Protein preparation contest: In-gel digestion best for plasma membrane proteins

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  • Published: May 21, 2012
  • Author: Steve Down
  • Channels: Sample Preparation
thumbnail image: Protein preparation contest: In-gel digestion best for plasma membrane proteins

Protein digestion

Three conventional procedures for the enzymatic digestion of plasma membrane proteins have been compared by scientists in the US, who found that in-gel digestion was the most efficient, finding the most plasma membrane and transmembrane proteins.

The analysis of protein mixtures by bottom-up processing is one of the classical proteomics techniques, providing a way of generating constituent peptides from which the identities of the respective proteins can be deduced. For some target tissues, like the plasma membrane, the proteins need some help getting into solution because many of them are hydrophobic.

The usual strategy is to use urea and the surfactant sodium dodecyl sulphate (SDS) as solubilising agents but these must be removed from the solution at a later stage before mass spectrometric analysis, because they suppress ionisation even at low concentrations.

Once solubilised, the peptides are normally generated from the proteins by digestion with an enzyme, usually trypsin, but sometimes chymotrypsin, pepsin, or one of several endoproteinases. Even with one enzyme, there are several ways to go about the digestion process and a team of scientists in the USA has undertaken a comparison of three modes of tryptic digestion, to see which is the most effective.

Catherine Fenselau and colleagues from the University of Maryland, College Park, Georgetown University Medical Center, Washington, DC, and the University of Maryland Medical School, Baltimore, compared in-gel, in-solution and on-filter digestion. One of their aims was to see which strategy was the best for removing SDS from solution without affecting the overall yield of peptides.

Digestions protocols

All three procedures were carried out on plasma membrane proteins isolated from the same set of human multiple myeloma cells. Washed cells were suspended in buffer and coated with cationic silica nanoparticles before being lysed under pressure by nitrogen cavitation. Following sedimentation in a sucrose solution, the membrane proteins were solubilised in SDS buffer ready for digestion.

For in-gel digestion, the proteins were separated by 1D gel electrophoresis and 17 individual bands were cut out from the gel for separate reduction, alkylation and digestion with trypsin.

In-solution digestion required precipitation of the proteins from the SDS solution before redissolution in a urea solution for reduction, alkylation and digestion with endoproteinase Lys-C then trypsin.

On-filter digestion was carried out on a 3 kDa molecular weight cutoff filter. The protein solution was mixed with the reducing agent dithiothreitol and applied to the filter where it was exchanged for a urea solution. The alkylating agent was added to the retentate followed by Lys-C then trypsin.

The peptides from each digestion procedure were analysed by HPLC-tandem-MS with electrospray ionisation on a high-resolution mass spectrometer. The data files were searched against the human IPI database with the requirement that at least two peptides were required for protein identification in order to increase confidence in the results.

Gel outperforms filter and solution techniques

The best performing technique by a long way was in-gel digestion, followed by on-filter and in-solution digestion, uncovering a total of 2580, 1009 and 967 peptides which identified 272, 118 and 106 proteins, respectively. This is the result that the researchers anticipated due to the initial protein fractionation that takes place on the gel before digestion.

However, they suspected that other factors were at play. When the relative abundances of the 79 proteins that were common to each technique were compared by spectral counting, 59 proteins analysed by in-gel digestion had the largest values. The lower value for in-solution digestion was attributed to protein losses during protein precipitation and resolubilisation whereas that for on-filter digestion was ascribed to incomplete resolubilisation and restricted enzyme access to the proteins on the filter.

The number of transmembrane domains identified via each technique varied in the same order as the number of proteins, with 106, 37 and 33 proteins containing at least one such domain found by in-gel, on-filter and in-solution digestion, respectively.

In a further comparison, the team found that the proteins identified by in-gel digestion were more hydrophobic than those from the other techniques, indicating that the process does a better job of getting those proteins into solution.

In proteomic studies of the plasma membrane, transmembrane and hydrophobic proteins are often under-represented, so any method which increases their numbers would be welcomed. Coupled with the fact that in-gel digestion identifies by far the greatest number of proteins, this should be regarded as the best technique of the three for studying plasma membrane proteins.

Related Links

Journal of Proteome Research 2012, 11, 3030-3034: "Comparative study of workflows optimized for in-gel, in-solution, and on-filter proteolysis in the analysis of plasma membrane proteins"

Article by Steve Down

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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