Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometry


It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Separation of protein from non-volatile buffer components.
Fig. 2: Comparison of OBE nMS using different size-exclusion columns.
Fig. 3: Effect of OBE on protein spectral quality.
Fig. 4: Deconvoluted mass spectra demonstrating the removal of non-volatile components from proteins in common biological buffers by OBE.
Fig. 5: Detection of overexpressed proteins from a clarified cell lysate after OBE with a self-packed P6 column.
Fig. 6: OBE coupled to different mass spectrometers.
Fig. 7: Limit of detection for OBE-MS on an EMR mass spectrometer.
Fig. 8: Experimental setup for OBE nMS.
Fig. 9: Implementation of OBE in structure-based protein screening.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.


  1. 1.

    Structural Genomics Consortium. et al. Protein production and purification. Nat. Methods 5, 135–146 (2008).

  2. 2.

    Arnau, J., Lauritzen, C. & Pedersen, J. Cloning strategy, production and purification of proteins with exopeptidase-cleavable His-tags. Nat. Protoc. 1, 2326 (2006).

  3. 3.

    Nallamsetty, S. & Waugh, D. S. A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag. Nat. Protoc. 2, 383–391 (2007).

  4. 4.

    Bondos, S. E. & Bicknell, A. Detection and prevention of protein aggregation before, during, and after purification. Anal. Biochem. 316, 223–231 (2003).

  5. 5.

    Vagenende, V., Yap, M. G. S. & Trout, B. L. Mechanisms of protein stabilization and prevention of protein aggregation by glycerol. Biochemistry 48, 11084–11096 (2009).

  6. 6.

    Kimple, M. E., Brill, A. L. & Pasker, R. L. Overview of affinity tags for protein purification. Curr. Protoc. Protein Sci. 73, 9.9.1–9.9.23 (2013).

  7. 7.

    Cavanagh, J., Benson, L. M., Thompson, R. & Naylor, S. In-line desalting mass spectrometry for the study of noncovalent biological complexes. Anal. Chem. 75, 3281–3286 (2003).

  8. 8.

    Waitt, G. M., Xu, R., Wisely, G. B. & Williams, J. D. Automated in-line gel filtration for native state mass spectrometry. J. Am. Soc. Mass Spectrom. 19, 239–245 (2008).

  9. 9.

    Ehkirch, A. et al. An online four-dimensional HIC×SEC-IM×MS methodology for proof-of-concept characterization of antibody drug conjugates. Anal. Chem. 90, 1578–1586 (2018).

  10. 10.

    Chen, Z. et al. Programmable design of orthogonal protein heterodimers. Nature 565, 106 (2019).

  11. 11.

    Fekete, S., Ganzler, K. & Guillarme, D. Critical evaluation of fast size exclusion chromatographic separations of protein aggregates, applying sub-2 μm particles. J. Pharm. Biomed. Anal. 78–79, 141–149 (2013).

  12. 12.

    Muddiman, D. C., Cheng, X., Udseth, H. R. & Smith, R. D. Charge-state reduction with improved signal intensity of oligonucleotides in electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 7, 697–706 (1996).

  13. 13.

    Mehmood, S. et al. Charge reduction stabilizes intact membrane protein complexes for mass spectrometry. J. Am. Chem. Soc. 136, 17010–17012 (2014).

  14. 14.

    Marcoux, J. et al. Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate. Protein Sci. 24, 1210–1223 (2015).

  15. 15.

    Townsend, J. A., Keener, J. E., Miller, Z. M., Prell, J. S. & Marty, M. T. Imidazole derivatives improve charge reduction and stabilization for native mass spectrometry. Anal. Chem. (2019) https://doi.org/10.1021/acs.analchem.9b04263.

  16. 16.

    Benesch, J. L. P., Ruotolo, B. T., Simmons, D. A. & Robinson, C. V. Protein complexes in the gas phase: technology for structural genomics and proteomics. Chem. Rev. 107, 3544–3567 (2007).

  17. 17.

    Sobott, F., McCammon, M. G., Hernandez, H. & Robinson, C. V. The flight of macromolecular complexes in a mass spectrometer. Philos. Trans. R. Soc. Math. Phys. Eng. Sci. 363, 379–391 (2005).

  18. 18.

    Wen, J., Zhang, H., Gross, M. L. & Blankenship, R. E. Native electrospray mass spectrometry reveals the nature and stoichiometry of pigments in the FMO photosynthetic antenna protein. Biochemistry 50, 3502–3511 (2011).

  19. 19.

    Rose, R. J., Damoc, E., Denisov, E., Makarov, A. & Heck, A. J. R. High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies. Nat. Methods 9, 1084–1086 (2012).

  20. 20.

    Loo, J. A. Studying noncovalent protein complexes by electrospray ionization mass spectrometry. Mass Spectrom. Rev. 16, 1–23 (1997).

  21. 21.

    Chowdhury, S. K., Katta, V. & Chait, B. T. Probing conformational changes in proteins by mass spectrometry. J. Am. Chem. Soc. 112, 9012–9013 (1990).

  22. 22.

    Winston, R. L. & Fitzgerald, M. C. Mass spectrometry as a readout of protein structure and function. Mass Spectrom. Rev. 16, 165–179 (1997).

  23. 23.

    Hall, Z. & Robinson, C. V. Do charge state signatures guarantee protein conformations? J. Am. Soc. Mass Spectrom. 23, 1161–1168 (2012).

  24. 24.

    Li, Y. & Cole, R. B. Shifts in peptide and protein charge state distributions with varying spray tip orifice diameter in nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 75, 5739–5746 (2003).

  25. 25.

    Bern, M. et al. Parsimonious charge deconvolution for native mass spectrometry. J. Proteome Res. 17, 1216–1226 (2018).

  26. 26.

    Marty, M. T. et al. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal. Chem. 87, 4370–4376 (2015).

  27. 27.

    Reid, D. J. et al. MetaUniDec: high-throughput deconvolution of native mass spectra. J. Am. Soc. Mass Spectrom. (2018) https://doi.org/10.1007/s13361-018-1951-9.

  28. 28.

    Ren, C. et al. Quantitative determination of protein–ligand affinity by size exclusion chromatography directly coupled to high-resolution native mass spectrometry. Anal. Chem. 91, 903–911 (2019).

  29. 29.

    Folta-Stogniew, E. Oligomeric states of proteins determined by size-exclusion chromatography coupled with light scattering, absorbance, and refractive index detectors. In New and Emerging Proteomic Techniques 97–112 (eds. Nedelkov, D. & Nelson, R. W.) (Humana Press, 2006). https://doi.org/10.1385/1-59745-026-X:97.

  30. 30.

    Lössl, P., van de Waterbeemd, M. & Heck, A. J. The diverse and expanding role of mass spectrometry in structural and molecular biology. EMBO J. 35, 2634–2657, https://doi.org/10.15252/embj.201694818 (2016).

  31. 31.

    Susa, A. C., Xia, Z. & Williams, E. R. Native mass spectrometry from common buffers with salts that mimic the extracellular environment. Angew. Chem. Int. Ed. Engl. 56, 7912–7915, https://doi.org/10.1002/anie.201702330 (2017).

  32. 32.

    Susa, A. C., Xia, Z. & Williams, E. R. Small emitter tips for native mass spectrometry of proteins and protein complexes from nonvolatile buffers that mimic the intracellular environment. Anal. Chem. 89, 3116–3122 (2017).

  33. 33.

    Nguyen, G. T. H. et al. Nanoscale ion emitters in native mass spectrometry for measuring ligand–protein binding affinities. ACS Cent. Sci. 5, 308–318, https://doi.org/10.1021/acscentsci.8b00787 (2019).

  34. 34.

    Clarke, D. J. & Campopiano, D. J. Desalting large protein complexes during native electrospray mass spectrometry by addition of amino acids to the working solution. Analyst 140, 2679–2686 (2015).

  35. 35.

    Iavarone, A. T., Udekwu, O. A. & Williams, E. R. Buffer loading for counteracting metal salt-induced signal suppression in electrospray ionization. Anal. Chem. 76, 3944–3950 (2004).

  36. 36.

    Flick, T. G., Cassou, C. A., Chang, T. M. & Williams, E. R. Solution additives that desalt protein ions in native mass spectrometry. Anal. Chem. 84, 7511–7517 (2012).

  37. 37.

    Cassou, C. A. & Williams, E. R. Desalting protein ions in native mass spectrometry using supercharging reagents. Analyst 139, 4810–4819 (2014).

  38. 38.

    Chen, Y., Mori, M., Pastusek, A. C., Schug, K. A. & Dasgupta, P. K. On-line electrodialytic salt removal in electrospray ionization mass spectrometry of proteins. Anal. Chem. 83, 1015–1021 (2011).

  39. 39.

    Wilson, D. J. & Konermann, L. Ultrarapid desalting of protein solutions for electrospray mass spectrometry in a microchannel laminar flow device. Anal. Chem. 77, 6887–6894 (2005).

  40. 40.

    Xu, N. et al. A microfabricated dialysis device for sample cleanup in electrospray ionization mass spectrometry. Anal. Chem. 70, 3553–3556 (1998).

  41. 41.

    Xiang, F., Lin, Y., Wen, J., Matson, D. W. & Smith, R. D. An integrated microfabricated device for dual microdialysis and on-line ESI-ion trap mass spectrometry for analysis of complex biological samples. Anal. Chem. 71, 1485–1490 (1999).

  42. 42.

    Zhang, Y., Fonslow, B. R., Shan, B., Baek, M.-C. & Yates, J. R. Protein analysis by shotgun/bottom-up proteomics. Chem. Rev. 113, 2343–2394 (2013).

  43. 43.

    Meng, F. et al. Processing complex mixtures of intact proteins for direct analysis by mass spectrometry. Anal. Chem. 74, 2923–2929 (2002).

  44. 44.

    Tian, Z. et al. Two-dimensional liquid chromatography system for online top-down mass spectrometry. Proteomics 10, 3610–3620 (2010).

  45. 45.

    Camacho-Carvajal, M. M., Wollscheid, B., Aebersold, R., Steimle, V. & Schamel, W. W. A. Two-dimensional blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach. Mol. Cell. Proteom. 3, 176–182 (2004).

  46. 46.

    Gingras, A.-C., Gstaiger, M., Raught, B. & Aebersold, R. Analysis of protein complexes using mass spectrometry. Nat. Rev. Mol. Cell Biol. 8, 645–654 (2007).

  47. 47.

    Bauer, A. & Kuster, B. Affinity purification-mass spectrometry. Eur. J. Biochem 270, 570–578 (2003).

  48. 48.

    Skinner, O. S. et al. Native GELFrEE: a new separation technique for biomolecular assemblies. Anal. Chem. 87, 3032–3038 (2015).

  49. 49.

    Skinner, O. S. et al. An informatic framework for decoding protein complexes by top-down mass spectrometry. Nat. Methods 13, 237–240 (2016).

  50. 50.

    Skinner, O. S. et al. Top-down characterization of endogenous protein complexes with native proteomics. Nat. Chem. Biol. 14, 36–41, https://doi.org/10.1038/nchembio.2515 (2017).

  51. 51.

    Catcott, K. C., Yan, J., Qu, W., Wysocki, V. H. & Zhou, Z. S. Identifying unknown enzyme–substrate pairs from the cellular milieu with native mass spectrometry. Chembiochem 18, 613–617 (2017).

  52. 52.

    Gan, J. et al. Native mass spectrometry of recombinant proteins from crude cell lysates. Anal. Chem. 89, 4398–4404 (2017).

  53. 53.

    Cveticanin, J. et al. Estimating interprotein pairwise interaction energies in cell lysates from a single native mass spectrum. Anal. Chem. 90, 10090–10094 (2018).

  54. 54.

    Ben-Nissan, G. et al. Rapid characterization of secreted recombinant proteins by native mass spectrometry. Commun. Biol. 1, 213 (2018).

  55. 55.

    Zhou, M., Jones, C. M. & Wysocki, V. H. Dissecting the large noncovalent protein complex GroEL with surface-induced dissociation and ion mobility–mass spectrometry. Anal. Chem. 85, 8262–8267 (2013).

  56. 56.

    VanAernum, Z. L. et al. Surface-induced dissociation of noncovalent protein complexes in an extended mass range orbitrap mass spectrometer. Anal. Chem. 91, 3611–3618 (2019).

  57. 57.

    Sobott, F., Hernández, H., McCammon, M. G., Tito, M. A. & Robinson, C. V. A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies. Anal. Chem. 74, 1402–1407 (2002).

  58. 58.

    Gasteiger, E. et al. ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31, 3784–3788 (2003).

Download references


The authors wish to thank R. Viner, A. Bailey and T. Zhang (Thermo Fisher Scientific) for assistance with the non-denaturing separations, M. Marty (University of Arizona) for assistance with UniDec, M. Bern and S.J. Skilton (Protein Metrics Inc.) for assistance with Intact Mass software, B. Rivera (Phenomenex) for helpful discussions and prototype Yarra columns, S. Thornton for assistance with figure production and S. Lai for careful proofing. The VP40 plasmid was a generous gift from the Ollmann Saphire research group (Scripps Research Institute). Work in the Wysocki laboratory was supported by National Institutes of Health Grant P41 GM128577, Ohio Eminent Scholar funds and a subaward from the University of Washington, Baker laboratory. The 15 T Bruker SolariXR FT-ICR instrument was supported by NIH Award Number Grant S10 OD018507. Design and preparation of proteins and protein complexes in the Baker laboratory was supported by the Howard Hughes Medical Institute and the generosity of Eric and Wendy Schmidt by recommendation of the Schmidt Futures program.

Author information

Z.L.V., F.B., M.J. and V.H.W. designed and technically developed the protocol. Z.C., S.E.B. and D.B. provided inspiration for the conception of the protocol and provided valuable ideas and feedback throughout the development and optimization of the protocol. Z.L.V., F.B., B.J.J., M.J. and A.S. performed experiments. Z.L.V. and F.B. wrote the manuscript with assistance from V.H.W. All authors discussed the results and commented on the manuscript.

Correspondence to Vicki H. Wysocki.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Chen, Z. et al. Nature 565, 106–111 (2019): https://doi.org/10.1038/s41586-018-0802-y

Pyles, H. et al. Nature 571, 251–256 (2019): https://doi.org/10.1038/s41586-019-1361-6

Schupfner, M. et al. Chembiochem 20, 2747–2751 (2019): https://doi.org/10.1002/cbic.201900323

Integrated supplementary information

Supplementary Fig. 1 Full mass spectra of the deconvoluted spectra shown in Fig. 2.

Cytochrome C (a), CRP (b) and NIST mAb (c) exchanged from PBS into 200 mM ammonium acetate using different columns (shown in legend). All spectra were acquired on an Exactive Plus EMR instrument. The y-dimension of each spectrum represents relative intensity.

Supplementary Fig. 2 Online separation of different proteins from non-volatile PBS components.

Mass spectra of proteins and protein complexes in PBS exchanged online into 200 mM ammonium acetate using a self-packed P6 column, acquired on an Exactive Plus EMR mass spectrometer. BSA (a), lysozyme (b), myoglobin monomer (M) and dimer (D) (c), cytochrome C (d), streptavidin tetramer (e), CRP pentamer (f), NIST mAb (g), concanavalin A monomer (M), dimer (D), and tetramer (Q) (h) and cholera toxin B pentamer (i). The most abundant charge state is indicated for each species. The y-dimension of each spectrum represents relative intensity.

Supplementary Fig. 3 Mass spectrum of GroEL tetradecamer acquired on a Q Exactive UHMR instrument after OBE using a self-packed P6 column.

5 µl of ~1 µM tetradecamer was injected onto the column. An in-source trapping desolvation voltage of −100 V was applied to improve desolvation and sensitivity. The spectrum was collected at a resolution setting of 6,000 (defined at m/z 400). The y-dimension represents relative intensity.

Supplementary Fig. 4 Comparison of online and offline buffer exchange.

A sample containing 4 µM BSA in 1× PBS was analyzed by OBE into 200 mM ammonium acetate with a self-packed P6 column (a and b), flow injection after one round of offline buffer exchange into 200 mM ammonium acetate using a P6 spin column (c and d) and e,f) flow injection after two rounds of offline buffer exchange into 200 mM ammonium acetate using a P6 spin column (e and f). a,c,e, A comparison of the BSA spectra shows that the overall signal intensity is lowest for the 1× offline buffer-exchanged sample and highest for the online buffer-exchanged sample. The differences in signal intensity are probably due to a combination of increased adducting on offline buffer-exchanged samples spreading the signal over a wider range of masses, as well as possible sample loss from offline sample handling. b,d,f, A zoom-in of the 13+ charge state shows the presence of multiple proteoforms (designated by mass difference) as well as non-covalent adduction (lower-intensity peaks). Differences in mass adduction are observed, with the 1× buffer-exchanged sample carrying the most adducts while the OBE sample carries the fewest adducts. The only difference between the analysis of the online and offline buffer-exchanged samples was the replacement of the OBE column with PEEK tubing of equal length for the offline buffer-exchanged samples. In all cases, the y-axis represents intensity normalized to the most abundant signal out of the three spectra.

Supplementary Fig. 5 Deconvoluted (zero-charge) mass spectra of the proteins and protein complexes shown in Supplementary Fig. 2.

BSA (a), lysozyme (b), myoglobin monomer (c), cytochrome C (d), streptavidin tetramer (e), CRP pentamer (f), NIST mAb (g), concanavalin A monomer (h) and cholera toxin B pentamer (i). Note that the x-axis scaling is different between each panel. Spectra were deconvoluted using Intact Mass software. The y-dimension represents relative intensity in each spectrum.

Supplementary Fig. 6 Full mass spectra of the deconvoluted spectra shown in Fig. 4.

Cytochrome C (a), CRP (b) and NIST mAb (c) exchanged from various non-volatile buffers into 200 mM ammonium acetate. All spectra were acquired on an Exactive Plus EMR instrument after removal of small-molecular-weight non-volatiles using a self-packed P6 column. The heterogeneity in c is due to the presence of variable glycoforms. The most abundant charge state is indicated for each set of spectra. The y-dimension of each spectrum represents relative intensity.

Supplementary Fig. 7 Data-dependent MS/MS of CRP pentamer in PBS online buffer-exchanged using a self-packed P6 column and acquired on a Q Exactive UHMR instrument.

A data-dependent HCD MS/MS method was used to collect dissociation data of CRP on the OBE time scale. a, Total ion chromatogram of the OBE data-dependent acquisition experiment. Each MS1 scan is designated by a purple line, and each MS2 scan is indicated by a purple line and labeled with the charge state of pentameric CRP that was isolated in the selection quadrupole and dissociated with 80 V of HCD. b, An example of a single-scan MS1 spectrum showing CRP pentamer (P). Each charge state highlighted in purple was isolated for MS/MS. c, An example of a single-scan MS2 spectrum where the 21+ charge state of CRP pentamer (P) was isolated and dissociated into monomer (M) and tetramer (Q) by HCD. The y-dimension of each spectrum represents relative intensity.

Supplementary Fig. 8 Switching valve and source setup for OBE.

a, Picture showing the connections to the switching valve for the OBE setup. b, Picture of the heated electrospray ionization (HESI) source on an Exactive Plus EMR instrument fitted with 10 ft. × 0.005 in. i.d. resistor tubing. The tubing acts as a resistor to reduce the ESI current and make it possible to spray from mobile phases with high ionic strength. Note that resistor tube length can be adjusted for the ionic strength of the mobile phase used.

Supplementary Fig. 9 Column packing setup for OBE.

Pictures of PEEK tubing inserted through the column packing station lid and into the vial containing the slurry (a), and packing station lid and PEEK tubing secured and bent into a scintillation vial to collect flow through (b) and a cross-section drawing of the column-packing station depicting the setup of the station as well as how the tubing is inserted into the slurry (c).

Supplementary information

Supplementary Information

Supplementary Figs. 1–9 and Supplementary Tables 1–4.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

VanAernum, Z.L., Busch, F., Jones, B.J. et al. Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometry. Nat Protoc (2020). https://doi.org/10.1038/s41596-019-0281-0

Download citation


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.