On-Line Concentration of Microheterogeneous Proteins by Capillary Electrophoresis Using SDS and PEO as Additives

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.

Effect of detergent on electromigration of proteins: CE of very low density lipoprotein receptor modules and viral proteins

“…Insofar this system seems predestined as unintentional ITP system under conditions aimed at carrying out zone electrophoresis. However, such setups are valuable for the online enrichment and preconcentration for better detectability, and are operated in practice as 'stacking systems' or 'transient isotachophoretic system' [9,11,13] (for the selection of appropriate electrolytes based on the model of electromigration, see ref. [31]).…”

LIF detection of peptides and proteins in CE

“…It should also be highlighted that sample concentration techniques have not been fully explored for protein and peptide analysis in CE. In this field, an interesting method of stacking which allows to introduce large volumes of protein samples in CE has appeared recently [184]. With an injection volume of ca.…”

CE with a new electrochemiluminescent detection system for separation and detection of proteins labeled with tris(1,10‐phenanthroline) ruthenium(II)

“…CE is able to detect both immunoreactive and immunounreactive urinary albumin, and therefore it is a good alternative for the assessing of urinary albumin concentration. However, the sensitivity of CE approaches is still a huge challenge for trace analysis of HSA in real urinary samples [52][53][54].…”