Integrated protein processing device

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Ezine

  • Published: Mar 22, 2010
  • Author: Steve Down
  • Channels: Sample Preparation
thumbnail image: Integrated protein processing device

Breaking down proteins into their constituent peptides for analysis by liquid chromatography-tandem mass spectrometry is one of the key steps in the bottom-up, or shotgun, approach to proteomics. Although it is now a well-established procedure, it is not always a straightforward one.

In some cases, the protein mixtures extracted from the original biological or biochemical medium are so complex that there are too many peptides for efficient HPLC separation. In addition, peptides produced from low-abundance proteins can be swamped by those from more abundant proteins. Consequently, it is now common practice to introduce a protein fractionation step before digestion. Typically, isoelectric focusing, 2D electrophoresis and multi-dimensional HPLC have been employed.

The combination of protein fractionation and digestion with subsequent peptide isolation is generally performed offline, typically taking several hours in total and representing one of the bottlenecks in proteomics analysis. However, a team of scientists in China has now developed an online procedure which cuts the total time to a few minutes without any loss of performance.

Lihua Zhang and co-researchers from the National Chromatographic R. & A. Center at the Dalian Institute of Chemical Physics and the Graduate School of Chinese Academy of Sciences, Beijing, based their procedure on an immobilised enzymatic reactor which they developed recently. The reactor comprised a capillary of internal diameter 100 µm containing trypsin immobilised on a hybrid silica monolith.

The reaction conditions for digestion require a pH of about 8.0 but this is not compatible with the solutions that are eluted from a typical HPLC run, which have a pH of about 3.0 and are rich in acetonitrile. Rather than carrying out the required adjustments offline, Zhang introduced an online buffer exchange device that used a hollow fibre membrane.

Evaluating the performance of the membrane revealed that the pH could be increased from 3.0 to 8.0 in 30 seconds for proteins dissolved in aqueous acetonitrile-urea and exchanged into aqueous ammonium bicarbonate buffer. This process reduced the acetonitrile content by about 90% and rendered the solution amenable to digestion.

The final step involved concentrating the proteins as they left the hollow fibre membrane before reaching the reactor. This was accomplished by placing a capillary coated with linear polyacrylamide between the membrane and the reactor. The back pressure generated from the reactor during continuous injection helped with buffer splitting and sample stacking.

The value of protein enrichment was illustrated by the analysis of myoglobin samples with and without enrichment in the integrated device. Online enrichment increased the peak intensities of 2 peptides by 11.5-fold and allowed the identification of 6 extra peptides. These improvements increased the sequence coverage from 19% to more than 60%.

In a demonstration of the whole integrated device, a mixture of cytochrome c, bovine serum albumin and myoglobin was separated on a C8 column. The three fractions were processed by traditional in-solution digestion followed by LC/MS/MS analysis, or with the integrated device.

The online device did not lead to improvements in sequence coverage, which was comparable by both routes. However, the total sample pretreatment time was reduced from 960 minutes to 5 minutes. For labs in which there are many samples to analyse and the pressure is on, the massive time savings will be highly beneficial.

In the longer term, the researchers recommended that their new integrated device for protein buffer exchange, enrichment and digestion should be linked to online protein separation and LC/MS analysis to produce a high-throughput system for proteome analysis.



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

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