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.
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The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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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.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Integrated supplementary information
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.
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.
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.
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.
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.
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).
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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