Quantifying Biomolecular Interactions Using Slow Mixing Mode (SLOMO) Nanoflow ESI-MS.

Area of Science:

• Biophysical Chemistry
• Analytical Chemistry
• Mass Spectrometry

Background:

• Electrospray ionization mass spectrometry (ESI-MS) is a label-free method for studying biomolecular interactions.
• Accurate quantification of binding affinities requires accounting for differing ESI-MS response factors (RFs) between free and bound species.
• Existing RF correction methods are often incompatible with nanoflow ESI (nanoESI) sources used in interaction studies.

Purpose of the Study:

• To develop a direct technique for determining both RFs and binding affinities (Kd) using static nanoESI.
• To overcome limitations of current ESI-MS methods for quantifying biomolecular interactions.

Main Methods:

• Introduction of slow mixing mode (SLOMO) nanoESI-MS.
• Continuous monitoring of interacting species and complexes under nonhomogeneous solution conditions.
• Determining relative RFs from changes in ion signals approaching or leaving steady-state.

Main Results:

• SLOMO allows simultaneous determination of RFs and Kd from a single static nanoESI measurement.
• Demonstrated reliability across diverse systems: peptide-antibiotic, protease-inhibitor, and protein oligomerization.
• Accurate affinity measurements for lectin-nanobody, lectin-glycoprotein, and lectin-glycolipid interactions.

Conclusions:

• SLOMO nanoESI-MS is a versatile and accurate method for quantifying biomolecular interactions.
• The technique overcomes previous limitations in RF correction for nanoESI.
• SLOMO has broad applicability for studying interactions relevant to human health and disease.