Unlikely partners: IEF coupled to ambient mass spectrometry

Direct gel analysis

The normal way of identifying proteins in biological mixtures is the bottom-up procedure, in which all of the proteins are jointly broken up into peptides, normally by enzymes, to be analysed by mass spectrometry. Although this is a well-established and successful process, it can be limited for different isoforms of a particular protein and its post-translational modifications.

The difficulty arises from the fact that structural confirmation is based on the sequences of a few peptides for each protein, so those sequences might not cover the stretches of protein that include the isoform variations. One alternative is the top-down strategy, in which intact proteins are observed. When this is used in conjunction with mass spectrometry, a high-resolution instrument is needed to measure the small differences in the m/z values of the parent compounds.

Some degree of protein separation can still be carried out before top-down analysis with electrophoretic techniques proving to be the most popular. However, direct linking to mass spectrometry, while desirable, has proved difficult. The conventional procedure requires protein staining on the gel following separation, then extraction in a buffer for enzymatic digestion and analysis.

Now, a team of scientists has devised an alternative procedure in which proteins that have been separated by isoelectric focusing (IEF) gel electrophoresis can be analysed from the gel directly. Hubert Girault and colleagues from the Swiss Federal Institute of Technology in Lausanne and Fudan University, Shanghai, deployed an ambient ionisation technique known as electrostatic spray ionisation to achieve the coupling.

Coupling electrostatic spray ionisation

Electrostatic spray ionisation was introduced in 2012 by the same research team. To achieve ionisation, an electrode is placed close to the sample but does not touch it. When a high voltage pulse is applied to the electrode, it charges the sample to produce a bipolar spray pulse of charged microdroplets. When the electrode is grounded, a pulse of oppositely charged species is generated to establish uncharged conditions on the sample. For a positive voltage, the two pulses constitute cations then anions.

In this proof-of-concept study, IEF was carried out in an immobilised pH gradient gel for a number of standard samples. IEF is a high-resolution separation technique, so proteins are well-separated from each other for the subsequent mass spectrometric analysis.

An electrode was placed below the gel’s plastic support and cycled between charging to 6.5 kV and grounding. Droplets of an acidic buffer were added to the gel to generate positive charges on the proteins or peptides which were easily extracted into the solution then transferred to the gas-phase under the high voltage.

In order to ensure good spatial resolution during analysis, a plastic cover containing small drilled holes was placed above the gel. The buffer was added to the holes, which were 1 mm in diameter, to control the droplet size. The holes were also used to help position the spray.

The ions that were released from the gel were analysed by a high-resolution linear ion trap mass spectrometer with a grounded inlet. For proteins that are coloured, it was easy to locate their positions on the gel, otherwise the gel was scanned by the mass spectrometer to locate and analyse each of the separated proteins.

Improved sensitivity and sequence coverage

For a solution of cytochrome c, the detection limit was 20 ng, equivalent to 1.63 pmol. However, this gave a mass spectrum comprising just one peak, the [M+9H]⁹⁺ ion, so it is better to have more sample present. With 200 ng, a range of peaks from 7+ to the 13+ were observed, allowing better detection and protein identification. For the peptide angiotensin I, the detection limit was 1.3 ng, equivalent to 1 pmol.

With improvements to the gel preparation and the ionisation system, the researchers think that the sensitivity could be improved more towards the fmol detection limits of this particular mass spectrometer.

A mixture of peptides generated by enzymatic digestion of bovine serum albumin was also analysed. Four areas of the gel, covering a pH range of 4-7, generated 28, 13, 19 and 13 peptides, respectively. When the peptides were identified from the molecular masses, they generated 74% sequence coverage of the parent protein. This compared to 48% and 47% when the digest was analysed by OFFGEL IEF separation-electrospray ionisation mass spectrometry and gel-based IEF with MALDI mass spectrometry, respectively. Clearly, the new technique has strong potential for protein identification in proteomics studies.

A further test involved an extract of Escherichia coli spiked with cytochrome c and myoglobin. Both of these proteins were detected but those from E. coli could not be identified at this stage.

The successful coupling of IEF with mass spectrometry eliminates several steps that are innate in other gel-based techniques, like removal of gel sections for digestion and chemical extraction. Its good sensitivity, spatial resolution, and provision of molecular weights warrant further investigation and optimisation and to see if protein isoforms can be detected routinely.