The utilization of Triton X-100 for enhanced two-dimensional liquid-phase proteomics.
One of the main challenges in proteomics lies in obtaining a high level of reproducible fractionation of the protein samples. Automated two-dimensional liquid phase fractionation (PF2D) system manufactured by Beckman Coulter provides a process well suited for proteome studies. However, the protein recovery efficiency of such system is low when a protocol recommended by the manufacturer is used for metaproteome profiling of environmental sample. In search of an alternative method that can overcome existing limitations, this study replaced manufacturer's buffers with Triton X-100 during the PF2D evaluation of Escherichia coli K12. Three different Triton X-100 concentrations-0.1%, 0.15%, and 0.2%-were used for the first-dimension protein profiling. As the first-dimension result was at its best in the presence of 0.15% Triton X-100, second-dimension protein fractionation was performed using 0.15% Triton X-100 and the standard buffers. When 0.15% Triton X-100 was used, protein recovery increased as much as tenfold. The elution reliability of 0.15% Triton X-100 determined with ribonuclease A, insulin, α-lactalbumin, trypsin inhibitor, and cholecystokinin (CCK) affirmed Triton X-100 at 15% can outperform the standard buffers without having adverse effects on samples. This novel use of 0.15% Triton X-100 for PF2D can lead to greater research possibilities in the field of proteomics.
Department of Microbiology, Chungbuk National University, 410 Sungbong-Ro, Heungduk-Gu, Cheongju 361-763, Republic of Korea.
Mina
M
Kim
Sang-Hee
SH
Lee
Jiho
J
Min
Fumihisa
F
Kobayashi
Hyun-Ju
HJ
Um
Yang-Hoon
YH
Kim
D001426:Bacterial Proteins / Q000302:isolation & purification*; D005591:Chemical Fractionation / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007768:Lactalbumin / Q000032:analysis*; D017830:Octoxynol / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*
D001426:Bacterial Proteins; D017830:Octoxynol; D007768:Lactalbumin