A cheap and portable peptide mass spectrometer can be constructed using existing nanopore technology and pores that were developed at University of Groningen, according to a paper published in Nature Communications yesterday. Mass spectrometers are invaluable for studying proteins, but they are both bulky and expensive, which limits their use to specialized laboratories.
In previous work, Giovanni Maglia, associate professor of chemical biology, showed that biological nanopores can be used to measure metabolites and to identify proteins and peptides. Molecules entering a pore or passing through it cause a change in an electric current across the pore. “A problem in measuring the mass of peptides is that they pass too quickly through even the smallest biological pore to get a reading,” explains Maglia.
Making smaller pores was a challenge. “Pores are made up from a number of monomers, so we initially modified the interaction between these monomers, but that didn't work.” The observation that mixing monomers with larger amounts of lipids resulted in a larger percentage of smaller pores gave Maglia and his team the idea to modify the interaction between monomers and lipids.
Maglia was then able to produce funnel-shaped pores, which, at their narrow end, only measured 0.84 nanometers. The next challenge was to ensure that peptides would pass through the pores, irrespective of their chemical composition. The charge causes water to flow through the pore, dragging the peptides along. But negatively charged peptides would be repelled by the negative charge at the thin funnel end. Maglia modified the charge by altering the acidity of the fluids used. “Eventually, we managed to find the right conditions by setting the acidity at a pH of exactly 3.8. This allows negatively charged peptides to pass through while maintaining a large enough water flow through the pores.”
Measurements across nanopores of different sizes show that the electric current is linear with the volume of the peptide passing through. These peptides ranged from 4 to 22 amino acids in length. The difference between the amino acids alanine and glutamate could be measured in this system, which meant the resolution is around 40 Dalton. “The resolution of conventional mass spectrometers is much better, but if we could get the system about forty times more sensitive, it would already be useful in proteomics research,” says Maglia.
The nanopore system has several unique selling points: it measures single molecules, the technology itself is already commercially available and it is relatively cheap. Furthermore, the nanopore system is portable. And, by using many different pores in a device, you can simultaneously measure differently sized peptides and even peptide modifications. “All of this means that a versatile and cheap mass spectrometer for peptide analysis is feasible,” says Maglia.
Image: A peptide enters the thin end of the nanopore, and there changes the current in proportion to its mass. By using differently sized nanopores, a range of peptide sizes can be measured. Image courtesy of G. Maglia / University of Groningen.