Protein enrichment and derivatisation: One device fits all

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  • Published: Dec 20, 2010
  • Author: Steve Down
  • Channels: Sample Preparation
thumbnail image: Protein enrichment and derivatisation: One device fits all

Seeing proteins at low concentration: fluorescence limitations

Rapid advances in protein analysis in recent years have led to the detection of increasing numbers of low-abundant proteins, often by fluorescence detection. The fluorescence derives from aromatic amino acid residues in the protein chain, such as phenylalanine, tryptophan and tyrosine but there are two key problems associated with this procedure.

In the first instance, there are a number of proteins that cannot generate fluorescence on their own, so they have to be converted to fluorescent forms for detection to be successful. Secondly, some proteins are found at such low levels that the derivatisation reactions are not effective, so that subsequent detection is unsuccessful. For instance, the common reagent fluorescein isothiocyanate (FITC) requires relatively high protein concentrations of 1-10 mg/mL.

Clearly, in these cases the protein must be enriched in some way to allow derivatisation to occur effectively and a team of scientists in China have come up with a novel solution. They have designed a device that combines protein enrichment and subsequent derivatisation in one.

One-stop protein device: a hollow success

Lihua Zhang and colleagues from the Dalian Institute of Chemical Physics, Dalian University of Technology, and the Graduate School of the Chinese Academy of Sciences, Beijing, based their device around a hollow fibre membrane. It was placed inside a centrifugal tube within a basic buffer solution of sodium carbonate-sodium bicarbonate.

Protein solution was injected continuously into the hollow fibre membrane which was sealed to allow the proteins to concentrate. The cellulose acetate membrane had a molecular weight cutoff at 3000 Da which trapped the proteins but allowed associated small molecules such as salts and denaturants to pass through into the buffer solution. The enrichment can continue as long as the protein concentration remains greater than its maximum solubility.

The device was tested with a solution of denatured bovine serum albumin, which was dissolved in the basic buffer solution before injection. The degree of enrichment, measured by HPLC with UV detection, peaked at a flow rate of 1.5 µL/min before falling sharply. Zhang attributed the fall off to a distortion of the membrane caused by the higher pressure, allowing protein molecules to escape.

For in situ derivatisation, the hollow fibre membrane was placed directly in a tube containing the reaction solution and warmed in the dark. The reagent was a water-soluble sulpho-3H-indocyanine dye which the research team had developed previously. It reacts with protein amino groups, especially those in the side chain of lysine residues, and is more reactive than conventional dyes such as FITC. It infiltrated the membrane to bring about reaction with the protein.

Following reaction, the derivatised protein was pumped into a vial for analysis by HPLC with fluorescence detection at excitation and emission wavelengths in the yellow region at 554 and 570 nm, respectively.

The fluorescence intensity was markedly increased by enrichment and derivatisation, resulting in a 200-fold enhancement of detection sensitivity. This was attributed to both the enrichment process and the absence of protein dilution during derivatisation, compared with conventional in-solution derivatisation.

The protocol was subjected to a more rigorous test with a denatured, reversed-phase HPLC fraction of Escherichia coli proteins. Conventional in-solution derivatisation produced no fluorescence signal at all, whereas the novel process resulted in two large HPLC peaks, confirming efficient enrichment and derivatisation.

The technique has the potential to be of universal application for the concentration and derivatisation of low-abundance proteins, with the superiority of executing both operations in the same device, while out-performing traditional methods.

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

 A novel device that accomplishes both protein enrichment and fluorescent derivatisation has been constructed and demonstrated by Chinese scientists. It is based on a hollow fibre membrane and will simplify the detection of low-abundant proteins

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