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On-line concentration of microheterogeneous proteins by capillary electrophoresis using SDS and PEO as additives.
J Proteome Res. 2006 Feb; 5(2):429-36.JP

Abstract

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.

Authors+Show Affiliations

Huang YF
Department of Chemistry, National Taiwan University, Taipei, Taiwan.
Hsieh MM
No affiliation info available
Tseng WL
No affiliation info available
Chang HT
No affiliation info available

MeSH

Electrophoresis, CapillaryErythrocytesFemaleFluorometryHumansLasersPolyethylene GlycolsSodium Dodecyl SulfateUrine

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16457610

Citation

Huang, Yu-Fen, et al. "On-line Concentration of Microheterogeneous Proteins By Capillary Electrophoresis Using SDS and PEO as Additives." Journal of Proteome Research, vol. 5, no. 2, 2006, pp. 429-36.
Huang YF, Hsieh MM, Tseng WL, et al. On-line concentration of microheterogeneous proteins by capillary electrophoresis using SDS and PEO as additives. J Proteome Res. 2006;5(2):429-36.
Huang, Y. F., Hsieh, M. M., Tseng, W. L., & Chang, H. T. (2006). On-line concentration of microheterogeneous proteins by capillary electrophoresis using SDS and PEO as additives. Journal of Proteome Research, 5(2), 429-36.
Huang YF, et al. On-line Concentration of Microheterogeneous Proteins By Capillary Electrophoresis Using SDS and PEO as Additives. J Proteome Res. 2006;5(2):429-36. PubMed PMID: 16457610.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - On-line concentration of microheterogeneous proteins by capillary electrophoresis using SDS and PEO as additives. AU - Huang,Yu-Fen, AU - Hsieh,Ming-Mu, AU - Tseng,Wei-Lung, AU - Chang,Huan-Tsung, PY - 2006/2/7/pubmed PY - 2006/3/30/medline PY - 2006/2/7/entrez SP - 429 EP - 36 JF - Journal of proteome research JO - J Proteome Res VL - 5 IS - 2 N2 - 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. SN - 1535-3893 UR - https://www.unboundmedicine.com/medline/citation/16457610/On_line_concentration_of_microheterogeneous_proteins_by_capillary_electrophoresis_using_SDS_and_PEO_as_additives_ DB - PRIME DP - Unbound Medicine ER -