WO2024035809A1

WO2024035809A1 - Soft landing molecular analysis systems and methods

Info

Publication number: WO2024035809A1
Application number: PCT/US2023/029891
Authority: WO
Inventors: Henry BENNER; Ben AGUILAR; Elizabeth Hecht MASSMAN; Alexis Lawrence ROHOU; Christopher Paul ARTHUR
Original assignee: Genentech, Inc.; Ion Dx, Inc.
Priority date: 2022-08-10
Filing date: 2023-08-09
Publication date: 2024-02-15

Abstract

This disclosure relates to novel systems and methods for soft landing of proteins and other macromolecules under atmospheric conditions at room temperature or under cryogenic conditions, and associated landing chamber apparatuses. For example, methods and systems herein may be useful in preparing samples for cryogenic electron microscopy and other imaging techniques.

Description

SOFT LANDING MOLECULAR ANALYSIS SYSTEMS AND METHODS

FIELD

BACKGROUND

Soft landing (SL) is a technique for deposition of a gas phase ion onto a surface, where the ion adheres without the formation of a covalent bond and without significant modification to its structure (14). Most commonly, a charged molecular ion is generated from a solid or solution material by an electrospray source, or alternatively by an ionization method that preserves noncovalent interactions. Electric and magnetic fields and gas flows may then be used to manipulate the trajectory of gas phase ions of an analyte of interest (15, 16). For example, ions may be filtered for those that fall within a particular m/z value, or mobility instruments may be used to select ions based on their collisional cross sectional (CCS) area. A landing surface is then used to collect the desired ions containing the analyte. In many cases, this process takes place under vacuum. Soft landing has been successful with small molecules, for instance, to prepare surface-modified chips and microarrays and the like. It has also been successful with certain polymers such that rationally designed monomers can be built onto chips to provide various coatings with applications in the optical coating and electronics fields (14, 17-24). It would be useful to employ soft landing techniques also with more complex molecules, such as proteins, polynucleotides, protein complexes, viruses, and the like, for example, for a variety of biological assays and imaging, such as cryo EM imaging. However, the delicate folding and structure of such molecules makes soft landing particularly challenging. (See, e.g., refs. 19, 22, 23, 25, 26, 27, 28.) For example, many efforts in protein soft landing imaging have stalled because of denaturation of proteins as a function of charge and landing time (18, 26, 28). Hence, there is a need in the field for improved systems for soft landing of proteins and other macromolecules and macromolecular complexes. SUMMARY

The present disclosure, inter alia, includes a new soft landing system compatible with macromolecular deposition that couples charge-reduced electrospray (58, 59) to an ion filtering mechanism such as differential mobility to atmospheric landing under variable temperature conditions. In some embodiments, the design incorporates a previously described electrospray device, such as a charge-reduction electrospray device (58, 59, 63), to a commercially available differential mobility analyzer (64-68), followed by two different custom landing chambers, one which operates under atmospheric conditions and one which operates under either cryogenic or atmospheric conditions. For example, landing under atmospheric conditions may be useful for a variety of work such as negative stain or protein surface deposition work. A cryogenic-compatible landing device may be useful for preparing samples for cryoelectron microscopy (cryo EM). The present disclosure also includes each of the landing chambers, and methods of using the systems herein.

Exemplary embodiments herein include the following, as well as others described elsewhere below in the sections that follow this Summary:

1. A system for gas phase separating charged protein ions on the basis of mobility, the system comprising: a. an electrospray source; b. a differential mobility analyzer (DMA); and c. an atmospheric landing chamber, wherein the landing chamber comprises a landing pedestal enclosed within the chamber, wherein the charged protein ions are capable of being deposited on the landing pedestal, wherein the landing chamber optionally further comprises a focusing lens, and wherein the electrospray source, DMA, and atmospheric landing chamber are connected to allow electrospray gas flow from the source, through the DMA, and to the landing chamber.

2. The system of embodiment 1, wherein the electrospray source is a charge reduced electrospray source.

3. The system of embodiment 1 or 2, wherein the pedestal holds a removable grid, such as a clipped or unclipped grid, upon which charged protein ions may be deposited.

4. The system of embodiment 3, wherein the grid comprises a carbon, graphene, graphene oxide-coated metallic film on a mesh surface such as a Cu, Ni, or Au mesh or other non-metallic support, such as silicon nitride. 5. The system of any one of embodiments 1-4, wherein the atmospheric landing chamber may be operated under ambient air, or under a gas such as nitrogen, carbon dioxide gas or a noble gas, wherein the air or gas has a relative humidity of less than 80%.

6. The system of any one of embodiments 1-5, wherein the landing chamber comprises a focusing lens.

7. The system of embodiment 6, wherein the focusing lens comprises an ion funnel with a preceding gas diffuser.

8. The system of any one of embodiments 1-7, wherein the system is capable of depositing protein ions of +1 to +5 charge on the landing pedestal.

9. The system of any one of embodiments 1-8, wherein the system is capable of depositing protein ions of from 3 to 100 nm diameter on the landing pedestal.

10. The system of any one of embodiments 1-9, wherein the system is capable of depositing protein ions in an atmospheric dry state, in droplet form, or as hydrated protein ions on the landing pedestal.

11. The system of any one of embodiments 1-10, wherein the DMA is a nano DMA (nDMA).

12. A system for gas phase separating charged macromolecular ions on the basis of mobility, the system comprising: a. an electrospray source; b. a differential mobility analyzer (DMA); and c. a cryogenic-compatible landing chamber, wherein the landing chamber comprises a landing pedestal enclosed within the chamber, wherein the charged macromolecular ions are capable of being deposited on the landing pedestal, wherein the landing chamber optionally further comprises a focusing lens, and wherein the electrospray source, DMA, and landing chamber are connected to allow electrospray gas flow from the source, through the DMA, and to the landing chamber.

13. The system of embodiment 12, wherein the electrospray source is a charge reduced electrospray source.

14. The system of embodiment 12 or 13, wherein the pedestal holds a removable grid, such as a clipped or unclipped grid, upon which charged macromolecular ions may be deposited. 15. The system of embodiment 14, wherein the grid comprises a carbon, graphene, graphene oxide-coated metallic film on a mesh surface such as a Cu, Ni, or Au mesh or other non-metallic support, such as silicon nitride.

16. The system of any one of embodiments 12-15, wherein the landing chamber comprises a focusing lens.

17. The system of embodiment 16, wherein the focusing lens comprises an ion funnel with a preceding gas diffuser.

18. The system of any one of embodiments 12-17, wherein the cryogenic-compatible landing chamber pedestal is in contact with a cooling unit in which a resistant heating element is submerged in a chilled fluid, such as liquid nitrogen or liquid ethane, stored in a dewar.

19. The system of any one of embodiments 12-18, wherein the cryogenic-compatible landing chamber may be operated in cryogenic conditions under air or under a gas whose dew point is less than a desired chilling temperature.

20. The system of any one of embodiments 12-19, wherein the system is capable of depositing macromolecular ions of +1 to +5 charge on the landing pedestal.

21. The system of any one of embodiments 12-20, wherein the DMA is a nano DMA (nDMA).

22. The system of embodiment 21, wherein the macromolecular ions are protein ions.

23. The system of any one of embodiments 12-22, wherein the system is capable of depositing macromolecular ions of 3-100 nm diameter on the landing pedestal.

24. The system of any one of embodiments 12-23, wherein the system is capable of depositing ions in a dry state, in droplet form, or as hydrated protein ions on the pedestal of the landing chamber.

25. The system of any one of embodiments 12-24, wherein the cryogenic-compatible chamber can be operated under either atmospheric or cryogenic conditions.

26. The system of any one of embodiments 12-25, wherein the pedestal comprises a central tube allowing flow of gas cooled by the cooling unit.

27. A cryogenic-compatible landing chamber for deposition of charged macromolecular ions generated by an electrospray source, wherein the landing chamber comprises a landing pedestal enclosed within the chamber, wherein the charged macromolecular ions are capable of being deposited on the landing pedestal, and further wherein the landing chamber is in contact with a cooling unit. 28. The cryogenic-compatible landing chamber of embodiment 27, wherein the cooling unit comprises a resistant heating element submerged in a chilled fluid such as liquid nitrogen or liquid ethane stored in a dewar.

29. The cryogenic-compatible landing chamber of embodiment 27 or 28, wherein the chamber further comprises an automatic temperature control element.

30. The cryogenic-compatible landing chamber of any one of embodiments 27-29, further comprising a means of connecting the landing chamber to a DMA or nDMA to allow flow of the charged macromolecular ions from the DMA or nDMA to the landing pedestal.

31. The cryogenic-compatible landing chamber of any one of embodiments 27-30, further comprising a focusing lens configured to allow electrospray gas flow from an electrospray source through the focusing lens and to the landing pedestal.

32. The cryogenic-compatible landing chamber of embodiment 31, wherein the focusing lens comprises an ion funnel with a preceding gas diffuser.

33. The cryogenic-compatible landing chamber of any one of embodiments 27-32, wherein the pedestal comprises a central tube allowing flow of gas cooled by the cooling unit.

34. The cryogenic-compatible landing chamber of any one of embodiments 27-33, wherein the chamber may be operated under either cryogenic or atmospheric conditions.

35. The cryogenic-compatible landing chamber of any one of embodiments 27-34, wherein the pedestal holds a removable grid, such as a clipped or unclipped grid, upon which charged macromolecular ions may be deposited.

36. The cryogenic-compatible landing chamber of embodiment 35, wherein the grid comprises a carbon, graphene, graphene oxide-coated metallic film on a mesh surface such as a Cu, Ni, or Au mesh or other non-metallic support, such as silicon nitride.

37. The system or cryogenic-compatible landing chamber of any one of embodiments 12- 36, wherein the chamber is fully enclosed during landing.

38. The system or cryogenic-compatible landing chamber of any one of embodiments 12- 36, wherein the bottom of the chamber is open to atmosphere during landing, and wherein the chamber is located above one or more cold traps, wherein the one or more cold traps are optionally made of metal, wherein the one or more cold traps are optionally in direct contact with liquid nitrogen, and wherein vaporized liquid nitrogen forms a nitrogen gas head space in the chamber during landing. A system for gas phase separating charged macromolecular ions on the basis of mobility, the system comprising: a. an electrospray source; b. a differential mobility analyzer (DMA); and c. a cryogenic-compatible landing apparatus, wherein the landing apparatus comprises a landing pedestal, wherein the charged macromolecular ions are capable of being deposited on the landing pedestal, wherein the landing pedestal is located above a first platform, the first platform being in contact with a liquid nitrogen source, and wherein the top of the landing pedestal is below or in line with a second platform, wherein the second platform and the first platform together are positioned so as to allow vaporized nitrogen gas from the liquid nitrogen source to displace atmospheric air at the top of the landing pedestal, wherein optionally the first and/or second platform is a cryo EM clipping station, and wherein the landing apparatus optionally further comprises a focusing lens, and wherein the electrospray source, DMA, and landing apparatus are connected to allow electrospray gas flow from the source, through the DMA, and to the landing apparatus. An atmospheric landing chamber for deposition of charged macromolecular ions generated by an electrospray source, wherein the landing chamber comprises a landing pedestal enclosed within the chamber, wherein the charged macromolecular ions are capable of being deposited on the landing pedestal, and further wherein the landing chamber may be operated under atmospheric conditions and may be operated under ambient air, or under a gas such as nitrogen, carbon dioxide gas or a noble gas, wherein the air or gas has a relative humidity of less than 80%. The atmospheric landing chamber of embodiment 40, further comprising a means of connecting the landing chamber to a DMA or nDMA to allow flow of the charged macromolecular ions from the DMA or nDMA to the landing pedestal. The atmospheric landing chamber of embodiment 40 or 41, further comprising a focusing lens configured to allow electrospray gas flow from an electrospray source through the focusing lens and to the landing pedestal. The atmospheric landing chamber of embodiment 42, wherein the focusing lens comprises an ion funnel with a preceding gas diffuser. The atmospheric landing chamber of any one of embodiments 40-43, wherein the pedestal holds a removable grid, such as a clipped or unclipped grid, upon which charged macromolecular ions may be deposited. The atmospheric landing chamber of embodiment 44, wherein the grid comprises a carbon, graphene, graphene oxide-coated metallic film on a mesh surface such as a Cu, Ni, or Au mesh or other non-metallic support, such as silicon nitride. A method of preparing and isolating a charged macromolecular ion comprising an analyte, the method comprising: a. ionizing a sample comprising a macromolecular analyte with an electrospray source to form charged macromolecular ions; b. separating the charged macromolecular ions on the basis of their collisional cross sectional area, for example, in a DMA or nDMA; and c. directing a charged macromolecular ion comprising the analyte to a landing pedestal located in a landing chamber or landing apparatus, wherein (i) the landing chamber or apparatus operates under atmospheric conditions or (ii) the landing chamber or apparatus operates under either atmospheric conditions or cryogenic conditions, and depositing the macromolecular ion comprising the analyte on the landing pedestal. The method of embodiment 46, wherein the macromolecular analyte is a polypeptide, nucleic acid, virus, or a complex comprising at least one polypeptide. The method of embodiment 46, wherein the macromolecular analyte is a polypeptide. The method of any one of embodiments 46-48, wherein the electrospray source is a charge reduced electrospray source. The method of any one of embodiments 46-49, wherein the ion is a +1 to +5 ion or a - 1 to -5 ion. The method of any one of embodiments 46-50, wherein the charged macromolecular ion is a +1 or -1 ion. The method of any one of embodiments 46-51, wherein the charged macromolecular ion is from 3 to 150 nm diameter, such as from 3 to 100 nm diameter, 25 to 150 nm diameter, 25 to 100 nm diameter, or 50 to 150 nm diameter. The method of any one of embodiments 46-52, wherein the charged macromolecular ion comprising the analyte is deposited on a removable grid on the landing pedestal, such as a clipped or unclipped grid, upon which charged macromolecular ions may be deposited. 54. The method of embodiment 53, wherein the grid comprises a carbon, graphene, graphene oxide-coated metallic film on a mesh surface such as a Cu, Ni, or Au mesh or other non-metallic support, such as silicon nitride.

55. The method of embodiment 53 or 54, wherein the method further comprises removing the grid, optionally wherein the grid is removed under a gas with a dew point less than the operating temperature of the landing chamber.

56. The method of any one of embodiments 46-55, wherein the landing chamber or apparatus comprises a focusing lens.

57. The method of embodiment 54, wherein the focusing lens comprises an ion funnel with a preceding gas diffuser.

58. The method of any one of embodiments 44-55, wherein the landing chamber or apparatus is an atmospheric landing chamber, such as described in any one of embodiments 1-11 or 40-45.

59. The method of embodiment 58, wherein the landing chamber is operated under ambient air, or under a gas such as nitrogen, carbon dioxide gas or a noble gas, wherein the air or gas has a relative humidity of less than 80%.

60. The method of any one of embodiments 46-57, wherein the landing chamber or apparatus is a cryogenic-compatible landing chamber or apparatus that may be operated under either atmospheric or cryogenic conditions, such as described in any one of embodiments 12-39.

61. The method of embodiment 60, wherein the landing pedestal comprises a central tube allowing flow of gas cooled by a cooling unit in contact with the landing chamber.

62. The method of embodiment 60 or 61, wherein the landing chamber is operated under cryogenic conditions.

63. The method of embodiment 60, wherein the landing chamber is operated under air or under a gas whose dew point is less than a desired chilling temperature, and/or wherein the landing chamber is fully enclosed during landing.

64. The method of any one of embodiments 46-63, wherein the charged macromolecular ion is deposited as a non-crystalline solid onto the landing pedestal.

65. The method of any one of embodiments 46-64, wherein the charged macromolecular ion is deposited on the landing pedestal as a charged droplet or a charged macromolecule comprising a hydration shell.

66. The method of any one of embodiments 46-65, further comprising removing the deposited charged macromolecular ion from the landing chamber or apparatus. 67. The method of embodiment 66, wherein removing the ion comprises removing a grid containing the deposited ion from the landing pedestal.

68. The method of embodiment 66 or 67, wherein the charged macromolecular ion is removed under a gas with a dew point less than the operating temperature of the landing chamber during the removal.

69. The method of any one of embodiments 66-68, further comprising submerging the removed deposited ion in liquid nitrogen.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims. All references cited herein are incorporated by reference into this disclosure in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

This disclosure also includes a number of figures, (Figs. 1-38), which help to illustrate particular embodiments or concepts described herein. These figures are summarized either in text provided with the figure or in the Examples section below. Abbreviations used within certain figures herein include the following: CAD = computer-aided design; SLD = soft landing device; nDMA = nano differential mobility analyzer; IM = ion mobility; EM = electron microscopy; MS = mass spectrometry; ESI = electrospray ionization; SEM = scanning electron microscopy.

Fig. 1 provides an overview of certain major components of a SLD described herein. In (1), a 210Po charge reduction ESI source (lonDX, Inc.) is coupled to (2) a nDMA (TSI, Inc.). Ions are directed into (3) a custom electrostatic focusing lens embedded within (4) a landing chamber. Different chambers were tested for atmospheric or atmospheric/cryogenic landing and various focusing lens were integrated during optimization of the SLD designs.

Figs. 2A-2B. To demonstrate the molecular weight range of proteins transmitted through the nDMA and detected on the landing pedestal during atmospheric conditions, proteins of MW 8-1.2 Mda were transmitted through the device. The starting concentration for all proteins shown is between 200-400 nM. However, for multimeric proteins the effective concentration of a certain oligomeric state would be some fraction of this starting concentration. Likewise, the transmission of certain proteins was significantly reduced due to nonspecific binding in the capillary infusion lines. Fig. 2A shows overlayed Gaussian fits from raw DMA voltage (kV) versus signal (V), while Fig. 2B shows a zoomed-in version of the data of Fig. 2A.

Figs. 3A-3B. The detection of (Fig. 3A) NSP4 or (Fig. 3B) ubiquitin proteins was achieved by coating the electrospray emitter with Sigmacote. Due to the small molecular weight of the proteins, a significant amount of electrospray oligomer artifact was additionally observed. The main fitted peaks of the monomer and dimers are shown in red, with the raw data shown in blue. Experiments of these and other small proteins infused with covalent coated capillary, bare fused silica, peek or buffer additives were generally not possible or extremely unreliable.

Fig 4 shows an exploded view of an atmospheric-only landing chamber.

Figs. 5A-5C show the pedestal top geometry for landing at atmospheric conditions. The top of the pedestals is made of brass, which is connected to an acetal plastic hollow ’A” tube. The total length of the hollow tube was adjusted for the atmospheric or the cryogenic landing chamber designs. Inside the tube is a wire to transmit current from the brass pedestal top, or from a grid seated on the brass pedestal top, to a detector. Fig. 5A shows the groove to fit autogrid style tweezers. Fig. 5B shows the groove to support tweezers for manipulating unclipped grids. Fig. 5C provides an illustration of a grid being inserted or removed from the pedestal top. The slot underneath the grid is to allow clearance for tweezers. The ridges ensure that the grid is securely seated and cannot fall off.

Figs. 6A-6C show three views of all components of the pedestal used for current detection at atmospheric conditions is shown. Figs. 6A and Fig. 6B show the same component in which the view is rotated, while Fig. 6C provides a sliced view. The acetal plastic hollow tube holds a wire connected to the brass top. A set-screw stopper can be adjusted to move the relative position of the pedestal to the focusing lens. The wire is connected to a BNC connector such that it may be subsequently connected to a detector.

Fig. 7 shows a sample SEM image taken at 10,000 x resolution of 20 nM particles landed. The particles per area calculated from this and other averaged images was 17.4 pm², translating to a total of 1.23 * 10⁸ on a 3mm grid. The total particles per grid based on sample current was calculated as 4.49* 10⁸, not accounting for the relative area of the grid to the pedestal. The calculation of particles based on the initial concentration was predicted to be 1.19 MO¹¹. Figs. 8A-8H show examples of the tested focusing lens that failed to work. Fig. 8A- 8F show simulations of the lenses tested with proteins transmitted at high or low voltage. Fig. 8A shows a lens structure, while Figs. 8B and 8C show simulations of testing with low voltage (Fig. 8B) and high voltage (Fig. 8C). Similarly, Fig. 8D shows a lens structure with Fig. 8E showing simulation of testing at low voltage and Fig. 8F showing simulation of testing at high voltage. At high voltage, the flow diameter is expanded, thus deflecting around the pedestal. The experimental results showing this trend are provided in Fig. 8G. A third focusing lens depicted in Fig. 8H, where a wire was threaded between the inlet and pedestal, was also not found to be effective, despite simulations suggesting otherwise.

Figs. 9A-9F. Fig. 9A shows a photo of the final ion focusing lens. Fig. 9B shows Simlon simulation of a protein transmitted through the lens from the chamber inlet to the pedestal at a DMA flow of 20 L/min and an aerosol in/out and ESI flow of 2 L/min. Fig. 9C shows a CAD drawing showing the funnel suspended in the cage in 3D. Fig. 9D shows a 2D view of the ion funnel seated in the cage. Fig. 9E shows a schematic of the atmospheric-only landing chamber, where the grey lines represent the ion funnel. Fig. 9F shows a CAD of the chamber inlet diffuser assembly, where a felt pad is inserted into a mesh backed ring.

Figs. 10A-10C. Fig. 10A provides a comparison of the change in the transmitted signal from 200 nM thyroglobulin dimer (660 kDa) was made based on current normalized at each focusing voltage setting, or by extracting the amplitude or area of the fitted peak. While the fits for amplitude and area from the same sweep scan showed good agreement, the area had significantly more error in the measurement. Thus, for testing subsequent proteins, either amplitude or normalized current was used. Thyroglobulin had an optimal transmission at 3000 or higher V. In Fig. 10B, avidin (67 kDa) was evaluated for peak transmission settings by the normalized pedestal method. Transmission was best from 0-1500 V, with a reduction observed at higher settings. In Fig. 10C, the 14-mer GroEl complex was evaluated for ideal transmission using the peak amplitude, where the optimal setting was approximately 4000 V. All plots in Figs. 10A-10C show standard deviation as the error bars.

Figs. 11 A-l ID. For the data shown in these figures, 200 nM aldolase was infused into the nDMA with a 50 pm ESI tip and analyzed at different nDMA settings through detection on the landing pedestal in the atmospheric system. The aldolase tetramer was detected at ~0.3 kV and the monomer at -0.17 kV. The ramp rate (RR) was calculated as the DMA voltage range divided by the ramp rate time (kV/s) and the flat time (FT) corresponded to the time the nDMA was held at baseline prior to initiating another scan. Fig. 11 A shows comparisons of the impact of FT on peak shape and position at two different RR. Regardless of RR, increasing the FT resulted in a rightward shift to the peak position, indicating a minimum FT was required for the nDMA to discharge. Fig. 1 IB shows that signal intensity was maximized at slow RR, and eventually converged around a true peak intensity. Fig. 11C shows a comparison of the CV in the amplitude and area of the peaks at different RR and focusing voltages. At a slow enough ramp rate, the CVs converged to values < 10 % and became similar to quantitation performed with a normalized pedestal reading at a constant transmission voltage (on/off analysis). Fig.

1 ID is a plot of the raw data for the experiments performed in Fig. 11C. The maximal focusing voltage between the pedestal constant transmission mode, on/off experiments began to converge with the peak fitting analysis at slow ramp speeds. Likewise, the area and amplitude of the peak began to converge at slower speeds.

Fig. 12 shows signal for 200 nM thyroglobulin in constant nDMA transmission mode that was taken at focusing voltage zero pre and post a focusing lens voltage sweep experiment. Even after 10 min, the signal measured post sweep was significantly higher. This indicated that some buildup of charge remained on the pedestal without being fully discharged to ground.

Figs. 13A-13G show data from landing of GroEl and tocilizumab. GroEl transmitted at nDMA sheath settings of (Fig. 13A) 10, (Fig. 13B) 40, and (Fig. 13C) 20 LPM, with standard deviation shown, with unbalanced flows. Where the aerosol in was held constant, and the aerosol out was changed. Tocilizumab was landed at balanced sheath flows but different rates of aerosol in/out and different sheath gas settings, as shown in Figs. 13D-13G.

Figs. 14A-14D show comparison of different focusing settings made by quantifying the fluorescence from 20 nM polystyrene particles landed. Shown are the images in greyscale. Fig. 14A shows the method for quantitation, where values are relative to a blank background. Fig. 14B shows fluorescence ranges across the line profile of the fluorescent grid. Fig. 14C shows an image of the 0V landed grid to the 5 kV focused landed grid. Fig. 14D shows an image of the 0V landed grid to the 2.5 kV focused landed grid.

Figs. 15A-15B show comparison of trastuzumab DAR 2 A488 landed for (Fig. 15 A) 60 minutes or (Fig. 15B) 180 minutes.

Figs. 16A-16B show a comparison of 20 nm fluorescent nanoparticles landed on a 150Cu ultra-thin carbon grid with (Fig. 16A) 0 V or (Fig. 16B) a rotating pedestal voltage between 0-3000 V applied to the grid for 70 min and 60 min, respectively. There was a approximate 2-fold improvement in the fluorescence when voltage was applied to the grid.

Figs. 17A-17B show trastuzumab A488 DAR2 selected by the nDMA and landed for 60 min on a (Fig. 17A) 0 V or (Fig. 17B) -1000 V grid. Between the grids, the punched-out grid normalized fluorescence increased ~50 % with voltage application. Trastuzumab Fab- A647 DAR 1 was selected from by the nDMA and landed for 60 min for (Fig. 17A) 0 V or (Fig. 17B) -1000 V grid. The fluorescence increased ~10 % with voltage application. The effects of voltage on grid had the largest effects on large molecules, perhaps because of their different trajectories into the landing chamber through the focusing lens, or because of their different electrostatic maps across the solvent accessible area. The fluorescence at observed at the two wavelengths confirmed that the nDMA selection in the gas phase was an effected way to purify the two proteins.

Figs. 18A-18B provide a demonstration of TEV enzymatic activity on a TEM grid. Fig. 18A shows the experimental workflow schematic, where 0.1 pM was landed, or 25 or 50 ng was spotted and stored at atmosphere for 60 min, moved to a well with substrate, and analyzed. Fig. 18B provides results showing that the time to substrate saturation is linear for spotted grids (N=3), with the 25 ng sample at saturation at 1325.6 s and the 50 ng sample at 2604.4 s. The landed TEV (N=2, 60 min landing, 0.1 pM solution) hit saturation at 2449.2 s, corresponding to 28 ng on grid.

Figs. 19A-19C show nDMA scans from 0-400 V of solutions of (Fig. 19A) 0.01% sucrose (Fig. 19B) 0.001% sucrose and (Fig. 19C) 0.0001% sucrose. The percentage of droplets of large size significantly increased with decreasing sucrose concentration.

Figs. 20A-20E show SEM scans of fluorescent nanoparticles on Cui 50 carbon UL grids at (Fig. 20A) 250x (Fig. 20B) lOOOOx and (Fig. 20C) 40000x magnification (with an inset zoom). Fig. 20D shows SEM or particles on a Quantifoil grids (Q2400CR1.3) at 20000x magnification after sputter coating samples prior to images. Fig. 20E is a histogram of the ImageJ analysis of the diameter of the nanoparticles landed in Fig. 20B, where the increased size likely relates to charging of the particles in the SEM, and the distribution likely reflects the true heterogeneity of the sample.

Figs. 21 A-E. Fig. 21 A shows “positive” stained particles of GroEl on a non-plasma- treated graphene oxide Quantifoil Rl.2/1.3, Gold, 400 mesh grid. Figs. 21B-21C show “negative” stained particles of GroEl on carbon coated Cu 150 mesh grid that was (in Fig. 2 IB) plasma treated or (in Fig. 21C) plasma treated and landed at a -500V applied gridvoltage. For the samples shown in Figs. 21B and C, 2D class averages were computed using cryoSparc for (Fig. 2 ID) plasma-treated grids or (Fig. 2 IE) plasma-treated + applied voltage grids, respectively.

Figs. 22A and 22B show photos of (Fig. 22A) the atmospheric-only and (Fig. 22B) the atmospheric and cryogenic system built for soft landing.

Fig. 23 provides an exploded view of the cryolanding chamber.

Figs. 24A-24B show a cryo-SLD cryolanding chamber and metal dish assembly, with landing chamber in (Fig. 24A) the closed position for landing, and in (Fig. 24B) the open position for grid manipulation.

Fig. 25 is a detail of the cryolanding chamber exhaust assembly, where the reversible flow for pedestal sheath gas enters through the tee.

Fig. 26 is a cryogenic pedestal view detail. Cold gas flows up through the center tube to cool the pedestal.

Figs. 27A and 27B provide (in Fig. 27A) a schematic and (in Fig. 27B) detail cutaway of the cooling system that uses a resistive heating element submerged in liquid nitrogen. Note that the tapered cap also serves as pressure relief in case of excessive pressure build-up.

Fig. 28 shows a detail of a banana dewar designed to sit around the landing chamber in the metal dish. Filling it with liquid nitrogen enables transfer of the grid from the pedestal to a grid holder.

Figs. 29A-B show details of the grid box holder for grid transfer (Fig. 29A), and of the grid holder sitting in the banana dewar (Fig. 29B).

Figs. 30A-30B show photos of the (Fig. 30A) hat to shield the cryogenic landing chamber during atmospheric mode for current measurement and (Fig. 3 OB) dome to trap liquid nitrogen headspace gas to provide a dry environment after lifting the landing chamber.

Fig. 31 shows a cryo-TEM image of lacey carbon grids landed for 30 min with GroEl at -20 C. The large dark spots represent condensate from grid transfer into the TEM. The smaller spots are GroEl. The concentration of GroEl on the C film, rather than within the holes, is an indicator that the graphene film has been ripped.

Figs. 32A-32B show cryo-EM images of GroEl (Fig. 32A) on a 2000mesh Cu grid with 1 layer graphene prepared by cold-landing for 20 min at -20° C or (Fig. 32B) on a UltrAuFoil R1.2/1,3 holey gold by traditional spotting and vitrification procedures. As shown in the insets in Fig. 32A, good contrast was observed, and the top-down ring/side barrel views were observed; also evident were locations where GroEl was landed on top of each other, which was not observed in Fig. 32B.

Fig. 33. Protein-less ice balls were analyzed to determine the structure of ice landed on-grid. The lack of diffraction spots in the averaged amplitude FFT spectrum suggested that a non-crystalline ice was formed.

Figs. 34A-34B show landing of ArgC under cryogenic conditions (see Example 3) at different concentrations to produce a standard curve); three different replicates are shown. An analysis of the ArgC concentration determined from lysozyme intensity is shown in Fig. 34B. The table in Fig. 34B shows the three repetitions of the landing from Fig. 34A with their respective agreement with the predicted signal.

Figs. 35A-35F show a series of landings of control (gold) and apoferritin (ApoF) protein performed in the system of Example 3 at -40 °C. Figs. 35A-C show the grid alone without buffer (Fig. 35 A), a control experiment with buffer only (Fig. 35B), and of buffer without DMA (Fig. 35C). Protein was landed with and without DMA transmission as shown in Figs. 35D and 35E, respectively. Fig. 35F shows landing of gold (Au) particles on the grid as a further positive control of the system. The Au particles were observed at -57C and were clearly identified on the basis of the high amount of scattering and their relative size (10 nm).

Figs. 36A-C show photographs of a second cryogenic soft landing system (Figs. 36A- B showing a grid positioned in the system and the whole system, respectively), and an example of GroEL landed at liquid nitrogen temperature at 73,000 magnification (Fig. 36C). As shown in Fig. 36C, GroEL landed at liquid nitrogen showed distinct shapes from warmer temperatures at 73,000 magnification.

Figs. 37A-37C. Figs. 37A-37B show a third cryogenic instrument design in closed (Fig. 37A) and open (Fig. 37B) positions, in which a landing pedestal is not contained in a chamber, but that is instead positioned between two platforms, one platform located below the landing grid but above a liquid nitrogen cooling element such as a tank or dewar, and a second platform surrounding and in line with the landing grid, wherein liquid nitrogen gas forms a headspace between the two platforms sufficient to expel the surrounding atmospheric gas so that condensation on the pedestal and grid is reduced. Fig. 37C shows landing of GroEL particles in this system. Limited GroEl particles were observed due to low current, but importantly, the amount of condensate was minimal demonstrating the potential of this approach.

Figs. 38A and 38B show data obtained from the second cryogenic landing system of Figs. 36A-B. Fig. 38A shows condensate levels at lOL/minute while Fig. 38B shows levels at 20L/minute. Fig. 38A compared to Fig. 38B shows that at 10 L/min flow, there are significantly larger ice crystals on the grid than at 20 L/min flow, when excluding the largest crystals which are a consequence of grid transfer steps.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Definitions

Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

In this application, the use of “or” means “and/or” unless stated otherwise. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim in the alternative only.

Terms such as “element” or “component” or “unit” may be used interchangeably to refer to parts of an apparatus or system.

As used herein, the transition term “consisting essentially of,” when referring to steps of a claimed process or method signifies that the process or method comprises no additional steps beyond those specified that would materially affect the basic and novel characteristics of the process or method. As used herein, the transition term “consisting essentially of,” when referring to a composition or product signifies that it comprises no additional components beyond those specified that would materially affect its basic and novel characteristics.

As used herein, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise.

As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

The term “soft landing” herein refers to the deposition of a gas phase ion onto a surface, where the ion adheres to the surface noncovalently and without significant structural modification, such as significant protein unfolding or mis-folding in the case of a protein ion.

An “analyte” herein refers to a molecule or substance to be analyzed, such as a small molecule, peptide, polypeptide, macromolecule, or a complex of any of the above.

The term “small molecule” as used herein refers to an organic molecule having a molecular weight of 50 Daltons to 2500 Daltons.

The term “peptide” as used herein refers to a chain of fifty amino acids or less linked by peptide bonds, including amino acid chains of 2 to 50, 2 to 15, 2 to 10, 2 to 8, or 6 to 14 amino acids.

A “polypeptide” or “protein” interchangeably herein typically refers to a chain of amino acids longer than that of a peptide.

A “macromolecule” as used herein refers to a molecule that is larger than a “small molecule” and includes, for example, biological polymers such as proteins, nucleic acids, and virus particles, and complexes comprising proteins and nucleic acids, as well as non- biological, synthetic polymeric molecules.

A “charged ion” herein, such as a “charged protein ion” or a “charged macromolecular ion” refers to a molecular ion that carries a charge, wherein the charge may be located on the molecule itself, or in or on the surface of a surrounding hydration shell. The charged ion may be in the form of a charged droplet, a charged molecule comprising a hydration shell (i.e., a hydrated ion), or a dry (i.e., anhydrous) ion. In a “charged droplet,” for example, the molecule may be surrounded by a aqueous shell, wherein either or both of the molecule and the surrounding aqueous shell may comprise the charge. Similarly, in the case of a “hydrated ion,” where the charged ion comprises a hydration shell, and thus is not anhydrous, either the molecule and/or the molecules of the aqueous shell may comprise the charge. Where the charged ion is in a “dry state” or “anhydrous state,” it does not contain a significant amount of surrounding water molecules.

“Cryogenic” herein is an adjective referring to the use of cold temperatures, such as due to cooling of equipment or samples with liquid nitrogen or in an appropriate freezing device that produces temperatures below 0 °C.

“Atmospheric” herein is an adjective referring to ambient air, i.e., the normal surrounding atmosphere, or its use. “Atmospheric conditions” herein refer to conditions under ambient pressure, or in which there is no device applied to deliberately change the pressure. “Non-atmospheric” conditions herein means conditions other than those at ambient pressure.

An “electrospray source” or “electrospray ionization source” or “ESI source” refers to an apparatus that applies electricity to a solution for the purposes of producing an aerosol comprising of charged droplets, usually containing an analyte, that may subsequently yield charged ions.

The term “charge reduction” as used herein refers to a device or method that can reduce the overall charge of a charged ion from an electrospray source.

A “charge reduced electrospray source” herein refers to an electrospray source that incorporates a charge reduction method or device.

A “differential mobility analyzer” or “DMA” refers to a device that separates charged ions on the basis of their collisional cross sectional area. In a DMA, ions travel within a sheath gas and take a trajectory directing them to an outlet when attracted to a center rod of opposite polarity at a given voltage. A “nano differential mobility analyzer” or “nDMA” is a type of DMA that uses a shortened center rod, and may be used, for example, to separate analytes with improved resolution.

A “chamber” in some embodiments herein refers to a space that is “enclosed.” A “chamber” herein can have any appropriate shape. As used herein, the term “enclosed,” such as referring to a landing pedestal in a chamber, means that the landing pedestal is configured to be inside the chamber space. A “fully enclosed” chamber herein refers to a chamber that is enclosed on all sides. In some other cases, the chamber may be enclosed on all but one side or face, or allowing for openings on one side or face, such as at the bottom face. In some cases, a chamber may be enclosed on either all but one side or on all sides, except for appropriate ports for tubing or connections that allow gas or charged ions to flow into and/or out of the chamber. Certain chambers herein are capable of operation under non-atmospheric conditions by being protected from entry of ambient air, either by being fully enclosed as noted above or by other means. In some embodiments, fully enclosing a chamber may help in keeping out ambient air so that the system is closed to atmosphere. In some embodiments, for example, a fully enclosed chamber may in some embodiments have an air-tight seal, such as via an O-ring. In other embodiments, a fully enclosed chamber does not have an airtight seal. For example, in certain cases an airtight seal might not be necessary to sufficiently shield a landing pedestal from ambient air. In other systems herein, a chamber may not be fully enclosed. For instance, it may be be enclosed, for example, on three sides and thus have one open side or face or an open portion on one side or face, such as at the bottom, or may otherwise not be fully enclosed, but, for example, nonetheless be capable of maintaining appropriate conditions on the landing pedestal and grid during operation, such as avoiding or reducing entry of ambient air.

A “pedestal” refers to a platform, such as a flat-shaped or grooved platform, which is placed on a rod or other support to hold it in place. A “landing pedestal” is a pedestal that can be used for deposition of charged ions in a system herein.

A “focusing lens” herein refers to a component of a system herein that may be useful in directing a particular charged ion toward a landing pedestal, and therefore increasing the ion flux in a certain area.

As used herein, the terms “deposition” or “depositing” or “landing” are used interchangeably when referring to placement of a charged ion on a landing pedestal.

A “grid” herein is a removable disk on the landing pedestal that is used for deposition of charged ions. The grid may be removed, for example, to be placed in a = microscope, such as an electron microscope, for imaging of the analytes. Types of “grid” include an “Autogrid®”, which is also known as a “clipped grid”, and grids made of any type of materials and with any type of film. An “Autogrid®” or “clipped grid” refers to a type of grid placed on the landing pedestal that has already been secured in place into a metal ring with a clip, such as a C-clip to enable automated handling by downstream instruments, such as a TEM. Other grids that do not have such features are “unclipped” grids.

A “cold trap” component of a system herein refers to a component chilled relative to the temperature of the landing chamber gas temperature, such as a component placed into contact with a cooling mechanism, such as a tank or dewar containing liquid nitrogen or liquid ethane or similar substance, and that is capable of collecting condensate from the surrounding air or gas. In some cases, a cold trap is included to help reduce condensation formation on the landing pedestal or grid during landing under cryogenic conditions when a landing chamber is used that is not fully enclosed during landing. In some cases, a cold trap comprises a metal component such as a metal grid holder.

A “non-crystalline solid” as used herein refers to a solid form in which molecules are at least partially randomly oriented, and thus, at least partially amorphous in structure. In some embodiments, cryoelectron microscopy requires a sample to be at least partially amorphous to avoid scattering of the electron microscopy beam and loss of signal.

Other terms may be defined in the sections that follow. Exemplary Systems and Components

Electrospray and Charge Reduction

Systems herein utilize an electrospray (or electrospray ionization, ESI) source to provide gas phase ions, such as charged macromolecular ions, such as charged protein ions. Examples of electrospray sources include a variety of commercially available electrospray devices, such as those adapted from mass spectrometry uses. In some cases, the electrospray source is a charge reduced electrospray source, while in other cases the overall system and methods are run so as to reduce the charge of the macromolecular ions generated in the electrospray ionization.

An exemplary charge reduced electrospray source, for example, is described in US Patent No. 6,649,907 Bl. In some embodiments, a sealed 210Po source, such as a 210Po static eliminator (e.g., from NRD, Long Island, NY or Amstat Industries, Inc., Lake Zurich, IL), can be used for charge reduction. 210Po decays into alpha particles, which yield a variety of charge reducing species through interactions with air and carbon dioxide. In some charge reduction systems, positively and negatively charged ions containing analyte enter a charge reduction chamber comprising 210Po decay particles, and with a sufficient electric field strength, may be reduced to as low as +1 and -1 charge and neutral charge species. (See also M. Scalf et al., Anal. Chem. 72(1): 52-60 (2000).) In other embodiments, a non-radioactive charge reducing device or an ESI source using an alternative alpha-particle emitting radioactive material may also be employed to reduce the charge of the charged ions.

In other embodiments, in lieu of a charge reduction electrospray source, one or more solvents or chemical additives known to reduce the positive or negative charge of ions generated from the electrospray source may be added. Examples include water, methanol, acetonitrile, and triethylamine.

In some embodiments, the charged ions to be deposited in the landing chamber have a charge of +1 to +10, such as +1 to +5. In some cases, the charged ions to be deposited in the landing chamber have a charge of +1 to +3. In some cases, the charged ions to be deposited in the landing chamber have a charge of +1 to +2. In some cases, the charged ions to be deposited in the landing chamber have a charge of +1. In some cases, the charged ions to be deposited in the landing chamber have a charge of -1 to -10, such as -1 to -5. In some cases, the charged ions to be deposited in the landing chamber have a charge of -1 to -3. In some cases, the charged ions to be deposited in the landing chamber have a charge of -1 to -2. In some cases, the charged ions to be deposited in the landing chamber have a charge of -1.

A low charge may be beneficial, for example, in ensuring that the charged ion in droplet form may be subjected to evaporation without causing a charged protein to be ejected and denatured at the air-water interface, for example. In addition, charge reduction occurs within microseconds, thereby minimizing the coulombic repulsion between droplets, resulting in fewer ion losses. Likewise, the low charge ensures the droplet may proceed through evaporation processes over tens to hundreds of milliseconds without causing a charged macromolecular ion to be ejected, which could result in denaturation at the air- water interface (71). Finally, given a homogenous charge state population, the collisional cross section of a droplet containing a protein is directly proportional to the analyte size (Stoke’s law), rather than the ratio of that size versus total charge (72).

Differential Mobility Analysis

Once the charged ions are generated, electrostatic fields may be applied to filter macromolecular charged ions generated by the electrospray source for ions that fall within a particular m/z value. In some embodiments, a differential mobility analyzer (DMA) may be applied to select ions based on their collisional cross sectional (CCS) area (64-66, 74). In a DMA, ions travel through a sheath gas towards a central rod of opposite polarity, resulting in specific trajectories related to an ion’s collisional cross sectional area. At a uniform charge, the CCS area is directly proportional to the size of the macromolecule to be analyzed. Furthermore, a particle diameter may be directly calculated from an nDMA voltage, where protein calibrants are not required due to the known first-order relationship (75). The extension of the DMA to separate very large particles (AAVs, types of aerosols) in the hundreds of nanometers of range has been previously demonstrated (76). The term “particles” herein, in the context of a charged macromolecular ion particle, means droplets or hydrated macromolecular ions or other discreet ion particles found in a vapor stream from electrospray, for example, and separated in a DMA.

In some cases, a DMA may be a commercially available DMA (e.g., TSI, Inc., Minnesota, USA). In some cases, a DMA may be a nano DMA (nDMA), which includes a shorter rod than a traditional DMA, and thus, may be useful for separating smaller macromolecular ions. In some embodiments, the DMA or nDMA is capable of separating particles of from 10 nm to 1000 nm in diameter, such as from 10 nm to 800 nm, from 10 nm to 500 nm, from 5 nm to 500 nm, from 3 nm to 300 nm, from 10 nm to 200 nm, from 5 nm to 200 nm, from 3 nm to 200 nm, from 10 nm to 100 nm, from 5 nm to 100 nm, or from 3 nm to 100 nm in diameter. Thus, in some embodiments, the system herein is capable of depositing particles of from 10 nm to 1000 nm in diameter, such as from 10 nm to 800 nm, from 10 nm to 500 nm, from 5 nm to 500 nm, from 3 nm to 300 nm, from 10 nm to 200 nm, from 5 nm to 200 nm, from 3 nm to 200 nm, from 10 nm to 100 nm, from 5 nm to 100 nm, or from 3 nm to 100 nm in diameter on the landing pedestal of the atmospheric or cryogenic-compatible landing chamber.

A DMA or nDMA may be connected to the electrospray source using conductive tubing. In general, the DMA or nDMA and the electrospray source should be in close proximity to each other, as longer tubing length could result in a longer delay time between ion deflection and current measurement.

Atmospheric Landing Chamber

In some embodiments, macromolecular ions, such as protein ions flow from the electrospray source through a DMA or nDMA or other device to filter the ions and isolate the ion containing the desired analyte, and to a landing pedestal on which the desired ions are landed. In some embodiments, the landing pedestal is enclosed within an atmospheric landing chamber. The atmospheric landing chamber allows the landing process to be run under atmospheric conditions, i.e., under atmospheric pressure conditions. In some cases, the system may be run under ambient air, while in other cases it may be run under a gas such as nitrogen, carbon dioxide, or a noble gas. In some cases, the relative humidity of the air or gas is less than 80%, for example, to avoid moisture retention by the charged ions. In some cases, the relative humidity is less than 75%, less than 70%, or less than 60%, or less than 50%. Thus, in contrast to certain prior systems, landing is performed under atmosphere rather than under a vacuum. Thus, in some embodiments, the system does not operate under vacuum and/or the landing chamber does not operate under vacuum, and thus, does not comprise a vacuum chamber. In some cases, the system is configured to land ions in an atmospheric dry state, in droplet form, or as hydrated protein ions on the landing pedestal. In some cases, the gas in the landing chamber is sufficiently dry as to remove water molecules from the charged macromolecular ions as they travel to the landing pedestal or once deposited on the landing pedestal.

Furthermore, in some embodiments, systems herein are placed in a room with controlled atmospheric conditions, such as controlled temperature and relative humidity so that the relative humidity of the ambient air that may be used in the system retains the appropriate relative humidity level for optimal operation of the system.

In some embodiments, the landing pedestal holds a removable grid onto which the charged ions are deposited. The grid may be a clipped grid, such as an Autogrid®, while in other cases it may be an unclipped grid. The grid may be made from any of a variety of materials suitable for holding macromolecular ions in dry or hydrated form. In some cases, the grid comprises a carbon, graphene, graphene oxide-coated metallic film on a mesh surface such as copper, nickel, or gold or on a non-metallic support material such as silicon nitride.

In some embodiments, the atmospheric landing chamber also comprises a focusing lens to further direct the charged ions to the landing pedestal. In some cases, the focusing lens comprises an ion funnel with a preceding gas diffuser. An example of such a focusing lens is provided in Fig. 9A-9F. In some cases, the focusing lens does not comprise a wire mesh ring, suspended ring, or grid wire (see Fig. 8). In some cases, the landing chamber also includes a temperature control device (e.g., from Omega Engineering, Norwalk, CT).

Cryogenic-Compatible Landing Chamber

In other embodiments, the landing chamber comprising the landing pedestal onto which the charged macromolecular ions are to be deposited is cryogenic-compatible. In some embodiments, the landing chamber is in contact with a cooling unit that is capable of cooling the landing pedestal to temperatures below zero degrees Celsius, such as, in some cases, -5 °C or lower, -10 °C or lower, -15 °C or lower, -20 °C or lower, -25 °C or lower, or -30 °C or lower. In some cases, the cooling unit comprises a heat resistant element submerged in a chilled fluid such as liquid nitrogen or liquid ethane or the like. In some cases, the chilled fluid is stored in a dewar. In some cases, the pedestal comprises a central tube allowing flow of gas cooled by the cooling unit. Other means of cooling the chamber may also be used in other embodiments. Examples include a Peltier stage cooling device and a cold finger condenser. For example, a Peltier device could be employed to direct cold gas into or around the pedestal. In some cases, the landing pedestal may be mounted on a Peltier stage.

The cryogenic-compatible landing chamber allows the landing process to be run under either cryogenic or atmospheric conditions, i.e., under atmospheric pressure conditions. In some cases, the system may be run under ambient air, while in other cases it may be run under a gas such as nitrogen, carbon dioxide, or a noble gas. In some cases, the cryogenic landing chamber may be operated in cryogenic conditions under air or under a gas whose dew point is less than the desired chilling temperature. In some cases, the system is configured to land ions in an atmospheric dry state, in droplet form, or as hydrated protein ions on the landing pedestal. In some cases, the gas in the landing chamber is sufficiently dry as to remove water molecules from the charged macromolecular ions as they travel to the landing pedestal or once deposited on the landing pedestal.

In some embodiments, the landing chamber is operated in cryogenic conditions under air or under a gas whose dew point is less than a desired chilling temperature. Furthermore, in some embodiments, systems herein are placed in a room with controlled atmospheric conditions, such as controlled temperature and relative humidity so that the relative humidity of the ambient air that may be used in the system retains the appropriate relative humidity level for optimal operation of the system. In some cases, the landing chamber also includes a temperature control device (e.g., from Omega Engineering, Norwalk, CT).

In some embodiments, the cryogenic-compatible landing chamber also comprises a focusing lens to further direct the charged ions to the landing pedestal. In some cases, the focusing lens comprises an ion funnel with a preceding gas diffuser. In some cases, the landing chamber also includes a temperature control device.

In some embodiments, when preparing cryogenic samples, such as for cryoEM analysis, it may be necessary to reduce the amount of condensate in or around the landed ions on the grid as much as possible in the system and/or when transferring the grids to a cryoEM device for analysis. In some embodiments, one may first dry the landing pedestal with dried air, such as from a hair dryer or similar device, in order to reduce the relative humidity, prior to chilling the landing chamber and conducting a landing experiment in the system. After landing, in some embodiments, a grid containing the landed ions may be transferred from the landing pedestal to a chilled holder, which may be of any appropriate size or shape to hold the grid, under a gas with a dew point less than the chilled temperature, such as nitrogen gas, carbon dioxide, or a noble gas. See, e.g., Figs. 27-29B for an example of one means of transferring a chilled landing grid. In some cases, the grid with the landed ions may be stored in chilled conditions for later manipulation, such as under liquid nitrogen.

The present application describes three systems for landing under cryogenic conditions. In one system, described for example in Figs. 23-26, a cryogenic-compatible landing chamber is fully enclosed during landing in order to reduce entry of surrounding atmosphere, which could cause condensation inside the chamber and onto the grid, for example, if it were allowed to enter to a significant degree. In this embodiment, the chamber is fully closed during the landing and attaches to the rest of the system via a seal that can be released after landing in order to manipulate the landed molecule on the grid. For example, in this embodiment, the seal is an O-ring seal that can be used to avoid entry of outside air into the chamber so that the gas conditions inside the chamber can be controlled to avoid condensation on the grid. For instance, in this example, the chamber is sealed shut during landing aside from the ports for entry of ions and sheath gas and the like. Figs. 23-26, for example, show an example of such a cryogenic-compatible chamber, which is designed to be closed on the top and sides, and to form an O-ring seal at the bottom. For example, Fig. 24A shows such a chamber in closed position for landing, where the chamber is fully enclosed during landing and the bottom of the chamber is in connection with a cooling unit that may comprise a resistant heating element submerged in a chilled fluid such as liquid nitrogen or liquid ethane stored in a dewar. Fig. 24B shows such a chamber in an open position, in which the bottom is unsealed and the chamber removed, so that one can manipulate the grid before or after landing. This system can be operated at temperatures down to -60 °C, or, in some cases, at a temperature of -5 °C or lower, -10 °C or lower, -15 °C or lower, -20 °C or lower, -25 °C or lower, or -30 °C or lower, or -40 °C or lower, or at temperature ranges between -60 °C and, for example, -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, or -40 °C, or at temperature ranges between -40 °C and, for example, -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, or -30 °C.

As an alternative to a fully enclosed chamber, a cryogenic-compatible chamber may be, in another design, partially open to the outside atmosphere during the landing process, such as at the bottom of the chamber, so long as condensation on or near the grid of the landing pedestal is sufficiently reduced. An example is shown in Figs. 36A-B. For example, in this design, in contrast to that of Figs. 23-26, the bottom of the chamber is not sealed, and therefore is open to atmosphere during landing, and the bottom of the chamber rests above at least one cold trap. In some cases, one or more further cold traps are added below the bottom of the chamber. The cold trap(s) may be made of metal, which preferentially attracts condensate from the surrounding air onto the cold trap(s). Below the cold trap(s) rests a tank or dewar containing liquid nitrogen. In some cases, the liquid nitrogen may be filled part-way up the cold traps so that the cold trap(s) are in contact with the liquid nitrogen but so that the cold trap(s) are only partly submerged in the liquid nitrogen and thus are also exposed to the air. In other cases, the cold trap(s) are not in direct contact with the liquid nitrogen.

Surprisingly, such a system unsealed at the bottom still was sufficient to control condensation on the landing pedestal and grid. This may be due in part to the movement of nitrogen gas from the liquid nitrogen located below the chamber up into the chamber itself due to its open bottom, sufficiently to retain a nitrogen headspace in the chamber, in part to condensate preferentially forming on the exposed surface of the cold trap(s), and in part due to the relatively dry gas flowing through the chamber, with a dew point much lower than the ambient air. For example, the nitrogen headspace may have lower humidity than the surrounding atmosphere, and may effectively displace the atmosphere from the chamber and also prevent outside air from entering the chamber to a significant degree during landing. For instance, depending on the gas chosen for the sheath gas, the dew point of the gas in the head space may be between -52 °C and -62 °C, and, while the dew point of the ambient air is expected to vary from day to day, it is often about 2 °C, which is considerably higher than that of the gas in the chamber head space. This system, compared with the fully closed chamber system described above, also can be operated at temperatures at or below -60 °C and in some cases down to liquid nitrogen temperature. As liquid nitrogen temperature is -196 °C, and as the system sits above the liquid nitrogen source, allowing for some heat to be lost, it is estimated that operating at liquid nitrogen temperature indicates an operational temperature of the landing pedestal would be higher than -196 °C, such as from -130 °C to -170 °C, or - 140 °C to -160 °C. Thus, this system may be more compatible with lower temperatures than the fully closed chamber system above. In some cases, this system can be operated at a temperature of -5 °C or lower, -10 °C or lower, -15 °C or lower, - 20 °C or lower, -25 °C or lower, or -30 °C or lower, or -40 °C or lower, or at a temperature of -60 °C or lower, such as -60 °C down to an operational liquid nitrogen temperature, such as -60 °C to -170 °C, -60 °C to -160 °C, -60 °C to -140 °C, -60 °C to -140 °C, or -60 °C to -120 °C. A third design (Fig. 37A-B) involves a landing pedestal that is not contained in a chamber, but that is instead positioned between two platforms, one platform located below the landing grid but above a liquid nitrogen cooling element such as a tank or dewar, and a second platform surrounding and in line with the landing grid, wherein liquid nitrogen gas forms a headspace between the two platforms sufficient to expel the surrounding atmospheric gas so that condensation on the pedestal and grid is reduced. To manipulate the grid after landing, the top platform can be removed. In one example of such a design, the two platforms are cryo-EM clipping stations that are designed to fit around the landing platform. Using clipping stations in this way may allow for simpler manipulation of molecules landed on the grid, as they can be moved to a location on the bottom clipping station platform for storage or analysis. An example of clipping stations that may be configured as platforms in this design are MiTeGen® clipping stations (Ithaca, NY, USA). A photograph of such a device in closed and open configuration is shown in Figs. 37A-B.

Additional Components

In some embodiments, the systems herein also include a computer processor, which may be used to store software for control one or more of the electrospray source, DMA, and/or landing chamber. In some embodiments, the processor is connected to the internet to allow monitoring online. The system may also comprise appropriate software for controlling one or more of the electrospray source, DMA, and/or landing chamber. Thus, for example, in some embodiments, a computer processor and appropriate software may be used to direct the electrospray of a starting sample, to adjust the electrospray voltage, to control gas flow and/or voltage in the DMA, and to control the temperature and/or atmospheric conditions in the landing chamber, or any combination of these features.

Some embodiments herein also include a means for storing a sample prior to electrospray ionization of the sample, such as a sample compartment or chamber in contact with the electrospray source, optionally connected via a means for introducing the sample into the electrospray source device. In some embodiments, a means for introducing the sample to the electrospray source is also included, such as a syringe or other pressure- controlled loading mechanism.

Methods of Performing Soft Landing

The present disclosure also includes methods of isolating a charged macromolecular ion using a system or landing chamber apparatus herein. For example, in some methods herein, a charged macromolecular ion comprising an analyte desired for later analysis is isolated by a method comprising (a) ionizing a sample comprising a macromolecular analyte with an electrospray source to form charged macromolecular ions; filtering the generated charged macromolecular ions or separating them on the basis of their collisional cross sectional area to isolate the ions comprising the analyte, for example, in a DMA or nDMA; and then directing the charged macromolecular ion comprising the analyte to a landing pedestal located in an enclosed landing chamber, and depositing the ion on the pedestal. In some cases, the landing chamber operates under atmospheric conditions, such as that described above. In other cases, the landing chamber operates under either atmospheric conditions or cryogenic conditions, or is otherwise cryogenic-compatible. In some methods herein, the landing chamber comprises a focusing lens, such as with an ion funnel with a preceding gas diffuser.

The macromolecular analyte may be any of a polypeptide, nucleic acid, virus, or a complex comprising at least one polypeptide, such as a protein-protein or protein-nucleic acid complex. In some cases, the macromolecular analyte is a polypeptide.

The electrospray source for performing the methods may be as described above, such as a charge reduced electrospray source. Alternatively charge reduction may be performed by appropriate solvent conditions.

For soft landing, it may be desirable for the ion containing the analyte to have as low a charge as possible. In some cases, the charged ions to be deposited in the landing chamber have a charge of +1 to +10 or of +1 to +5. In some cases, the charged ions to be deposited in the landing chamber have a charge of +1 to +3. In some cases, the charged ions to be deposited in the landing chamber have a charge of +1 to +2. In some cases, the charged ions to be deposited in the landing chamber have a charge of +1. In some cases, the charged ions to be deposited in the landing chamber have a charge of -1 to -10, such as -1 to -5. In some cases, the charged ions to be deposited in the landing chamber have a charge of -1 to -3. In some cases, the charged ions to be deposited in the landing chamber have a charge of -1 to -2. In some cases, the charged ions to be deposited in the landing chamber have a charge of -1. In some cases, the charged macromolecular ion is a +1 or -1 ion.

In some cases, the charged macromolecular ion particles have a diameter of from 10 nm to 1000 nm, such as from 10 nm to 800 nm, from 10 nm to 500 nm, from 5 nm to 500 nm, from 3 nm to 300 nm, from 10 nm to 200 nm, from 5 nm to 200 nm, from 3 nm to 200 nm, from 10 nm to 100 nm, from 5 nm to 100 nm, or from 3 nm to 100 nm. Thus, in some embodiments, the charged macromolecular ions are from 10 nm to 1000 nm in diameter, such as from 10 nm to 800 nm, from 10 nm to 500 nm, from 5 nm to 500 nm, from 3 nm to 300 nm, from 10 nm to 200 nm, from 5 nm to 200 nm, from 3 nm to 200 nm, from 10 nm to 100 nm, from 5 nm to 100 nm, or from 3 nm to 100 nm in diameter.

In some cases, the charged macromolecular ion comprising the analyte is deposited on a removable grid on the landing pedestal, such as a clipped or unclipped grid, upon which charged macromolecular ions may be deposited. In some cases, the grid comprises a carbon, graphene, graphene oxide-coated metallic film on a mesh surface such as a Cu, Ni, or Au mesh or other non-metallic support, such as silicon nitride. Where a grid is used, the method may further comprise removing the grid. In some cases, the grid is removed under a gas with a dew point less than the operating temperature of the landing chamber, so as to avoid condensation on the deposited macromolecular ion sample on the grid to the extent possible. The method, in some embodiments, further comprises submerging the removed deposited ion in liquid nitrogen, for example, in preparation for analysis. In some embodiments, the charged macromolecular ion adheres to the landing pedestal or grid without formation of a covalent bond. In some embodiments, it adheres to the landing pedestal or grid without significant loss of structure, complexation, or folding modifications compared to its native state in solution.

In some methods herein, the method is performed with an atmospheric landing chamber such as that described above and elsewhere in the Examples and figures herein. In some cases, the landing chamber is operated under ambient air under atmospheric conditions. In other cases, it is operated under a gas such as nitrogen, carbon dioxide gas or a noble gas, wherein the air or gas has a relative humidity of less than 80%. In some cases, the method is performed in a controlled temperature and humidity room in order to control the ambient air temperature and humidity.

In other cases, the landing chamber is a cryogenic-compatible landing chamber that may be operated under either atmospheric or cryogenic conditions, such as that described above and in the Examples and figures herein. In some cases, the landing chamber is operated under cryogenic conditions. In some cases, the landing pedestal is chilled by a central tube allowing flow of gas cooled by a cooling unit in contact with the landing chamber. In some cases, the cryogenic-compatible landing chamber is operated under air or under a gas whose dew point is less than a desired chilling temperature.

In some cases, when performing methods herein, the DMA or nDMA transmission voltage needed for a particular analyte is known or can be accurately estimated prior to performing the method, for example, based on the calculated particle diameter for the analyte. (See Table 1 for examples.) In other cases, prior to performing the deposition experiments, it may be necessary to perform a scan over the expected transmission voltage range and to collect a spectrum of sample ions over the range of voltages in order to select the optimal voltage for use in the DMA or nDMA during the method.

In some cases, the charged macromolecular ion is deposited on the landing pedestal as a charged droplet or a charged macromolecule comprising a hydration shell. When preparing samples for cryo EM analysis, in particular, it is beneficial for the deposited charged molecular ions to be deposited as a non-crystalline solid onto the landing pedestal, particularly where the charged molecular ions are deposited as droplets or in hydrated form, such that water molecules surrounding the analyte molecules are arranged in an amorphous and non-crystalline manner. In some embodiments, particular solution components are added to the sample in order to encourage the deposited ions to form a non-crystalline solid on the landing pedestal or grid, or that otherwise are known to help prevent formation of crystalline water. Examples include solvents such as methanol or ethanol or certain compounds such as silicon or silicon oxide.

EXAMPLES

Soft Landing for Preparation of Cryo-EM Samples

Cryogenic electron microscopy (cryo-EM) is a powerful technology for elucidating multiple conformers of targets, observing noncovalent protein interactions, and for enabling drug discovery. Using the state of the art techniques, “well behaved” proteins can routinely achieve about 2.5-3 A resolution, making Cryo- EM a competitor to X-ray crystallography in terms of structure (1). The advantages of electron microscopy over crystallography include the capacity to look at many conformers of a protein, to look at proteins that do not crystallize easily (especially membrane proteins), and to preserve noncovalent interactions. Images are limited by a number of factors, including: the number of single particles (per structure) identified, the distribution of three-dimensional protein orientations observed, the electron transparency of the grid, the thickness and uniformity of the ice layer, the phase of ice generated, and the heterogeneity of the sample (and compatibility with upstream purification methods) (2). The totality of these problems creates a lower molecular cutoff, where proteins < 50 kDa are rarely detected in their monomeric form (3), and no protein <35 kDa has been solved (4). Nearly all of these problems derive from the sample preparation process of depositing a solution of protein on the grid, and subsequently freezing it (2). Transformative technologies are in demand to replace the current sample preparation method, improve grid and image quality, and to push the boundaries of Cryo-EM (2).

Cryo-EM relies on a surface layer of water to both capture molecules in a random solution state orientation and to shield the molecules from radiation damage (1). Vitrification techniques have largely remained static since the early 1980’s (5). The most common technique is to flash freeze a sample in liquid ethane following careful blotting of the protein sample on the grid. The faster the freezing and deposition, the reduced time for proteins to migrate to the air-water interface, where they are prone to adopt a certain orientation (limiting 3-D reconstruction) or denature. Likewise, the speed and temperature of freezing impacts the phase of ice formed on the grid, where a low-density amorphous ice is preferred for its electron transparency advantages (5). Vitrification became automated with the introduction of the Vitrobot robot (Thermo Fisher Scientific, Waltham, MA), which is used in nearly all modern Cryo-EM facilities.

Alternative preparation systems often seek to improve on the vitrification process by more evenly distributing protein-containing droplets across a grid. Approaches include piezoelectric nebulization of protein (6), depositing nanoliter quantities of protein via inkjet printers (7), or relying on newer liquid microfluidic handlers or methods to deposit picolitres of protein (8-11). Most of these methodologies remain confined to individual academic laboratories, although recently Spotiton has emerged to commercialize aspects of these advancements. Other approaches include grid re-designs with self-wi eking behavior (12), or modifications to the protein buffers. While the techniques generally result in improvement to the signal-to-noise, they have failed to overcome the totality of the problems in the EM, and are not easy to implement at scale.

Soft landing (SL) could theoretically provide a revolutionary solution for Cryo-EM grid preparation. The conventional design of a soft landing device consists of three main features (15). First, a source is used to generate a gas-phase ion from a solution or solid material. Nearly all sources are directly adapted from their original applications of generating ions for mass spectrometry analysis, with electrospray ionization proving to be most common as it is a soft and non-pulsed technique at atmosphere (16). Second, ion optics apply electrostatic fields to select, focus and/or accelerate ions through the instrument. These can look like traditional quadrupoles (17), ion funnels (18, 19), or a variety of emerging designs (20-22). Alternatively, mobility instruments can be used to select ions based on collisional cross sectional (CCS) area (23-26). Third, an analyzer, containing the landing surface, is employed to detect the ions transmitted (yielding a mass spectrum) and deposit the ions, a fraction of which are cited here (27-36). The majority of these devices take place under vacuum.

Much of the successful soft landing literature to date has focused on the landing of polymers such that rationally designed monolayers can be built onto chips, with applications for optical coatings and the electronics industry (13, 29-31, 33, 37- 40). Small molecule deposition has been robustly demonstrated and used for surface- modified chip generation (13, 41-43), microarray experiments (33, 44, 45), and for purification from reaction mixtures (46-48). Protein deposition is an emerging area for bioassays, and this approach has been successful because of the retained gross structure and enzymatic activity of the large molecules (27-29, 33, 39). A smaller subset of the work has been dedicated to understanding and attempting to preserve the structure of peptides and proteins with only partial success to the tens of angstrom level. Starting in 2011, the Cooks group led protein landing approaches performed under ambient conditions (49). Enzymatic activity and protein mass were examined, gross and fine structure were not investigated, and no further work on ambient landing for negative stain or Cryo-EM has been published on atmospheric systems. In 2010 and 2014, TEM in the Robinson group was used to confirm that the quaternary structure of GroEL and ferritin landed under vacuum mimicked native conditions (34, 36). Later on, sample transferred under vacuum from the deposition chamber to an electron holographic microscope showed at the nanometer scale that many orientations of single proteins were resolved and structurally intact and that aggregates could be distinguished from each other (38, 50). Yet many of the efforts in protein soft landing imaging have stalled because of protein denaturation shown to be a function of charge and landing time (28, 34, 38). Recent work from the Cooks, Robinson and Coon labs have shown gross structure may be maintained with glycerol application to grids (27, 51-53) an approach that works for negative staining but is not ideal for Cryo-EM.

To our knowledge, there is a single successful soft landing technique that preserves fine protein structure, rather than just gross structure, yet is rarely cited because it was published with the terminology “voltage assisted spray” (54, 55). The approach takes advantage of ultra-short time-course spray deposition (within tens of milliseconds) onto a moving surface plunged into liquid ethane. The spray deposited yielded significantly larger charged droplets than those traditionally by electrospray. The work was designed for imaging products from protein reactions accelerated by being performed in droplet-based, micro systems (56). The resulting structures were shown to be comparable to traditionally prepared Cryo-EM images, demonstrating that soft landing can be successful under atmospheric conditions, with highly solvated ions/charged droplets, and when frozen within a certain time course. Deposition with extended time (hundreds of milliseconds), led to denaturation as previously reported (27, 51-53).

The present Examples describe a new soft landing device that couples charge-reduced electrospray (57, 58) to differential mobility to atmospheric landing under variable temperature conditions. All precedent for linking differential mobility systems to imaging has been done for the purpose of protein recovery or hydrodynamic radius validation of particles as observed by SEM or TEM (59-61), with samples never looked at by negative stain or cryo- EM. The design described below incorporates a previously described charge-reduction electrospray device (57, 58, 62) to a commercially available nano differential mobility analyzer (63-67) followed by two different custom landing chambers. For negative stain or protein surface deposition work, an atmospheric chamber was built, and for Cryo-EM work, a first-in-kind atmospheric chilled landing chamber was designed. The only other attempt known to land on chilled grids is performed with a beam doser, as described here, under vacuum (53). The following results and discussion detail the theoretical concept, engineering, and application testing of this new soft landing device (SLD), with comparisons drawn to relevant systems and prior art.

An SLD (Fig 1) was constructed to comprise four primary components, a charge reducing electrospray source, a nano differential mobility analyzer (nDMA) (TSI Inc., Shoreview, MN), a focusing lens, and a landing chamber.

Example 1: Electrospray source, nDMA, and focusing lens

A charge reduced electrospray source was built using principles previously described (57, 58, 62, 68, 69) . The source includes an inlet for a capillary with a pulled or ground electrospray emitter stabilized by differentially controlled air and carbon dioxide. Techniques for electrospray are extensively described in the literature (16). The ESI tips sit just outside a charge reduction chamber, where a 210Po static eliminator (NRD, LLC, Long Island, NY) decays into alpha particles, which yield a variety of charge reducing species through interactions with the air and CO2. With the application of voltage at a liquid junction, positive and negatively charged liquid droplets enter this field, and, given sufficient field strength, are reduced to -1, +1, and neutral charged species.

Charge reduction theoretically offers a number of benefits to soft landing. The charge reduction occurs within microseconds, thereby minimizing the coulombic repulsion between droplets, resulting in a fewer ion losses. Likewise, the low charge ensures the droplet may proceed through evaporation processes over tens to hundreds of milliseconds without causing a charged protein to be ejected, which could result in denaturation at the air-water interface (70). Finally, given a homogenous charge state population, the collisional cross section of a droplet containing a protein is directly proportional to the protein size (Stoke’s law), rather than the ratio of that size versus total charge (71).

A comparison was made for the theoretical deposition energy of a +1 protein at atmosphere versus a +20 protein under vacuum in a Q Exactive™ UHMR (Thermo Fisher Scientific, Bremen, DE). At atmosphere, the +1 ion would land with 8 x 10'⁶ eV, versus 63 eV for a +20 ion under vacuum moving at 226 m/s, such as in the recent example of Westphall et al. 2022 (27). Energy would have to be reduced under vacuum to a velocity of 100 m/s to fall under the classical “soft” definition of < 35 eV; without moving to lower charge, there is a limitation to how far velocity can be reduced due to the optics and gas expansion acceleration that occurs in a vacuum based system (72). Thus, the current design is nearly 6-orders of magnitude softer than those at the state-of-the-art, and inversely lower to the charge state of ions deposited by the time-resolved velocity spray atmospheric system (0-50-fold reduced).

Differential mobility analyzers offer a low-resolution technique for separating charged droplets/analytes on the basis of collisional cross sectional area (63-65, 73). Ions travel through a sheath gas towards a central rod of opposite polarity, resulting in specific trajectories related to an ion’s collisional cross sectional area. All experiments described here are performed to detect positive-mode ions. The particle diameter may be directly calculated from the nDMA voltage, where protein calibrants are not required due to the known first-order relationship (74). As shown in Fig 2, proteins ranging from 8 - 2000 kDa and 20 nm polysyrene particles were detected by the nDMA, where their associated hydrodynamic radiuses were extracted (Table 1). The extension of the DMA to separate very large particles (AAVs, types of aerosols) in the hundreds of nanometers of range has been previously demonstrated and compatibility is expected (75). While large proteins could be observed with standard fused silica capillary, proteins in the hundreds of kilodalton range required either polyacrylamide or methylated coated capillary to prevent nonspecific interactions. For proteins less than 45 kDa, Sigmacote (Sigma Aldrich, St. Louis, MO) was added to prevent binding to the capillary (Fig 3). No changes to the mean mobility or the hydrodynamic distribution was observed, and follow up work to observe if any chemical adducts are present is required by mass spectrometry.

Table 1 shows the measured protein nDMA transmission voltages, the calculated particle diameters, and the predicted molecular weight for several different protein and protein complex examples. Note that the extrapolation of mass from diameter is much weaker than the calculation of particle diameter directly, and in particular will hold less true for protein complexes. Table 1

Example 2: Atmospheric landing chamber and focusing lens

A landing chamber designed for room temperature atmospheric landing was constructed (Fig 4). The landing chamber was connected to the DMA outlet by rubber tubing, where ions were propelled through the line by a gas flow. The sheath gas exhaust from the DMA was plumbed into the landing chamber, such that it provided a secondary flow of inert gas to fill the chamber. Ions were initially directed through the landing chamber to a glass rod filled with steel wool soldered to a wire that transmitted signal via connection to a detector. The glass rod/pedestal was eventually replaced with a brass rod designed with a special seat to hold unclipped grids or clipped Autogrids®, with space to insert a tweezer to move grids (Fig 5-6). In a different format, brass rods with a smooth top were used, and copper or other types of conductive tape were used to secure grids to the pedestal. Ions were tested for landing directly onto the pedestal or a grid-on-pedestal setup by measuring the transmitted current. Initial experiments were performed with 20 nm green A488 fluorescent particles (FluoSpheres™, Thermo Fisher Scientific, Sunnyvale, CA) deposited onto a CF150-Cu-UL grid (EMS, Hatfield, PA). Based on the initial concentration, 1.19 x 10¹¹ parti cles/pm² were estimated as being infused, and 4.49 x 10⁸ parti cles/pm² were calculated based on the current. This corresponded to 300-1000-fold losses between infusion and landing, which could be explained by the ionization efficiency of yielding +1 ions and inherent losses during transmission. There was a 72% reduction in the number of particles calculated from the current and from those detected by SEM (Figure 7). This was near the expected difference in area of the pedestal to the grid, where the grid comprised 30% of the area transmitting current.

The transmission of particles was evaluated with the addition of different focusing lenses. Focusing lenses represent a mature area of research (18-22) in the field of mass spectrometry, and thus different designs were tried based on prior knowledge. While models in SIMION (76) can attempt to predict the trajectories based on gas flows and electric fields, it is often difficult to exactly replicate what is observed experimentally. As shown in Fig 8, the transmission of tocilizumab was not significantly changed with the first three focusing designs of wire mesh rings, suspended rings (without mesh), or grid wires. Interestingly, a blocking effect was observed at high voltages on both ring designs. The fourth design integrated an ion funnel with a preceding gas diffuser (Fig 9), resulting in an approximately 2-3-fold increase across all proteins tested (Fig 10). Optimized focusing voltage was a function of the protein/particle mass/mobility, where higher voltages were required to transmit larger species. Different current measurement approaches were investigated to enable quantitation, where the pedestal current differential, normalized at each voltage, was considered the most reliable approach, followed by a comparison of the absolute amplitude of a protein across a voltage sweep scan (Fig 11). It was observed that for each protein, to obtain reliable current measurements, the nDMA voltage ramp rate and the nDMA voltage flat time (time to return to ground) must both be above certain thresholds, or else the peak shape and signal became unreliable (Fig 11). For measurements made at a constant nDMA voltage and across variable focusing lens voltages, normalization was required at each new focusing lens setting, demonstrating that the pedestal was not held at a true ground. The pedestal’s ability to build a charge throughout the course of experiments was further demonstrated by measuring the transmitted current pre and post a focusing voltage sweep. There was a significant difference in the two voltages detected pre/post experiment despite a grounding wire on the pedestal, suggesting that charge may be building within the chamber and unable to dissipate efficiently (Fig 12). Thus, when pedestals were later constructed of all metal for cryogenic landing, rather than for current measurement and atmospheric landing, the pedestals were additionally grounded.

The significance of the gas flow rate became evident after the integration of the focusing lens and thus was further explored. Traditionally a nDMA is operated with equal and balanced “aerosol in” and “aerosol out” flows (Fig 13) (77). Modifying the two ratios can alter the efficiency of the transmission, potentially affecting the transmission of the nDMA and decreasing the resolution of the mobility separation. Despite these theoretical concerns, GroEl nDMA sweep experiments with both balanced and unbalanced “aerosol in” and “aerosol out” flows were conducted as a way to both validate the mass flow controller readings, and to assess the experimental effects (Figure 13). The experiments were performed at three nDMA sheath flows, 10 L/min, 20 L/min, and 40 L/min, where resolution improved with increasing flows, with the biggest change from 10 to 40 L/min. The changes in the relative aerosol in/out ratios had the largest effects at low sheath flows. With an “aerosol in” of 2 L/min, the GroEl complex (-800 kDa) decreased by 50% from 2-3.25 L/min aerosol out rates at 10 L/min (Fig 13 A), but was unaffected by these changes at 40 L/min (Fig 13B). The effects of the “aerosol out” rate were also a function of the size of the analyte. GroEl at high concentrations formed an electrospray-artifact noncovalent dimer (2xl4-mer complexes, -1.6 MDa) and was also found in its native 14-mer state. Degraded products were also observed, including the 7-mer (single heptameric ring) and monomeric and dimeric species. The rate of change across the increasing “aerosol out” flows, at a controlled “aerosol in flow” was a function of analyte size, with the rate of change for the 14mer greater than the 7mer (Fig 13C). Interestingly, the GroEl dimer (2xl4-mer) did not change as a function of aerosol in/out, instead the signal more reflected differences in ESI stability. This suggests the possibility that the dimer is formed after exiting the nDMA, rather than through co-ionization in a single ESI droplet. This theory will be discussed in greater detail in the Cryo-EM results section. When the flow rates were balanced, providing optimal transmission, differences could be observed in the amount of protein transmitted (Fig 13D-G). Higher resolution at 40 LPM likely came at a cost to the signal of tocilizumab. At 20LPM, a 2 LPM aerosol in/out flow resulted in consistently higher signal compared to 1 LPM or no sheath flow.

Confirmation of particle landing onto carbon coated Cu or Au mesh unclipped grids was next performed by fluorescence microscopy experiments using 20 nm fluorescent polystyrene nanoparticles, trastuzumab-A488 DAR 2 and trastuzumab Fab-A647 DAR 1. For 20 nm particles, fluorescence was not detected without employ of the focusing lens, and semi-quantitated results confirmed that increasing the focusing lens voltage yielded more particles (Fig 14). An increase in the landing time also resulted in an increase to the absolute fluorescence detected (Fig 15). The flux through the landing instrument was then tested when a voltage was applied to the pedestal. It was found that grids with thinner films did not transmit the voltage efficiently, but that increases in the number of particles landed were observed on films with sufficiently thick films (Fig 16). With the application of voltage, particles preferentially landed on the film over the wire mesh, whereas without voltage, a more even distribution was observed. Very bright spots of particles were observed on the grids. It is not clear if this represents spots where the grid was deformed in a concave manner, places where the film was thicker/thinner, or a stochastic function of particles coalescing. A mixed sample of trastuzumab and FAB was also tested to demonstrate the functionality of the nDMA gasphase mobility selection (Fig 17). Given that the analytes were tagged with different fluorophores, signal would only be detected if the correct analyte was landed.

Fluorescence monitoring also offered an opportunity to assess the enzymatic activity of a landed protein. Two assays were designed using either the TEV protease (Abeam, Cambridge, UK) or the NSP4 protease (produced in house). Fluorescent measurements were taken with an on-grid time course reaction in a 96-well plate reader (Fig 18), and quantitation performed was based on a 5-FAM standard assay. The landing experiment demonstrated that the TEV protease was active and that it was linearly related, as expected, to the amount landed. The predicted amount on the grid was 50 ng, in close agreement to the calculated 28 ng on the grid. While differences in the amount could be related to the activity, this value is within the differences observed in other predicted-to-detected experiments (for example SEM work). As TEV is a robust protease, the enzyme NSP4 was next selected for evaluation. NSP4 is a 27 kDa protein that was discovered to nonspecifically bind to polyacrylamide coated capillaries, methylated capillaries, fused silica capillaries of interior diameters 20 - 100 pm, polyimide capillaries, and peek capillaries, regardless of the buffer ionic strength or detergents added. Recently, it was discovered that application of Sigmacote (Sigma Aldrich, St. Louis, MO) to the fused silica lines could limit this binding, and a mobility spectra was obtained for the enzyme (Fig 3 A). Experiments are planned with noncovalent and covalent inhibitors in addition to the native enzyme to best quantify landing activity and to demonstrate that low abundant conformers can be selected and landed from a heterogeneous mixture.

The hydration level of the proteins landed is expected to be critical to maintaining the fine structure and enzymatic activity. Droplets produced in this charge reduction system are expected to be predominantly governed by evaporation, rather than fission processes, thus allowing their end droplet size to be predicted. In the nDMA, particle diameter may be directly determined (78). Droplet evaporation rates are a function of the initial droplet size, surrounding gas flow rate, the ambient humidity, pressure, temperature, ionic strength, and surface tension (79-84). The dew point of the air used as the sheath gas in the instrument was measured at -62° C and the SLD was used in a room at ambient temperature between 20-23° C. When an electrospray source was introduced, resulting in an increase in the humidity of the sheath gas, the air post-nDMA was measured with a dew point of -50° C. The initial droplet size is a function of the electrospray emitter diameter and flow rate of the liquid. The buffers in the system may be modulated with various detergents (such as DDM, UDM, LMNG, etc.), small molecules (such as histidine, sucrose, etc.), salts (such as imidazole, ammonium acetate, calcium carbonate, etc.) at varying concentrations, and solvents (such as acetonitrile, isopropanol, etc.). To demonstrate the range in accessible sizes, experiments with different solvent components and flow rates to generate different initial sized ESI droplets were performed, and the distribution of diameters is shown in Fig 19. Across different sucrose conditions, droplets of size 0-13 nm could be formed. The ability to tune the end droplet size through multiple methods allows the system to work for proteins that may be stable in smaller hydration shells, membrane proteins which require large amounts of detergent to remain soluble, and complexes, which may demand increased solvation.

The atmospheric version of this soft landing device holds the potential to benefit different areas of analytical research. By coupling electrospray to a nDMA, gas phase purification of complexes, protein mixtures, or other heterogenous systems, which may not be compatible with traditional purification methods, such as LC, can be achieved. Likewise, specific conformers or PTMs have the potential to be enriched using this gas phase approach. Assuming a spherical model, particle diameters may be directly calculated from the nDMA measurements, enabling an orthogonal piece of information to be used in identifying image features. Lastly, deposition of active proteins can be used to build biophysical assays or for the purposes of directly purifying material for later use.

The first imaging instrument used to examine atmospheric SLD grids was a scanning electron microscope, used here for the purposes of validating the number of particles landed and the particle size detected (Fig 20). As mentioned earlier, the number of particles detected closely correlated with the current detection when taking into account the area on the pedestal occupied by the grid. From the images, the Ferret’s diameter may be calculated using ImageJ (85). It is expected that charging of polystyrene molecules in the SEM led to an increase in the particle diameter detected, and when accounting for this, the results are in good agreement with the nDMA measurements. Also shown are two grid types, demonstrating compatibility across grids with different support structures. Lastly, the number of particles overlapping each other was calculated to determine if there was a bias for specific landing spots. The images aligned with the expected rate of ~4% of species landing on top of another species simply based on random probability.

A different imaging technique that can be performed on samples at atmosphere is negative stain electron microscopy. In this technique, contrast between the protein and a stained grid is used to evaluate the analyte’s gross resolution structure, yielding information about tertiary and quaternary structure (86). Prior attempts for negative stain EM at atmosphere and vacuum have demonstrated that proteins denature on landing (27, 28, 34, 51). These effects may be mitigated through the surface application of glycerol (27, 46, 52). Interestingly, we found that there was a need to plasma treat all grids prior to landing in order for any stain to apply, and for the contrast to significantly increase. While plasma treatment is common in traditional negative stain preparation, it is rarely discussed in the context of soft landing. Application of plasma to the grids switched the images from appearing in positive to negative mode (Fig 20). For non-plasma treated grids landed and stained with any other reagent other than uranyl acetate, the contrast was extremely low, and in many cases, protein could not be observed (data not shown). As shown in Fig 21, the proteins were observed with poor resolution, supporting prior work that showed denaturation without the application of glycerol. We also investigated if UDM or sucrose in the electrospray buffer could mitigate denaturation, but saw no differences between the negative stain images with different buffers (data not shown). It is possible the inclusion of other buffers, including glycerol directly in the electrospray, could ameliorate the denaturation on landing. Importantly, the class averages of these images did demonstrate that GroEl was landed at least in a top down and side view conformation, although no features could be discerned. Likewise the proteins were detected at the expected abundance levels based on the current readback.

Example 3: Cryogenic landing chamber

The ultimate achievement of a soft landing device would be compatibility with CryoEM. Many of the challenges of Cryo-EM would be solved by soft landing, including preferential orientation, heterogeneity, and contrast challenges. Given that a protein was sufficiently hydrated, rather than nearly dry as under vacuum systems, issues associated with gas-phase denaturation, where a native protein begins to turn “inside-out” in the mass spectrometer (87), could also be avoided. While a hydrated protein would still have air-water interfaces, soft landing is a fast technique. A traditional mass spectrometer’s cycle time is in the tens of microseconds for a time-of-flight instrument (88) and in the tens of milliseconds in an Orbitrap mass spectrometer (where cycle time excludes measurement of the image current in the Orbitrap) (89). For a differential mobility instrument, transient times are dependent on sheath, aerosol flows, and an ion’s collisional cross sectional area, and would therefore range from the high hundreds of microseconds to single millisecond range (90). In each of these cases, these transient times are below the expected time of migration for a protein in a droplet, which is largely thought to be 100-500 ms, depending on the protein, in traditional EM (91). Prior work related to “voltage assisted spray” has demonstrated that nearly no denaturation of apoferritin occurred when sprayed for 36 ms prior to freezing (55). While there are significant energy deposition advantages to landing at atmosphere (described above), such a system also creates its own challenges. Given the propensity of samples to denature when landing at room temperature, we set about to develop a first in kind cryogenic landing chamber, something that has never been explored in the soft landing literature at atmosphere. We conceived an approach centered around two concepts. In the first, we hypothesized that proteins with nanometer sized water shells would form amorphous ice under relatively warm conditions (0 to - 60 C), although it was not clear at which density level. Nanolayer ice formation has been studied in a limited manner in nanolayer bulk solutions (92-98), and never in the case of droplets moving through a chilled field. Currently, Cryo-EM’s standard vitrification must use liquid ethane preferably (5), or at least liquid nitrogen (99), temperatures to cool protein liquid films fast enough to form low density amorphous ice, so this would represent a major advancement. In the second hypotheses, we believed that vacuum systems were not required to land proteins on a dry chilled surface, rather that an inert atmosphere within a landing chamber would suffice, and that grid extraction could be achieved by external atmosphere controls.

Pictured in Fig 22 are photos of the device configured for atmospheric use (Fig 22A) or cryogenic use (Fig 22B). The device remained in the same configuration through the outlet of the nDMA between the two systems. The atmospheric landing chamber was replaced with a similar chamber where the bottom of the chamber was open and grooved for an O-ring seal (Fig 23). The new cryo-landing chamber was seated on a sliding and locking rod (Fig 24). The inlet was connected to the nDMA “aerosol out” with rubber tubing, and the sheath gas was connected to the Swagelok union port at the top of the chamber. The dew point of the “aerosol out” gas from the nDMA, with an ESI spray flowing or off, was measured at -62 C or -50 C, respectively. Given a need to further dry the gas and reduce the dew point, a separate desiccant system was built and installed, though is currently not in use. The open-ended landing chamber slides down into place on an O-ring in a seated metal dish. In the center of the O-ring, a gas line assembly was made to allow gas to enter or exist the system (Fig 25). The gas line assembly was covered in three layers of foam insulation to minimize heat transfer to the environment. The gas assembly was made of metal from the liquid nitrogen rubber dewar cap up to the point where a right angle turn to reach the landing pedestal was required, at which point it was replaced with doubly insulated rubber tubing. Multiple types of insulation were tested to achieve the best insulation possible. Special cryogenic pedestals were designed (Fig 26) which allowed for a chilled gas to circulate and cool the landing surface. A cooling system was developed by submerging a resistant heating element in liquid nitrogen stored in a dewar (Figure 27). Real time correction of the temperature was made through a powered temperature control unit that supplied voltage to the element and monitored the temperature.

Operation of the cryogenic unit was critical to preventing buildup of condensate on the chilled grid/pedestal. As such, the critical points of the user operation include those described here. The nDMA gas was kept on but the nDMA voltage was turned off to prevent protein transmission. Prior to turning on the chilling gas, the cryo-landing chamber was slid up, and the rubber tubing between the metal gas line assembly and the pedestal was disconnected. A hair dryer was used to warm the metal dish/pedestal/assembly to room temperature and to ensure all parts were fully dry. A clipped or unclipped grid was loaded onto the pedestal using EM tweezers. The cryo-landing chamber was slid down, pressed into the O-ring, and locked in place. After a minute of allowing the nDMA exhaust gas to purge any atmosphere from the cryo-landing chamber, the rubber tubing was connected to the metal cooling gas assembly and the heating control was set to the desired temperature (for the following experiments -20 C). When temperature was reached (~30 sec), the nDMA transmission voltage was turned on, and landing was allowed to proceed for the desired time length (for the following experiments, generally 20 min). After landing was finished, the nDMA voltage was turned off. A “banana” dewar custom designed to fit the cryogenic metal dish was filled with liquid nitrogen (fig 28). A grid/ AutoGrid box was seated into a custom box holder (fig 29), that was then placed in the dewar. The dewar was transferred with tongs or a special tool into the metal dish around the cryo-landing chamber (Fig 24). It was given ~l-2 min to fill the air with N2 headspace. The cryo-landing chamber was then unsealed from the O-ring, but not lifted. An independent N2 gas supply was connected via rubber tubing to the tee previously acting as the exhaust, and turned on at a low level (<4 L/min) as a reverse sheath gas. The cryo-landing chamber was then lifted, and a cap was slid in place to trap the liquid nitrogen headspace from evaporating (Fig 30B). Tweezers were used to pickup the grid and then rotate it such that the surface of the grid was parallel to the direction of the reverse sheath gas. The grid was quickly transferred into the grid box holder under liquid nitrogen, and subsequently stored. The timing of these steps as well as the orientation of the various caps used were critical to preventing condensate from occurring on the surface of the grid/pedestal. Given a desire to perform a system check prior to cryogenic landing, a set of cryogenic metal dish compatible pedestals for use at atmosphere that connected to a detector were also constructed. A custom hat was made for shielding to replace the door that was previously used in the atmospheric setup (Fig 30A). The design has the same and enhanced functionality of the atmospheric device previously described. The device voltages and control were set either manually through power controllers, or through a computer interfaced to a national instrument control box.

Initially, Cryo-EM results with landing under cryogenic conditions proved challenging. It was observed that nearly all of the graphene or graphene oxide films, regardless of mesh size, ripped during landing when tested with lacy carbon or Quantifoil® AutoGrids (data partially shown, Fig 31). Interestingly, these grids did not rip in the atmospheric only system. Any application of a plasma treatment to the grid surface significantly exacerbated this problem. On these grids, we were able to observe that GroEl was landing with minimal condensate developing (any condensate shown is at levels expected with the standard grid transfer steps into the Glacios™ EM). 1 layer graphene over 2000 mesh Cu grids showed the most robust behavior with only about 50% (versus near 100%) of the films ripped. On these grids, GroEl was deposited over the graphene film with multiple different orientations, and the barrel versus top-down views could be distinguished. Interestingly, GroEl was also observed as multimers within a single frozen droplet, but not in the form of a complex. These orientations are highlighted in the inlay in Figure 32A. In addition to the GroEl-like particles and multimer particles, smaller GroEl species, potentially representing GroEl subunits were observed. These may be a contaminant, a grid feature, or a result of landing. Consequently, 2D averages were not obtained due to a lack of a single homogeneous sub population. The contrast observed in the cryo-EM images was qualitatively at least as good, if not above, that observed in control experiments (Figure 32B). Importantly, class averages were next performed on the protein-less ice balls that were not condensate (Figure 33). Without an x-ray experiment, it is impossible to determine the exact ice structure. But the lack of a high-intensity ring at the 1/3.7 A spatial frequency corresponding to the (002) diffraction spots of cubical and hexagonal ice in the averaged 2D amplitude spectrum of all ice balls suggests that some form of amorphous (unknown if low or high density) ice was achieved, and thus landing at -20 C, rather than under much colder conditions, is a suitable approach for soft landing cryo-EM.

In sum, we have demonstrated a new system through which proteins, embedded in +1 droplets, may be gas phase separated on the basis of mobility and landed. Proteins were successfully detected over a range of molecular weights and with noncovalent species bound by using focusing lenses to improve transmission. We demonstrated comparable negative stain results to the state-of-the art systems currently used for glycerol-free room temperature soft landing. We showed that landing could be performed chilled with no deleterious condensate effects. Low temperature freezing yielded non-crystalline ice suitable for EM. With continued development and refinement, we expect that we will be able to look at fine structure with much more detail in the coming weeks to months of experiments. With this cryogenic soft landing device described, we are hopeful to see many positive impacts regarding gas-phase purification/enrichment and improvement in contrast (especially enabling small protein imaging).

Example 4: Landing of protease ArgC at room temperature

The cyrogenic-compatible landing system of Example 3 was used to land the protease ArgC at room temperature. ArgC is dependent on an auto-catalytic cleavage prior to enzymatic activity, and this ability to cleave into a noncovalent dimer is contingent on the correct structure. Thus, a successful landing of ArgC indicates that structure-sensitive proteins are able to survive the landing process with high activity. ArgC (clostripain) was obtained from Sigma. To achieve a method yielding a reproducible assay with a linear standard curve, considerations included reproducibility in extracting protein off landing surfaces of different materials, the volume of the reaction, and the type of analyzer. Ultimately, 3 mm sized plastic was used for landing, as this surface allowed a reduced, but live current to be read during landing, and had less sticking than a carbon grid. The ArgC was landed or spotted onto the surface at different concentrations (for a standard curve); three different replicates are shown. See Fig. 34A. The grids were maintained for ~20 min at room temperature, and then substrate (lysozyme) in a digest buffer was added to the system. After a 4 hour incubation, the reaction was quenched, and sample was injected for LC-MS analysis. The intensity of lysozyme was used as the response variable to determine the amount of ArgC landed. An analysis of the ArgC concentration determined from the lysozyme intensity is shown in Fig. 34B. The table in Fig. 34B shows the three repetitions of the landing from Fig. 34A with their respective agreement with the predicted signal.

Example 5: Landing of Apoferritin under cryogenic conditions

A series of landings of control and apoferritin (ApoF) protein were performed in the system of Example 3 at -40 °C. Results are shown in Fig. 35. Specifically, Figs. 35A-C show the grid alone without buffer (Fig. 35 A), a control experiment with buffer only (Fig. 35B), and of buffer without DMA (Fig. 35C). Protein was landed with and without DMA transmission as shown in Figs. 35D and 35E, respectively. The control experiments of Figs. 35A-C were used to observe if the protein spots shown in Fig. 35D were unique to the sample. The ApoF protein spots of Fig 35D were clearly observable on the grid, showing that landing in the system was successful, but the spots did not converge to a structure. As can be seen in the figure, the particles observed in the ApoF sample were not observed in the ammonium acetate or the ’’blocked” (i.e., no DMA) transmission control. Fig. 35F shows landing of gold (Au) particles on the grid as a further positive control of the system.. The Au particles were observed at -57C and were clearly identified on the basis of the high amount of scattering and their relative size (10 nm).

Example 6: Second cryogenic-compatible chamber system

New hardware was developed for landing at liquid nitrogen temperatures. In comparison to the system of Example 3, the metal landing chamber dish with a Styrofoam dewar was removed. A hole of about 2/16 inches was then drilled in the center of the dewar, which contains liquid nitrogen during operation, in order to accommodate the landing pedestal. A tight seal around the pedestal prevented the liquid nitrogen from leaking, and allowed the pedestal to be moved up and down in height without the liquid nitrogen leaking. The tee valve exhaust holding the pedestal was shortened and the exhaust was capped with a nut. A photograph of this system is shown in Figs. 36A-B, and an example of GroEL landed at liquid nitrogen temperature at 73,000 magnification is shown in Fig. 36C.

Before operation, the system was brought to room temperature and dried. The grid was placed on the pedestal and raised into place. At least two cold traps were added to the system. Cold traps consisted of large pieces of metal that were raised higher than the liquid nitrogen fill line such that any condensate would preferentially condense onto them. The landing chamber was placed above the metal cold traps. The landing chamber was operated in an open-to-atmosphere position, such that the bottom of the chamber, which is open to atmosphere, sat right above the cold traps. The liquid nitrogen was manually filled during landing, which ranged from 5-20 min in time. At the start of the landing time, the focusing lens was turned on and the DMA was set to transmit at the desired protein transmission voltage. After landing, the DMA voltage was turned off and the focusing lens was set to off. The pedestal was pulled down until it hovered above the liquid nitrogen fill line. The tweezers were prechilled, and used to grab the grid and dunk it in liquid nitrogen as the chamber was simultaneously lifted. The grid was then stored as previously described.

The key features that differentiate this second landing system from the cryogenic- compatible system described in Example 3 are that the landing chamber is open to atmosphere at the bottom instead of being closed to atmosphere, and the temperature of freezing that could be achieved. Remarkably, no increase in condensate on the grid was observed despite the chamber being open to atmosphere at the bottom, which is attributed to atmosphere being pushed away by the liquid nitrogen head space & the sheath gas. For example, because liquid nitrogen in the dewar below the chamber vaporizes, it forms a nitrogen gas head space inside the chamber above. Furthermore, the “open-to-air” landing approach should have an approximate 2-fold decrease in the landing kinetic energy. As the gas is no longer directed into a constricted exhaust port, it is no longer accelerated to the same degree, and thus the particles move slower prior to landing.

Further data, shown in Figs. 38A-B, indicate that a combination of the sheath flow and the liquid nitrogen headspace in the chamber are important in controlling condensate in the open-bottom, unsealed chamber system. For example, Fig. 38A compared to Fig. 38B shows that at 10 L/min flow, there are significantly larger ice crystals on the grid than at 20 L/min flow, when excluding the largest crystals which are a consequence of grid transfer steps.

Example 7: Third cryogenic-compatible landing system

A further alternative system for landing was also designed that was relatively easy to operate, and that does not include a focusing lens component. It consisted of using two MiteGen® (Ithaca, NY, USA) clipping stations stacked on top of each other, and a 6 mm grid pedestal positioned halfway up the hole of the second station. A photograph of the system in closed and open configurations is shown in Figs. 37A-B. The landing pedestal consisted of a 6 mm threaded rod held in place with a thread adapter on a PTFE plate, and pushed through a foam dewar. By positioning the rod halfway up the second (top) station, the walls of the holder acted as boundaries to keep the grid secure during landing, to ensure that liquid nitrogen did not fill the void, and to limit condensate. Into the top of the hole of the second (top) clipping station, tubing from the nDMA was inserted, such that there was about 2 mm between the ion exit and the grid. The holes of each clipping station were therefore directly on top of each other, so the grid was located in line with the entry of the aerosol out gas flow. The top clipping station conveniently acted as a cold trap for the system, and liquid nitrogen was filled to a level a few millimeters below the height of the pedestal. During grid removal, the top station was lifted, and the landing grid was simultaneously pushed onto the bottom station before it was picked up with tweezers and stored. Because, in this system, clipping stations were used as platforms surrounding the landing pedestal and grid, this system has the possibility of being more easily automated such that, after landing, the grid may be moved to a location on the bottom station, for example, for storage. Fig. 37C shows GroEL particles from landing in this system. While only few particles were observed, the amount of condensate was minimal, demonstrating the potential of this system design.

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Claims

What is claimed is:

2. The system of claim 1, wherein the electrospray source is a charge reduced electrospray source.

3. The system of claim 1 or 2, wherein the pedestal holds a removable grid, such as a clipped or unclipped grid, upon which charged protein ions may be deposited.

4. The system of claim 3, wherein the grid comprises a carbon, graphene, graphene oxide-coated metallic film on a mesh surface such as a Cu, Ni, or Au mesh or other non-metallic support, such as silicon nitride.

5. The system of any one of claims 1-4, wherein the atmospheric landing chamber may be operated under ambient air, or under a gas such as nitrogen, carbon dioxide gas or a noble gas, wherein the air or gas has a relative humidity of less than 80%.

6. The system of any one of claims 1-5, wherein the landing chamber comprises a focusing lens.

7. The system of claim 6, wherein the focusing lens comprises an ion funnel with a preceding gas diffuser.

8. The system of any one of claims 1-7, wherein the system is capable of depositing protein ions of +1 to +5 charge on the landing pedestal.

9. The system of any one of claims 1-8, wherein the system is capable of depositing protein ions of from 3 to 100 nm diameter on the landing pedestal.

10. The system of any one of claims 1-9, wherein the system is capable of depositing protein ions in an atmospheric dry state, in droplet form, or as hydrated protein ions on the landing pedestal. The system of any one of claims 1-10, wherein the DMA is a nano DMA (nDMA). A system for gas phase separating charged macromolecular ions on the basis of mobility, the system comprising: a. an electrospray source; b. a differential mobility analyzer (DMA); and c. a cryogenic-compatible landing chamber, wherein the landing chamber comprises a landing pedestal enclosed within the chamber, wherein the charged macromolecular ions are capable of being deposited on the landing pedestal, wherein the landing chamber optionally further comprises a focusing lens, and wherein the electrospray source, DMA, and landing chamber are connected to allow electrospray gas flow from the source, through the DMA, and to the landing chamber. The system of claim 12, wherein the electrospray source is a charge reduced electrospray source. The system of claim 12 or 13, wherein the pedestal holds a removable grid, such as a clipped or unclipped grid, upon which charged macromolecular ions may be deposited. The system of claim 14, wherein the grid comprises a carbon, graphene, graphene oxide-coated metallic film on a mesh surface such as a Cu, Ni, or Au mesh or other non-metallic support, such as silicon nitride. The system of any one of claims 12-15, wherein the landing chamber comprises a focusing lens. The system of claim 16, wherein the focusing lens comprises an ion funnel with a preceding gas diffuser. The system of any one of claims 12-17, wherein the cryogenic-compatible landing chamber pedestal is in contact with a cooling unit in which a resistant heating element is submerged in a chilled fluid, such as liquid nitrogen or liquid ethane, stored in a dewar. The system of any one of claims 12-18, wherein the cryogenic-compatible landing chamber may be operated in cryogenic conditions under air or under a gas whose dew point is less than a desired chilling temperature. The system of any one of claims 12-19, wherein the system is capable of depositing macromolecular ions of +1 to +5 charge on the landing pedestal. The system of any one of claims 12-20, wherein the DMA is a nano DMA (nDMA). The system of claim 21, wherein the macromolecular ions are protein ions. The system of any one of claims 12-22, wherein the system is capable of depositing macromolecular ions of 3-100 nm diameter on the landing pedestal. The system of any one of claims 12-23, wherein the system is capable of depositing ions in a dry state, in droplet form, or as hydrated protein ions on the pedestal of the landing chamber. The system of any one of claims 12-24, wherein the cryogenic-compatible chamber can be operated under either atmospheric or cryogenic conditions. The system of any one of claims 12-25, wherein the pedestal comprises a central tube allowing flow of gas cooled by the cooling unit. A cryogenic-compatible landing chamber for deposition of charged macromolecular ions generated by an electrospray source, wherein the landing chamber comprises a landing pedestal enclosed within the chamber, wherein the charged macromolecular ions are capable of being deposited on the landing pedestal, and further wherein the landing chamber is in contact with a cooling unit. The cryogenic-compatible landing chamber of claim 27, wherein the cooling unit comprises a resistant heating element submerged in a chilled fluid such as liquid nitrogen or liquid ethane stored in a dewar. The cryogenic-compatible landing chamber of claim 27 or 28, wherein the system further comprises an automatic temperature control element. The cryogenic-compatible landing chamber of any one of claims 27-29, further comprising a means of connecting the landing chamber to a DMA or nDMA to allow flow of the charged macromolecular ions from the DMA or nDMA to the landing pedestal. The cryogenic-compatible landing chamber of any one of claims 27-30, further comprising a focusing lens configured to allow electrospray gas flow from an electrospray source through the focusing lens and to the landing pedestal. The cryogenic-compatible landing chamber of claim 31, wherein the focusing lens comprises an ion funnel with a preceding gas diffuser. The cryogenic-compatible landing chamber of any one of claims 27-32, wherein the pedestal comprises a central tube allowing flow of gas cooled by the cooling unit. The cryogenic-compatible landing chamber of any one of claims 27-33, wherein the chamber may be operated under either cryogenic or atmospheric conditions. The cryogenic-compatible landing chamber of any one of claims 27-34, wherein the pedestal holds a removable grid, such as a clipped or unclipped grid, upon which charged macromolecular ions may be deposited. The cryogenic landing chamber of claim 35, wherein the grid comprises a carbon, graphene, graphene oxide-coated metallic film on a mesh surface such as a Cu, Ni, or Au mesh or other non-metallic support, such as silicon nitride. An atmospheric landing chamber for deposition of charged macromolecular ions generated by an electrospray source, wherein the landing chamber comprises a landing pedestal enclosed within the chamber, wherein the charged macromolecular ions are capable of being deposited on the landing pedestal, and further wherein the landing chamber may be operated under atmospheric conditions and may be operated under ambient air, or under a gas such as nitrogen, carbon dioxide gas or a noble gas, wherein the air or gas has a relative humidity of less than 80%. The atmospheric landing chamber of claim 37, further comprising a means of connecting the landing chamber to a DMA or nDMA to allow flow of the charged macromolecular ions from the DMA or nDMA to the landing pedestal. The atmospheric landing chamber of claim 37 or 38, further comprising a focusing lens configured to allow electrospray gas flow from an electrospray source through the focusing lens and to the landing pedestal. The atmospheric landing chamber of claim 39, wherein the focusing lens comprises an ion funnel with a preceding gas diffuser. The atmospheric landing chamber of any one of claims 37-40, wherein the pedestal holds a removable grid, such as a clipped or unclipped grid, upon which charged macromolecular ions may be deposited. The atmospheric landing chamber of claim 41, wherein the grid comprises a carbon, graphene, graphene oxide-coated metallic film on a mesh surface such as a Cu, Ni, or Au mesh or other non-metallic support, such as silicon nitride. A method of preparing and isolating a charged macromolecular ion comprising an analyte, the method comprising: a. ionizing a sample comprising a macromolecular analyte with an electrospray source to form charged macromolecular ions; b. separating the charged macromolecular ions on the basis of their collisional cross sectional area, for example, in a DMA or nDMA; and c. directing a charged macromolecular ion comprising the analyte to a landing pedestal located in an enclosed landing chamber, wherein (i) the landing chamber operates under atmospheric conditions or (ii) the landing chamber operates under either atmospheric conditions or cryogenic conditions, and depositing the macromolecular ion comprising the analyte on the landing pedestal. The method of claim 43, wherein the macromolecular analyte is a polypeptide, nucleic acid, virus, or a complex comprising at least one polypeptide. The method of claim 43, wherein the macromolecular analyte is a polypeptide. The method of any one of claims 43-45, wherein the electrospray source is a charge reduced electrospray source. The method of any one of claims 43-46, wherein the ion is a +1 to +5 ion or a -1 to -5 ion. The method of any one of claims 43-46, wherein the charged macromolecular ion is a +1 or -1 ion. The method of any one of claims 43-48, wherein the charged macromolecular ion is from 3 to 100 nm diameter. The method of any one of claims 43-49, wherein the charged macromolecular ion comprising the analyte is deposited on a removable grid on the landing pedestal, such as a clipped or unclipped grid, upon which charged macromolecular ions may be deposited. The method of claim 50, wherein the grid comprises a carbon, graphene, graphene oxide-coated metallic film on a mesh surface such as a Cu, Ni, or Au mesh or other non-metallic support, such as silicon nitride. The method of claim 50 or 51, wherein the method further comprises removing the grid, optionally wherein the grid is removed under a gas with a dew point less than the operating temperature of the landing chamber. The method of any one of claims 43-52, wherein the landing chamber comprises a focusing lens. The method of claim 53, wherein the focusing lens comprises an ion funnel with a preceding gas diffuser. The method of any one of claims 43-54, wherein the landing chamber is an atmospheric landing chamber. The method of claim 55, wherein the landing chamber is operated under ambient air, or under a gas such as nitrogen, carbon dioxide gas or a noble gas, wherein the air or gas has a relative humidity of less than 80%. The method of any one of claims 43-54, wherein the landing chamber is a cryogenic- compatible landing chamber that may be operated under either atmospheric or cryogenic conditions. The method of claim 57, wherein the landing pedestal comprises a central tube allowing flow of gas cooled by a cooling unit in contact with the landing chamber. The method of claim 57 or 58, wherein the landing chamber is operated under cryogenic conditions. The method of claim 59, wherein the landing chamber is operated under air or under a gas whose dew point is less than a desired chilling temperature. The method of any one of claims 43-60, wherein the charged macromolecular ion is deposited as a non-crystalline solid onto the landing pedestal. The method of any one of claims 43-61, wherein the charged macromolecular ion is deposited on the landing pedestal as a charged droplet or a charged macromolecule comprising a hydration shell. The method of any one of claims 43-62, further comprising removing the deposited charged macromolecular ion from the landing chamber. The method of claim 63, wherein removing the ion comprises removing a grid containing the deposited ion from the landing pedestal. The method of claim 63 or 64, wherein the charged macromolecular ion is removed under a gas with a dew point less than the operating temperature of the landing chamber during the removal. The method of any one of claims 63-65, further comprising submerging the removed deposited ion in liquid nitrogen.