Large Molecule Capture

Large Molecule Capture

MIK-MS may be used to enrich molecules from complex solutions. Say for instance a substance in a cellular extract appears active, though that substance is unknown. Using the SKi Pro system together with SKi Sensors one can capture many more molecules than with any planar system.

Capture large sketch

Figure 1 Sketch of how proteins are enriched using MIK-MS. The top shows what is expected from the eluent at each step.

Protein Enrichment Concept

A protein which exists in a complex environment may be enriched, i.e. increased in concentration relative to other molecules, by using its binding properties, in a fully automated fashion.

Shown in figure 1, is the NPOI signal as a function of time. This signal increases, stabilizes and then decreases, during, respectively, the capture, associated and chemically induced, dissociation phases of the binding interaction.

Shown above the figure are whan is expected from the eluent of the sample and reference channels at each phase. During capture, one expects an nearly equal amount of molecules from both sample and reference as not every molecule is bound. During associated, for a tight binding interaction as shown here, one expects next to nothing to be released. However during dissociated, one expects only bound molecule from the sample channel.

Protein Enrichment Data

In this experiment an antibody molecule was immobilized onto the SKi Sensor surface using carboxyl coupling. A protein antigen was introduced, allowed to stay on the surface for a time, and then dissociated with 100 mM phosphoric acid. The NPOI signal and MS signal are shown together in figure 2. For the MS signal, a QTrap type mass spectrometer is used to measure the eluent from the various phases of the interaction shown in figure 1, after tryptic digestion.

The level of signal from the MS, shows that approximately 40% of the original signal was recovered from the initial fraction but all non-binding elements are removed, which has now isolated that protein which was responsible for the binding signal in the first place.

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MIK-MS sketch

Figure 2 NPOI and MS data from an antibody-antigen measurement. The extracted-ion chromatagram data (XIC) is from an LC-MS run after tryptic digestion of the eluents.