On-line concentration of microheterogeneous proteins by capillary electrophoresis using SDS and PEO as additives.
In this paper, we describe a method for analyzing large-volume protein samples using capillary electrophoresis in conjunction with laser-induced fluorescence detection (CE-LIF). To improve the stacking and separation efficiencies of proteins, we added either 0.01% sodium dodecyl sulfate (SDS) or 0.01% poly(ethylene oxide) (PEO) to the Tris-borate solutions (pH 10.0) used to prepare the protein samples. After injection of the large-volume samples (ca. 1.0 microL, 0.1 microM), the proteins migrate against the electroosmotic flow (EOF) and enter the PEO zone; this process causes them to slow and stack at the boundary between the PEO and sample zones. As a result, the limits of detection (LODs) at a signal-to-noise (S/N) of 3 for most proteins are sub-nM to several nM. For instance, the LOD (S/N = 3) for alpha-lactalbumin is 0.48 nM, which is an 84-fold sensitivity enhancement over the traditional method. By applying a short plug of 0.2% SDS prior to sample injection, a greater number of peaks, representing the microheterogeneity of the proteins, were resolved and the stacking efficiency of the proteins increased slightly. This method allowed us to detect 12 peaks when injecting a large volume of sample containing six model proteins (0.1 microM). We also analyzed the microheterogeneities of the proteins by using CE with UV-Vis absorption detection when injecting a large volume of sample containing six model proteins (1.0 microM) in the presence of a 1.0% SDS plug. The practical method is validated by the detection of human serum albumin in a urine sample, obtained from a healthy female, without sample pretreatment; its concentration was 0.18 microM. We further demonstrate the capability of this method to detect low amounts of proteins through the detection of 45 nM hemoglobin after injecting ca. 1.0 microL of ultradilute lysed red blood cells. The experimental results indicate that our proposed method has great potential for use in diagnosis and proteomics applications.
Huang
Yu-Fen
YF
Department of Chemistry, National Taiwan University, Taipei, Taiwan.
Hsieh
Ming-Mu
MM
Tseng
Wei-Lung
WL
Chang
Huan-Tsung
HT
D019075:Electrophoresis, Capillary; D004912:Erythrocytes / Q000737:chemistry; D005260:Female; D005470:Fluorometry; D006801:Humans; D007834:Lasers; D011092:Polyethylene Glycols / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D014556:Urine / Q000737:chemistry
D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols