Native Mass Spectrometry

Versatile technique to screen and characterize compound interaction

Native mass spectrometry (native MS) is a robust and reliable tool for the analysis of non-covalent complexes. This label-free analytical technique also referred to as non-covalent, non-denaturing or supramolecular MS, utilizes electrospray to ionize and transfer non-covalent assemblies from solution to the gas phase. Accurate measurement of the molecular mass of an intact complex provides valuable insights into biomolecule interaction, including stoichiometry, reversibility, site-specificity, affinity and polar contributions to the binding.


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Native MS is widely applicable to a variety of biomolecular assemblies including multi-protein and nucleic acid systems, as well as to small molecules complexed to proteins or nucleic acids.

Combining medium throughput screening capability with the delivery of high-information-content, native MS complements other methods SPR, NMR, X-ray crystallography and ITC commonly used in drug discovery programs for hit finding and hit-to-lead optimization. Experimental conditions require optimization so there is no substitute for expertize and experience to routinely produce reliable data.

Reliable native MS data requires:

  1. Sample preparation, QC
  2. Mass spectrometer – operational parameters
  3. Automation

1) Sample preparation and QC

Sample preparation is the key to high quality mass spectra. Proteins stored in cocktails of non-volatile components, salts, buffers and surfactants are incompatible with electrospray ionization. Buffer exchange against a volatile ammonium-based buffer is a prerequisite for protein native MS.

Quality control of the desalted sample under denaturing and non-denaturing conditions provides information of sample homogeneity and molecular mass, conformation (folded versus unfolded), and oligomerization state. Analysis under native conditions may also uncover the presence of natural or fortuitous ligands that happen to have co-purified with the protein sample.

2) The mass spectrometer

Electrospray ionization (ESI) is the best-suited source of non-covalent complex ions from aqueous solutions.

For native MS applications, ESI sources are advantageously coupled to time-of-flight (TOF) mass analyzers which combine speed, resolution, sensitivity and broad mass range.

The analysis of non-covalent complexes using ESI-TOF instruments requires fine tuning of acceleration voltage and gas pressure to provide optimal ion desolvation and transmission while preserving the integrity of non-covalent complexes [Vivat Hannah et al., Future Medicinal Chemistry 2010, 2, 35].

3) Automation

Hyphenation of ESI-TOF mass spectrometers with a Triversa Nanomate® automated chip-based nanoESI robotic interface achieves remarkable throughput allowing daily screening of 384 compounds or cocktails, consuming only 50-200 pmol of protein per well. Subsequent raw data processing has been significantly accelerated by in-house developed programs allowing automated hit compound assignment.

Features and benefits


  • Protein, nucleic acid or compound labeling/immobilization: NO
  • Purified, homogeneous and soluble targets: YES


  • Sensitivity: detection of nanomolar complex concentrations.
  • Selectivity: species are clearly identified through accurate mass measurement (no data modeling or fitting is required); multiplexing – the ability to provide evidence in a single mass spectrum of several assemblies present together in solution.
  • Speed: analyses take only few minutes of data acquisition per sample affording medium throughput capability.


  • Hydrophobic complex: partial or complete in source dissociation (false negatives)
  • Screening for low affinity binders (Kd > 100 µM): false positives due to non-specific aggregation of compounds during the ESI process.
  • Buffer compatibility is limited to volatile ammonium-based buffers
  • Not easily applicable to whole membrane proteins such as GPCRs