Alexander Ivanov, Associate Professor at Northeastern University, and his lab are exploring different separation modalities in liquid-phase separation. They are looking into polymerizing monolithic columns and using porous layer open tubular columns to enhance the analysis of biological molecules.
Porous layer open tubular (PLOT) columns consist of porous, sponge-like columns that are polymerized in a few silica capillaries. The outer diameter of the column is just 20 microns. Meanwhile, the inner diameter can be between 5 and 10 microns. So, these very small and conventional column specs can be used in multiple labs.
Ivanov introduced another modality of separation called capillary electrophoresis. Capillary electrophoresis can be coupled with the Moini interface, making it adaptable for peptide, protein, and glycan analysis. The goal of this pairing is to achieve single-cell level analysis. By decreasing the flow rate, they hope to increase the signal.
The high field asymmetric waveform ion mobility spectrometry (FAIMS) interface functions as an ion mobility-based filtering device and reduces background ions and increases signal-to-noise. Overall, the interface has shown massive noise reduction and signal improvement. Ivanov explained that this leads to better identification of peptides and proteins.
An experiment on ion transfer efficiency showed that the FAIMS interface increases the signal to noise ratio significantly compared to controls and with certain optimisations, it was able to identify double the number of peptides and proteins. Ivanov added that by dropping the flow rate from 64 to 12 nanolitres per minute, they dramatically improved signal-to-noise ratio and detection sensitivity.
At a single-cell level, FAIMS and capillary electrophoresis detected as few as 1 – 10 cells. Ivanov also covered attempts at single-cell top-down proteomics—analysing intact proteins without digestion. Ivanov said: “What was interesting also was that the technique showed very good reproducibility and between replicas and very sharp peaks.” This reproducibility was achievable from as few as 200 cells.
Meanwhile, in glycan analysis, the team developed a non-derivatized CE-MS approach. Ivanov said that his reasoning behind this was that labelling methods are often inefficient for glycans. The alternative CE-MS approach successfully analysed glycans from as few as 50 cells, and even down to single cells, showing clear, quantifiable patterns in response to treatments like growth factors and cytokines.
