Native mass spectrometry is an expanding approach for studying intact biomolecular structure in the near-native state, especially proteins and protein complexes, in the gas phase . The Loo group  at UCLA, Robinson group  at Oxford, and Heck group  in the Netherland are among the leaders in this field. Two key conditions are required for a successful native MS experiment, ionization of the target sample preserved in a volatile buffer and an instrument capable of high m/z measurement.
a. Ionization: The preferred buffer that maintains a protein or protein complex in the native state is not amenable to ionization of the protein in mass spectrometry. It has to be exchanged to a medium that mimics the original buffer to keep the protein folded and volatile during the ionization process. Aqueous solutions of ammonium acetate (NH4OAc) are usually chosen for this purpose. The concentration of NH4OAc ranges from 5 mM to 1 M depending on the protein system.
b. Instrumentation: TOF instrument coupled with collisionally activated dissociation (CAD) has played a dominant role in the field. Recently, TOF with surface-induced dissociation (SID) , FTICR , and Orbitrap  have demonstrated unique capabilities for providing structural information. Instruments available for native MS at Washington University include a Bruker MaXis 4G Q-TOF and a Bruker SolariX 12 T FTICR in the MS Resource, and a Waters Synapt G2 ion mobility Q-TOF in the Photosynthetic Antenna Research Center.
Below is a brief description of performing such an experiment.
Step 1: Buffer exchange
Buffer exchange is performed by centrifugation with two techniques. One employs a molecular weight cut-off (MWCO) filter, the other a micro gel column. They can be used separately or in combination. Usually 25 uL of the original sample solution is loaded onto the filter, followed by addition of 200 uL 200 mM NH4OAc solution. This filter is then placed in a centrifuge, counter balanced, and spun to get rid of the buffer solution. The cycle of adding NH4OAc solution and centrifugation is repeated about five to six times. To ensure a sample has the least components from the original buffer, the filtered sample is then loaded to a micro gel column to remove small molecules. After this step, the sample is ready for mass spectrometry. If a protein is prone to aggregation, the multiple cycles of dilution-concentration process can be problematic. In such cases, micro gel column is usually used exclusively. The final concentration of the proteins solution loaded for MS analysis is about 10 uM.
Step 2: Instrument calibration
To calibrate the instruments for a broad range up to m/z values of tens of thousands, cesium salts are often used i(e.g., cesium perfluoroheptanoate or cesium iodide). For example, 100 mM stock solutions of cesium bicarbonate in water and perfluoroheptanoic acid in acetonitrile are prepared and diluted to a concentration of 5 mM in 50/50 water/acetonitrile right before an experiment. This calibration solution serves also to optimize the instrument parameters to reach the best performance around the target m/z region of the protein sample.
Step 3: Native electrospray ionization
A syringe pump with nanoliter flow rate and metal-coated glass tips are used to directly introduce the buffer-exchanged protein sample, similar to nano-ESI, into the gas phase for mass spectrometry analysis. Because the protein is close to its native state in NH4OAc solution, the surface area is smaller than that of its denatured counterpart in the normal acidic ESI buffer containing half organic and half water. Thus, a protein or protein complex in native ESI will have fewer charges than in normal ESI. Taking a 150 kDa antibody  as an example (Figure 1), native ESI generates a narrow distribution of about five charge states around 25+ at m/z 6000, while normal ESI generates a wide charge state distribution around 60+ at m/z 2500. Ionization is key for a successful experiment and native MS is sometimes called native ESI.
Step 4: Spectra acquisition
Depending on the analytical purpose for a specific protein sample, we can employ the three instruments, Q-TOF, FTICR, and Synapt G2, alone or in combination. Mass spectra can be acquired to obtain the following structural information:
Native ESI provides a narrow charge state distribution of the protein ions. Deconvolution of these charge states determines the intact mass of the target protein/complex, and the stoichiometric information can be deduced. This kind of experiment and collisionally activated dissociation (CAD) can be accomplished on all three instruments. CAD results provide insights into the connectivity of the constituent components of a complex.
Electron capture dissociation (ECD) top-down sequencing on the FTICR instrument, often combined with CAD, is able to probe the flexible regions of a protein or protein complex. This correlates well with the B-factor in X-ray crystallography, a parameter that reflects the extent of atom displacement in the solid crystal .
The Waters Synapt G2 Q-TOF has an ion mobility cell to measure the collision cross section of protein ions. This offers structural information in another dimension – the shape and size of a protein in the gas phase.
- H. Zhang, W. Cui, M. L. Gross, R. E. Blankenship, Native mass spectrometry of photosynthetic pigment-protein complexes, FEBS Lett. 2013, 587:1012-20.
- J. A. Loo, Studying noncovalent protein complexes by electrospray ionization mass spectrometry, Mass Spectrom. Rev. 1997, 16: 1-23.
- J. L. Benesch, B. T. Ruotolo, D. A. Simmons, C. R. Robinson, Protein complexes in the gas phase: technology for structural genomics and proteomics, Chem. Rev. 2007, 107: 3544-67.
- A. J. Heck, R. H. Van Den Heuvel, Investigation of intact protein complexes by mass spectrometry, Mass Spectrom. Rev. 2004, 23: 368-89.
- M. Zhou, C. M. Jones, V. H. Wysocki, Dissecting the large noncovalent protein complex GroEL with surface-induced dissociation and ion mobility-mass spectrometry, Anal. Chem. 2013, 85: 8262-7
- W. Cui, H. W. Rohrs, M. L. Gross, Top-down mass spectrometry: recent developments, applications and perspectives, Analyst. 2011, 136(19):3854-64.
- R. J. Rose, E. Damoc, E. Denisov, A. Makarov, A. J. Heck, High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies, Nat. Methods. 2012, 9: 1084-6.
- H. Zhang, W. Cui, M. L. Gross, Mass spectrometry for the biophysical characterization of therapeutic monoclonal antibodies, FEBS Lett. 2013, doi: 10.1016/j.febslet.2013.11.027.