable conditions for removal of high-abundance proteins in plasma is 60% TCA/acetone (4 times sample volume), especially for the plasma of obesity.
Shenzhen Center for Chronic Disease Control, Shenzhen 518020, China. junwangwh96@gmail.com
Jun
J
Wang
Liru
L
Feng
Wei
W
Yu
Jian
J
Xu
Hui
H
Yang
Xiaoli
X
Liu
D000096:Acetone / Q000737:chemistry; D000293:Adolescent; D000328:Adult; D001798:Blood Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D011232:Chemical Precipitation; D006801:Humans; D008297:Male; D009765:Obesity / Q000097:blood*; D040901:Proteomics / Q000379:methods*; D014238:Trichloroacetic Acid / Q000737:chemistry; D055815:Young Adult
D001798:Blood Proteins; D000096:Acetone; D014238:Trichloroacetic Acid