Coupling Isoelectric Focusing Gel Electrophoresis to Mass Spectrometry by Electrostatic Spray Ionisation

Gel electrophoresis has been used for decades as a high-resolution separation technique for proteins and protein isomers, but limited in the coupling with MS because of low throughput and poor automaticity compared with LC-MS. In this work, we have developed an ambient ionisation strategy, electrostatic spray ionisation, for in-situ ionisation of proteins or peptides inside a surfactant-free polyacrylamide gel. The samples can be first separated by isoelectric focusing in a gel and then quickly in-situ detected by scanning the gel with the electrostatic spray ionisation mass spectrometry. With this strategy, nanograms of proteins or peptides inside a band are enough to be ionised for MS detection. This method for protein/peptide spots visualization is sensitive providing sample molecular weight information whilst avoiding spot staining and chemical extraction procedures that can introduce contaminants and sample loss. Proof-of-principle results have demonstrated that the electrostatic spray ionisation can produce sample ions from a complex background and with a spatial resolution matching the isoelectric focusing, therefore a good choice to couple directly isoelectric focusing gel electrophoresis with mass spectrometry.