Proteomic analysis of mammalian basic proteins by liquid-based two-dimensional column chromatography.
To develop a standard method for separating highly basic proteins in mammalian cells, we established a 2-D LC separation system coupled with chromatofocusing/nonporous RP column chromatography (CF/NPRPC) in a ProteomeLab PF2D system. After standardizing conditions for 2-D LC, a 2-D liquid protein map of uninfected macrophage proteins with pH range 8.3-11.3 was constructed, and then compared with a macrophage protein map made after infection with Candida albicans. The results demonstrate that 2-D LC offers both high resolution and reproducibility for separation of highly basic, macrophage proteins. After protein identification using a nano 2-D LC-MS/MS Proteomics Solution System, quantitative determination of the changes in the differentially expressed proteins (e.g., galectin-3) in C. albicans-infected macrophages was also accomplished by measuring the peak area of the chromatogram in 2-D LC. The result from this measurement of galectin-3 expression shows a 3.41-fold decrease in the infected macrophage cells, which was further confirmed by that from the RT-PCR of mRNA of galectin-3. Thus, 2-D LC coupled with CF/NPRPC could be applicable to common analysis of highly basic proteins in a high-throughput manner.
Shin
Yu-Kyong
YK
Department of Biochemistry, Yonsei Proteome Research Center and Biomedical Proteome Research Center, Yonsei University, Sudaemoon-ku, Seoul, Korea.
Lee
Hyoung-Joo
HJ
Lee
Joon Seok
JS
Paik
Young-Ki
YK
D000818:Animals; D002176:Candida albicans / Q000472:pathogenicity; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D037502:Galectin 3 / Q000235:genetics / Q000378:metabolism; D007525:Isoelectric Focusing; D008264:Macrophages / Q000378:metabolism* / Q000382:microbiology; D051379:Mice; D010449:Peptide Mapping; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured
D037502:Galectin 3; D020543:Proteome; D012333:RNA, Messenger