WO2023015002A1 - Methods and compositions for systems-wide crosslinking mass spectrometry - Google Patents

Abstract

Methods of in situ labeling for systems-wide protein-protein interaction analysis using ultra-rapid cryo-fixation together with freeze-substitution, and compositions therefor.

Description

METHODS AND COMPOSITIONS FOR SYSTEMS-WIDE CROSSLINKING MASS SPECTROMETRY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/230,678, filed August 6, 2021, which is incorporated herein by reference in its entirety and for all purposes.

FIELD OF THE INVENTION

[0002] Provided herein is a system for covalent labeling of proteins in situ, using cryogenic cooling and freeze-substitution principles, which expands the selection of reagents that can be used for protein labeling by maintaining a low-water or water-free environment. It allows for labeling reactions in a temperature-programmed fashion, from room temperature down to -80°C, with high temporal sampling capability.

BACKGROUND

[0003] The cell is driven by macromolecular interactions involving many thousands of proteins in an ever-changing network. It is the central effort of molecular biology to uncover the organization and structure of proteins in the native environment of the cell, to better understand cellular function and disease mechanisms. Proteomics, as a method for global analysis of all proteins, can identify and quantify proteins, but it generally struggles to determine spatial associations.

[0004] In situ molecular interactions drive all the cellular processes that support life, and these interactions are primarily mediated by proteins. A protein possesses a 3D structure that is determined by its primary sequence, but also by the numerous interactions it maintains with other proteins, DNA, RNA, metabolites and exogenous small molecules. The resulting network of interactions is critical to protein function, and this network itself has a 3D structure and dynamicity.

[0005] Laboratory methods for measuring interactions in the cell exist, but these generally (a) lack throughput and (b) do not always return spatial information. For example, sophisticated microscopy methods such as FRET can determine whether two protein molecules are close in space (<10 nm) but this does not necessarily mean that the molecules directly interact. In addition, because the method involves genetic engineering of fluorescently-tagged proteins in a binary manner, the method is difficult to scale to the entire proteome. Most optical/microscopy techniques are hampered by similar concerns. [0006] There remains a need for methods and systems for determining spatial associations of proteins based on in vivo conditions.

SUMMARY

[0007] The instant technology generally relates to methods, apparatuses, and reagents for use in the in situ covalent labeling for systems-wide protein-protein interaction analysis, using ultra-rapid cryo-fixation together with freeze-substitution.

[0008] In an aspect, provided herein, is a method of analyzing cellular protein interactions by mass spectrometry, including: freezing a cell by ultra-rapid freezing; contacting the frozen cell with an organic solvent and a chemical tag under conditions to remove cellular water and cause covalent labeling of cellular protein by the chemical tag; analyzing the labeled cellular protein by mass spectrometry to determine cellular protein interactions.

[0009] In an aspect, provided herein, is a method of analyzing cellular protein interactions by mass spectrometry, including: freezing a cell by ultra-rapid freezing; contacting the frozen cell with an organic solvent and a chemical tag under conditions to remove cellular water and cause crosslinking of cellular protein by the chemical tag; analyzing the crosslinked cellular protein by mass spectrometry to determine cellular protein interactions.

[0010] In an aspect, provided herein, is a method of determining protein interactions within a cell, the method including: freezing a cell by ultra-rapid freezing; contacting the frozen cell with an organic solvent and a chemical tag under conditions to remove cellular water and cause covalent labeling (e.g., crosslinking) of cellular protein by the chemical tag; analyzing the labeled (e.g., crosslinked) cellular protein by mass spectrometry to determine protein interactions.

[0011] In embodiments, the ultra-rapid freezing is performed by plunge freezing. In embodiments, the ultra-rapid freezing is performed by spray freezing. In embodiments, the ultra-rapid freezing is performed by self-pressurized rapid freezing. In embodiments, the ultrarapid freezing is performed by high pressure freezing.

[0012] In embodiments, the ultra-rapid freezing is performed by a spray freeze apparatus.

[0013] In an aspect, provided herein, is a spray freeze apparatus including a cryogenic reservoir; a heat sink within the cryogenic reservoir; a copper cup within the heat sink, the copper cup containing a cryogen; and a membrane suspended within the copper cup, such that when frozen cells are in contact with the membrane, the frozen cells are immersed in the cryogen. [0014] In an aspect, provided herein, is a system for freezing of cells for analysis by mass spectrometry, the system including a spray freeze apparatus containing a cell solution in the cell reservoir.

[0015] In an aspect, provided herein is a freeze substitution device. The freeze substitution device may include an organic solvent pump fluidly connected to a reaction chamber via an inlet tube. The reaction chamber may be within a chiller. The chiller may be temperature controlled. The reaction chamber may comprise a porous membrane. The porous membrane may be extended perpendicular to the reaction chamber such that the porous membrane collects/retains frozen cells under flow. In embodiments, an outlet tube extends from an end of the reaction chamber. In embodiments, an autosampler is fluidly connected to the organic solvent pump. In embodiments, the chemical tag is added to the cells with the autosampler.

[0016] In an aspect, provided herein, is a composition of labeled (e.g., crosslinked) cells, wherein the covalent labeling (e.g., crosslinking) method includes: freezing a cell by ultra-rapid freezing; and contacting the frozen cell with an organic solvent and a chemical tag under conditions to remove cellular water and cause covalent labeling (e.g., crosslinking) of cellular protein by the chemical tag.

[0017] In an aspect, provided herein, is a mass spectrometer containing a composition of cells that are labeled (e.g., crosslinked) by a covalent labeling (e.g., crosslinking) method that includes: freezing a cell by ultra-rapid freezing; contacting the frozen cell with an organic solvent and a chemical tag under conditions to remove cellular water and cause covalent labeling (e.g., crosslinking) of cellular protein by the chemical tag.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1A shows a freeze substitution to introduce FAMD-diazirine into human A549 cells. FIG. 1A shows the preservation of structure, followed by photolytic coupling using 355 nm UV light. Negative: no reagent used in freeze substitution. Photolysis generated fluorescein-protein conjugates (green) showing labeling throughout the cell. DAPI labeled DNA (blue) marks the nucleus. P-W: photolysis of entrained FAMD-diazirine, followed by washout of uncoupled reagent using -80°C acetone. W-P: washout followed by photolysis. W : washout. Scale bar=20 pm.

[0019] FIG. IB is the structure of FAMD-diazirine.

[0020] FIGs. 2A and 2B show a freeze substitution at -80°C to introduce 0.5 mM NHS-FAM into human A549 cells with preservation of structure, followed by fixation in methanol at various temperatures. Fluorescence images (FIG. 2A) demonstrate increased labeling at higher reaction temperatures (after freeze substitution), supported by quantification of labeling (FIG. 2B). Scale bar=20 pm.

[0021] FIG. 3 shows covalent labeling of human A549 cells under freeze-substitution conditions with propionic anhydride. Three reaction temperature were profiled, and the addition of a base catalyst in freeze substitution (here triethylamine) was explored. Whether freeze-substitution using neat acetone prior to introducing the propionic anhydride (“predesiccation”) conferred any advantage on labeling yield was also assessed. Quantitation was by proteomics, using mass spectrometry to detect what fraction of the whole proteome was modified with the reagent or not.

[0022] FIGs. 4A and 4B show an example embodiment of a spray freeze apparatus for ultrafast cryogenic cooling of cells in liquid ethane (FIG. 4A) and the effect of pressure (y- axis) on droplet size (x-axis: diameter in microns) (FIG. 4B). The device provides control over the size of the droplets containing the cells, based on gas pressure. Y axis shows droplet diameter in microns.

[0023] FIG. 5 shows an example embodiment of a solvent exchanger for freezesubstitution, based on a fixed temperature design using dry ice (-80°C), allowing for both continuous and stop-flow solvent exchange in a water-free design.

[0024] FIG. 6 shows a schematic for an example embodiment of a freeze substitution device.

[0025] FIG. 7 shows a detailed schematic of an embodiment of a cryotagging procedure.

[0026] FIG. 8A shows an exemplary schematic of gas nebulization to efficiently spray freeze suspended cells. FIG. 8B is a photograph of E. coli droplets. Droplets (containing a red dye) were sprayed into oil to prevent evaporation and allow measurement.

[0027] FIG. 9 is a graph of the droplet size versus cell density for fast freezing. Droplet size is independent of cell density.

[0028] FIG. 10 shows reactivity as a function of temperature for different cryotag types. Percent labeling of the proteome measured by bottom-up quantitative proteomics from E. coli experiments. This process is an efficient mimic of the cell-based freeze substitution process, in order to profile reaction chemistries with greater efficiency.

[0029] FIG. 11 shows exemplary crosslinking of E. coli with ethylenediaminetetraacetic dianhydride under cryo-coupling conditions. Select MS/MS spectrum shows a single peptide with a K to K crosslink. [0030] FIGs. 12A - 12C show labeling in a two-step reaction, involving preactivation with a carboxylic acid-targeting agent (pentafluorophenyl trifluoroacetate) to create a mixed anhydride in the proteome, which is then reacted with simple diamines in a crosslinking step, or internally quenched with a nearby free amine somewhere else in the proteome to create a zero-length crosslink. See scheme in (FIG. 12 A) to illustrate the process, and an MS/MS spectral example of a diamine-based crosslinker in (FIG. 12B), and an internally quenched crosslink in (FIG. 12C).

[0031] FIGs. 13A and 13B show cryotagging is effective at very low temperatures. Cryotagging with hexanoic anhydride is effective in whole cells even at -40°C. Yields are comparable to labeling at room temperature, albeit with a longer reaction time (FIG. 13 A). The preference for lysine is not changed at the different reaction temperatures (FIG. 13B).

[0032] FIGs. 14A and 14B show the cryotagging procedure works equally well on whole E.coli cells as shown with cryotagging reagent pentafluorophenyl trifluoroacetate, and demonstrates the necessity of controlling water content. The presence of water strongly reduces cryotagging efficiency and labeling of whole cells is almost as efficient as labeling denatured, bead-bound proteins (FIG. 14A). The reagent labels free lysines, aspartic acid and glutamic acid, primarily (FIG. 14B). Sensitivity to protein structure is demonstrated as the bead-bound proteome shows less lysine labeling than whole cells because they are bound to the bead surface. This experiment also demonstrates that the bead-based labeling is an effective tool for simulating whole-cell labeling.

DETAILED DESCRIPTION

[0033] After reading this description it will become apparent to one skilled in the art how to implement the present disclosure in various alternative embodiments and alternative applications. However, all the various embodiments of the present invention will not be described herein. It will be understood that the embodiments presented here are presented by way of an example only, and not limitation. As such, this detailed description of various alternative embodiments should not be construed to limit the scope or breadth of the present disclosure as set forth herein.

[0034] Before the present technology is disclosed and described, it is to be understood that the aspects described below are not limited to specific compositions, methods of preparing such compositions, or uses thereof as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. [0035] The detailed description divided into various sections only for the reader’s convenience and disclosure found in any section may be combined with that in another section. Titles or subtitles may be used in the specification for the convenience of a reader, which are not intended to influence the scope of the present disclosure.

Definitions

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0037] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0038] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0039] The term “about” when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by ( + ) or ( - ) 10%, 5%, 1%, or any subrange or subvalue there between. Preferably, the term “about” when used with regard to an amount means that the amount may vary by +/- 10%.

[0040] “Comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0041] The term “covalent labelling” refers to one or more links between any probe, polymer, protein, or biological molecule, where the link is formed by at least one covalent bond. Covalent labeling may be, for example, monovalent labeling or crosslinking. [0042] The term “crosslinking” refers to the use of a probe to link a polymer, protein, or biological molecule to at least a second polymer, protein, or biological molecule, usually by changing the chemical properties of the polymer, protein, or biological molecule. The term “crosslink” refers to the bond between the polymer, protein, or other biological molecule.

[0043] The term “apparatus” refers to machinery or technical equipment for use to perform a particular activity or for a particular purpose.

[0044] The term “nebulizer” refers to an apparatus for use in generating droplets or a mist from a liquid, suspension, or solution.

[0045] The term “plunge freezing” refers to the process of ultra-rapid cooling of a cell or sample by plunging into a cryogen at a cryogenic temperature.

[0046] The term “spray freezing” refers to the process of ultra-rapid cooling of a cell or sample by spraying into a cryogen at a cryogenic temperature.

[0047] The term “self-pressurized freezing” refers to the process of ultra-rapid cooling of a cell or sample contained within a capillary or similar sample holder by plunging into a cryogen at a cryogenic temperature.

[0048] The term “high pressure freezing” refers to the process of ultra-rapid cooling of a cell or sample by plunging into a cryogen at a cryogenic temperature under pressures greater than 2000 bar.

[0049] The term “cryogenic material” or “cryogen” refers to any material or substance used to produce very low temperatures.

[0050] The term “cryogenic temperature” refers to a temperature where all cellular motions and metabolism is effectively stopped or strongly reduced, relative to the timescale of the subsequent chemical processes conducted upon the cell.. One example of a common cryogenic temperature cut-off is about -80°C (the temperature of dry ice, or solid carbon dioxide). A cryogenic temperature range can also be between the temperatures of -20°C to - 100°C. A cryogenic temperature can also be 77 K, the temperature of liquid nitrogen.

[0051] Conceptually, crosslinking mass spectrometry (XL-MS) is a powerful approach to directly identify protein associations in situ. However, current methods have not led to the anticipated abundance of linkages, and the long chemical reactions can undermine the validity of detected protein-protein linkages. Crosslinking proteins in situ is fundamentally limited by reagent hydrolysis in cellular water, forcing the use of stable and slow acting crosslinkers (e.g. NHS esters).

[0052] Cryoelectron microscopy methods developed for ultrastructure preservation displace cellular water with organic solvents containing chemical fixatives, at ultralow temperatures. Thus, ultra-rapid cryo-fixation in tandem with freeze-substitution can be extended to systems- wide XL-MS experiments.

Methods and Systems

[0053] Provided herein are methods, devices, and systems including a spray-freezing device for ultra-rapid cryo-fixation of cells, and a device for freeze-substitution, where MS-friendly labeling agents can be introduced and reacted at cryogenic temperatures. The removal of water allows the user to survey conventional and higher-reactivity compounds (including anhydrides and acyl chlorides moieties), including modified monovalent reagents to profile cell structure preservation, reaction specificity and yield and then select novel crosslinkers on the basis of these profiles. Reaction products can be analyzed through cellular fluorescent microscopy or by bottom-up proteomics methods (e.g., Orbitrap Eclipse nanoLC system). Data analysis can be performed using PEAKS Studio, ProteomeDiscoverer, for example, with label-free quantitation for yield measurements. Analysis of crosslinks can performed using CRIMP 2.0 (Mass Spec Studio), for example.

[0054] In embodiments, is a method of analyzing cellular protein interactions by mass spectrometry, including: freezing a cell by ultra-rapid freezing; contacting the frozen cell with an organic solvent and a chemical tag under conditions to remove cellular water and cause covalent labeling (e.g., crosslinking) of cellular protein by the chemical tag; and analyzing the labeled (e.g., crosslinked) cellular protein by mass spectrometry to determine cellular protein interactions.

[0055] In embodiments, is a method of determining protein interactions within a cell, the method including: freezing a cell by ultra-rapid freezing; contacting the frozen cell with an organic solvent and a chemical tag under conditions to remove cellular water and cause covalent labeling (e.g., crosslinking) of cellular protein by the chemical tag; and analyzing the labeled (e.g., crosslinked) cellular protein by mass spectrometry to determine protein interactions.

[0056] In some embodiments, the chemical tag is an anhydride, acyl chloride, or activated ester. In some embodiments, the chemical tag is an anhydride. In some embodiments, the chemical tag is an acyl chloride. In some embodiments, the chemical tag is an activated ester. [0057] In embodiments, the ultra-rapid freezing is performed by plunge freezing. In embodiments, the ultra-rapid freezing is performed by spray freezing. In embodiments, the ultra-rapid freezing is performed by self-pressurized rapid freezing. In embodiments, the ultrarapid freezing is performed by high pressure freezing. [0058] In some embodiments, plunge freezing is performed by plunging a watercontaining sample or cell into a liquid cryogen in order to freeze a sample at a rapid rate. The rapid rate of freezing during plunge freezing ultimately obtains ice crystals less than 5 nm in size. In some embodiments, the cryogen is ethane. In some embodiments, the cryogen is liquid methane. In some embodiments, the cryogen in liquid propane. In some embodiments, the cryogen is a mixture of liquid ethane and liquid propane. In some embodiments, the cryogen is a mixture of liquid methane and liquid propane. In some embodiments, the plunge freezing is handled by an automatic plunge freezer.

[0059] In some embodiments, spray freezing is performed by spraying a sample or cell into a liquid cryogen with a nebulizer. In some embodiments, the nebulizer sprays into a nonferrous reservoir cooled by a liquid cryogen. In some embodiments, the nebulizer is an air brush. In some embodiments, the nebulizer is an ultrasonic nebulizer. In some embodiments, the nebulizer is attached to the outlet of a flow cytometer cell sorter.

[0060] In some embodiments, self-pressurized rapid freezing is performed by plunge freezing a sample or cell in a sealed capillary tube.

[0061] In some embodiments, high pressure freezing is performed by rapidly freezing a sample or cell under high pressure (greater than 2000 bar). In some embodiments, the formation of ice crystals is prevented and vitreous ice is formed within the sample or cell.

[0062] In some embodiments, the cells are frozen at a rate between of about 1 Kelvin/second (K/s) to about 10000 K/s. In some embodiments, the cells are frozen at a rate between of about 100 Kelvin/second (K/s) to about 10000 K/s. In some embodiments, the cells are frozen at a rate between of about 1000 Kelvin/second (K/s) to about 10000 K/s. In some embodiments, the cells are frozen at a rate of about 10000 K/s. In some embodiments, the cells are frozen at a rate of about 1000 K/s. In some embodiments, the cells are frozen at a rate of about 100 K/s. In some embodiments, the cells are frozen at a rate of about 10 K/s. In some embodiments, the cells are frozen at a rate of about 1 K/s. The rate of cell freezing may be any value or subrange within the recited range, including endpoints.

[0063] In some embodiments, the cells are frozen using a spray freezing device. In some embodiments, the spray freezing device generates droplets that are less than 100 microns in diameter. In some embodiments, the droplets are less than 50 microns in diameter. In some embodiments, the spray freezing device generates droplets that are between about 0.2 microns and about 100 microns in diameter. In some embodiments, the spray freezing device generates droplets that are between about 1 micron and about 100 microns in diameter. In some embodiments, the spray freezing device generates droplets that are between about 10 microns and about 100 microns in diameter. In some embodiments, the spray freezing device generates droplets that are between about 0.1 microns and about 50 microns in diameter. In some embodiments, the spray freezing device generates droplets that are between about 1 micron and about 50 microns in diameter. In some embodiments, the spray freezing device generates droplets that are between about 10 microns and about 50 microns in diameter. The diameter of the droplets may be any value or subrange within the recited ranges, including endpoints.

[0064] In some embodiments, the spray-freezing device includes a cryogenic reservoir; a heat sink within the cryogenic reservoir; a non-ferrous cup within the heat sink, the non-ferrous cup containing the cryogen; and a membrane suspended within the cup, such that when the frozen cells are in contact with the membrane, the cells are immersed in the cryogen. In some embodiments, the membrane is a mesh. In some embodiments, the non-ferrous metal is copper. [0065] In some embodiments, the cryogen is selected from carbon dioxide, nitrogen, oxygen, argon, helium, methane, ethane, propane, or hydrogen. In some embodiments, the cryogen is nitrogen. In some embodiments, the cryogen is ethane. In some embodiments, the cryogen is propane. In some embodiments, the cryogen is carbon dioxide.

[0066] In embodiments, the spray-freezing device further includes a cell reservoir, wherein the cell reservoir contains a cell suspension for freezing; a pressured gas supply; and a channel between the cell reservoir and the pressured gas supply, wherein the cell reservoir and the pressured gas supply are in fluid contact via the channel; wherein the cell suspension contains the cell. In embodiments, the gas in the pressured gas supply includes an inert gas. In embodiments, the inert gas is nitrogen. In embodiments, the gas in the pressured gas supply includes a nitrogen/air mixture.

[0067] In some embodiments, the pressured gas supply is at a pressure of about 5 psi to about 50 psi. In some embodiments, the pressured gas supply is at a pressure of about 10 psi to about 30 psi. In some embodiments, the pressured gas supply is at a pressure of about 5 psi. In some embodiments, the pressured gas supply is at a pressure of about 10 psi. In some embodiments, the pressured gas supply is at a pressure of about 15 psi. In some embodiments, the pressured gas supply is at a pressure of about 20 psi. In some embodiments, the pressured gas supply is at a pressure of about 25 psi. In some embodiments, the pressured gas supply is at a pressure of about 30 psi. In some embodiments, the pressured gas supply is at a pressure of about 35 psi. In some embodiments, the pressured gas supply is at a pressure of about 40 psi. In some embodiments, the pressured gas supply is at a pressure of about 45 psi. In some embodiments, the pressured gas supply is at a pressure of about 50 psi. The pressure may be any value or subrange within the stated ranges, including endpoints. [0068] In embodiments, the cell suspension is aerosolized by contacting the cell suspension with the pressured gas supply. In embodiments, the cell is sprayed onto the membrane by the pressured gas supply. In embodiments, the cell is frozen by contacting the cell aerosol with the cryogen.

[0069] In embodiments, the cryogenic reservoir contains a cryogenic material. In some embodiments, the cryogenic material comprises carbon dioxide, nitrogen, oxygen, argon, helium, or hydrogen. In some embodiments, the cryogenic material is at a temperature below about -80°C.

[0070] In embodiments, the cells are sorted using fluorescent activated cell sorting (FACS) prior to freezing. In embodiments, a FACS nozzle forms droplets of the cell suspension prior to freezing. In embodiments, the FACS nozzle forms droplets between about 10 microns and about 200 microns in diameter. In embodiments, the FACS nozzle forms droplets between about 30 microns and about 100 microns. In embodiments, the FACS nozzle aerosolizes the cell suspension prior to freezing. The diameter of the droplets may be any value or subrange within the recited ranges, including endpoints.

[0071] In embodiments, the cryogenic reservoir is comprised of a heat sink within the cryogenic reservoir; a non-ferrous container within the heat sink, the non-ferrous container containing the cryogen. In embodiments, the non-ferrous container is immersed in a cryogenic material (e.g., liquid nitrogen). In embodiments, the cryogen is stirred. In embodiments, the stirring includes magnetic stirring of the cryogen. In embodiments, the non-ferrous container is a copper container.

[0072] In embodiments, the frozen cells are contacted with an organic solvent. In embodiments, the organic solvent includes a chemical tag. In embodiments, the organic solvent is combined with the frozen cells and the cryogen. In embodiments, the organic solvent/frozen cells/cryogen mixture is warmed such that the cryogen is removed by boiling of the cryogen. In embodiments, the organic solvent/frozen cells/cryogen mixture is warmed above the boiling point of the cryogen. In embodiments, the cells remain frozen in the organic solvent. In embodiments, the organic solvent/frozen cells/cryogen mixture is kept colder than the boiling point of the organic solvent.

[0073] In some embodiments, the frozen cells and organic solvent form a cell slurry. In some embodiments, the volume of the cell slurry is between about 0.01 and 10 ml. In some embodiments, the volume of the cell slurry is between about 0.01 and 5 ml. In some embodiments, the cell slurry volume is between about 4 ml to about 5 ml. In some embodiments, the cell slurry volume is between about 3 ml to about 4 ml. In some embodiments, the cell slurry volume is between about 3 ml to about 2 ml. In some embodiments, the cell slurry volume is between about 2 ml to about 1.5 ml. In some embodiments, the cell slurry volume is between about 1.5 ml to about 1 ml. In some embodiments, the cell slurry volume is between about 1 ml to about 0.9 ml. In some embodiments, the cell slurry volume is between about 0.9 ml to about 0.8 ml. In some embodiments, the cell slurry volume is between about 0.8 ml to about 0.8 ml. In some embodiments, the cell slurry volume is between about 0.7 ml to about 0.6 ml. In some embodiments, the cell slurry volume is between about 0.6 ml to about 0.5 ml. In some embodiments, the cell slurry volume is between about 0.5 ml to about 0.4 ml. In some embodiments, the cell slurry volume is between about 0.4 ml to about 0.3 ml. In some embodiments, the cell slurry volume is between about 0.3 ml to about 0.2 ml. In some embodiments, the cell slurry volume is between about 0.2 ml to about 0.1 ml. In some embodiments, the cell slurry volume is between about 0.1 ml to about 0.09 ml. In some embodiments, the cell slurry volume is between about 0.09 ml to about 0.08 ml. In some embodiments, the cell slurry volume is between about 0.08 ml to about 0.07 ml. In some embodiments, the cell slurry volume is between about 0.07 ml to about 0.06 ml. In some embodiments, the cell slurry volume is between about 0.06 ml to about 0.05 ml. In some embodiments, the cell slurry volume is between about 0.05 ml to about 0.04 ml. In some embodiments, the cell slurry volume is between about 0.04 ml to about 0.03 ml. In some embodiments, the cell slurry volume is between about 0.03 ml to about 0.02 ml. In some embodiments, the cell slurry volume is between about 0.02 ml to about 0.01 ml. The cell slurry volume may be any value or subrange within the recited ranges, including endpoints.

[0074] In embodiments, the chemical tag is at a concentration of about 0 mM to about 100 mM in the organic solvent. In embodiments, the chemical tag is at a concentration of about 0.01 mM to about 100 mM in the organic solvent. In embodiments, the chemical tag is at a concentration of about 1 mM to about 100 mM in the organic solvent. In embodiments, the chemical tag is at a concentration of about 0 mM to about 50 mM in the organic solvent. In embodiments, the chemical tag is at a concentration of about 0.01 mM to about 50 mM in the organic solvent. In embodiments, the chemical tag is at a concentration of about 1 mM to about 50 mM in the organic solvent. In embodiments, the chemical tag is at a concentration of about 100 mM or less in the organic solvent. In embodiments, the chemical tag is at a concentration of about 50 mM or less in the organic solvent. In embodiments, the chemical tag is at a concentration of about 45 mM or less in the organic solvent. In embodiments, the chemical tag is at a concentration of about 40 mM or less in the organic solvent. In embodiments, the chemical tag is at a concentration of about 35 mM or less in the organic solvent. In embodiments, the chemical tag is at a concentration of about 30 mM or less in the organic solvent. In embodiments, the chemical tag is at a concentration of about 25 mM or less in the organic solvent. In embodiments, the chemical tag is at a concentration of about 20 mM or less in the organic solvent. In embodiments, the chemical tag is at a concentration of about 15 mM or less in the organic solvent. In embodiments, the chemical tag is at a concentration of about 10 mM or less in the organic solvent. In embodiments, the chemical tag is at a concentration of about 5 mM or less in the organic solvent. In embodiments, the chemical tag is at a concentration of about 1 mM or less in the organic solvent. The concentration may be any value or subrange within the recited ranges, including endpoints.

[0075] In embodiments, contacting the frozen cell with an organic solvent happens with continuous solvent exchange. In embodiments, contacting the frozen cell with an organic solvent happens with stop-flow solvent exchange.

[0076] In embodiments, a solvent exchanger is used for the solvent exchange.

[0077] In embodiments, the solvent exchanger includes a solvent reservoir containing the organic solvent and the chemical tag, and a reaction chamber, wherein the solvent reservoir and reaction chamber are in fluid communication, such that the organic solvent flows into the reaction chamber; and wherein the solvent reservoir and reaction chamber are kept below freezing.

[0078] In embodiments, the reaction chamber contains the frozen cell(s) and contains less than 5 % (v/v) water to organic solvent. In some embodiments, the reaction chamber contains less than 1% (v/v) water to organic solvent. In some embodiments, the reaction chamber contains about 0% to about 5% (v/v) water to organic solvent. The percentage may be any value or subrange within the recited ranges, including endpoints.

[0079] In embodiments, the solvent reservoir and reaction chamber are kept below the freezing point of water by immersion in a cryogenic material.

[0080] In embodiments, the cryogenic material is contained in a chiller. In some embodiments, the chiller contains solid carbon dioxide (dry ice). In some embodiments, the chiller contains solid carbon dioxide and an organic solvent. In some embodiments, the organic solvent is acetone. In some embodiments, the organic solvent is ethanol. In some embodiments, the organic solvent is methanol. In embodiments, the chiller is maintained at a temperature between about -80°C and about 0°C.

[0081] In embodiments, the heat sink is heated while it is immersed in the cryogenic material. [0082] In embodiments, the solvent exchanger further comprises an inlet for an inert gas. In some embodiments, the inert gas is nitrogen.

[0083] In embodiments, the solvent exchanger further includes a vacuum connected to the reaction chamber.

[0084] In embodiments, the spray freezer contains a cell reservoir; a pressured gas supply; and a channel between the cell reservoir and the pressured gas supply, wherein the cell reservoir and the pressured gas supply are in fluid contact via the channel.

[0085] In embodiments, the spray freezer further contains a cryogenic material in the cryogenic reservoir. In some embodiments, the cryogenic material comprises carbon dioxide, nitrogen, oxygen, argon, helium, or hydrogen.

[0086] In some embodiments, the spray freezer includes a solvent exchanger.

[0087] In embodiments, provided is a system for freezing of cells for analysis by mass spectrometry, the system including a spray freeze apparatus containing a cell solution in the cell reservoir.

[0088] In embodiments, provided is a mass spectrometer containing a composition of cells that are covalently labeled (e.g. crosslinked) by a labeling (e.g. crosslinking) method that includes: freezing a cell by ultra-rapid freezing; contacting the frozen cell with an organic solvent and a chemical tag under conditions to remove cellular water and cause crosslinking of cellular protein by the chemical tag. In some embodiments, the cellular proteins contain a chemical tag post-labeling. In some embodiments, the cellular proteins are extracted from cellular debris. In some embodiments, the cellular proteins are denatured. In some embodiments, the cellular proteins are digested with an enzyme. In some embodiments, the enzyme is trypsin. In some embodiments, the enzyme is pepsin. In some embodiments, enzymatic digestion of the cellular proteins generate peptides. In some embodiments, the digested peptides are further analyzed via LC-MS/MS. In some embodiments, digested peptides contain a chemical tag. In some embodiments, the digested peptides do not contain a chemical tag. In some embodiments, the same peptide within the same sample generated by the labeling method can contain a chemical tag or can be untagged.

[0089] Any cell types may be used in the methods and systems described here, including, without limitation, prokaryotic cells and eukaryotic cells. For example, the cells may be animal cells, plant cells, yeast cells, fungal cells, or protozoan cells. The cells may be archaea cells or bacterial cells. The cells may be mammalian cells, insect cells, etc. In embodiments, the cells are human cells. In embodiments, the cells are a cell line. In embodiments, the cells are yeast cells. Chemical Reactions

[0090] In embodiments, freeze substitution and introduction of chemical tags can be performed at the same time. In embodiments, the freeze substitution may be performed first, followed by addition of the chemical tags. When done sequentially, freeze substitution and addition of chemical tags may be performed at different temperatures, and optionally in different solvents. In embodiments, a freeze substitution may be performed with any solvent at -20°C or lower, for example, at -80°C. In embodiments, the freeze substitution may use short-chain ketones such as acetone, or short-chain alcohols such as methanol or ethanol. [0091] Examples of chemical tagging reagents include two reactive groups, with a variable length spacer between them, said spacer to range from 0 to 40 angstroms in length (e.g., less than 20 angstroms). The spacer length may be any value or subrange within the recited ranges, including endpoints. The spacer could optionally contain a functional group for the introduction or use of an enrichment “handle.” The handle may include affinity tags (such as biotin, FLAG, His-tags, or HA tags) or “click” chemistries (such alkynes or azides) for introducing affinity after installing the crosslinks on the proteins.

[0092] Each chemical tag may contain two reactive groups that can be chemically reacted with protein in non-aqueous conditions at temperatures less than 0 degrees Celsius, invoking accelerants and/or catalysts that would not otherwise be tolerable in aqueous solution reactions. In embodiments, the reaction temperature is less than -20°C, such as less than -40°C. The two reactive groups may be homobifunctional or heterobifunctional.

[0093] The solvents can be selected from solvents typically used in organic chemistry, including but not limited to, acetic acid, acetone, acetonitrile, benzene, 1 -butanol, 2 -butanol, 2-butanone, /-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-di chloroethane, di ethylene glycol, diethyl ether, diglyme (di ethylene glycol dimethyl ether), 1,2-dimethoxy-ethane (glyme, DME), dimethyl -formamide (DMF), dimethyl sulfoxide, 1,4-di oxane, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane methanol, hexamethylphosphoramide (HMPA), hexane methyl /-butyl ether (MTBE), methylene chloride, A-methyl-2-pyrrolidinone (NMP), nitromethane, pentane, petroleum ether (ligroine), 1 -propanol, 2-propanol, pyridine, tetrahydrofuran (THF), toluene, tri ethyl amine, o-xylene, m-xylene, -xylene, or any combination thereof. In embodiments, the organic solvent includes acetic acid. In embodiments, the organic solvent includes acetone. In embodiments, the organic solvent includes acetonitrile. In embodiments, the organic solvent includes benzene. In embodiments, the organic solvent includes 1 -butanol. In embodiments, the organic solvent includes 2-butanol. In embodiments, the organic solvent includes 2- butanone. In embodiments, the organic solvent includes /-butyl alcohol. In embodiments, the organic solvent includes carbon tetrachloride. In embodiments, the organic solvent includes chlorobenzene. In embodiments, the organic solvent includes chloroform. In embodiments, the organic solvent includes cyclohexane. In embodiments, the organic solvent includes 1,2- di chloroethane. In embodiments, the organic solvent includes diethylene glycol. In embodiments, the organic solvent includes diethyl ether. In embodiments, the organic solvent includes diglyme (diethylene glycol dimethyl ether). In embodiments, the organic solvent includes 1,2-dimethoxy-ethane (glyme, DME). In embodiments, the organic solvent includes dimethyl-formamide (DMF). In embodiments, the organic solvent includes dimethyl sulfoxide. In embodiments, the organic solvent includes 1,4-di oxane. In embodiments, the organic solvent includes ethanol. In embodiments, the organic solvent includes ethyl acetate. In embodiments, the organic solvent includes ethylene glycol. In embodiments, the organic solvent includes glycerin. In embodiments, the organic solvent includes heptane methanol. In embodiments, the organic solvent includes hexamethylphosphoramide (HMPA). In embodiments, the organic solvent includes hexane methyl /-butyl ether (MTBE). In embodiments, the organic solvent includes methylene chloride. In embodiments, the organic solvent includes A-methyl-2-pyrrolidinone (NMP). In embodiments, the organic solvent includes nitromethane. In embodiments, the organic solvent includes pentane. In embodiments, the organic solvent includes petroleum ether (ligroine). In embodiments, the organic solvent includes 1 -propanol. In embodiments, the organic solvent includes 2- propanol. In embodiments, the organic solvent includes pyridine. In embodiments, the organic solvent includes tetrahydrofuran (THF). In embodiments, the organic solvent includes toluene. In embodiments, the organic solvent includes triethyl amine. In embodiments, the organic solvent includes o-xylene. In embodiments, the organic solvent includes m-xylene. In embodiments, the organic solvent includes /?-xylene. Any one or more of the listed solvents may be expressly excluded.

[0094] In embodiments, the chemical tags can be any moiety that can covalently couple to protein in an irreversible manner under the stated conditions of temperature and solubility in the organic solvent.

[0095] In some embodiments, the chemical tag is homobifunctional. In some embodiments, the chemical tag is selected from one or more of compounds 1 - 6, or a derivate thereof. In some embodiments, the chemical tag is compound 1. In some embodiments, the chemical tag is compound 2. In some embodiments, the chemical tag is compound 3. In some embodiments, the chemical tag is compound 4. In some embodiments, the chemical tag is compound 6.

[0096] In some embodiments, the chemical tag is heterobifunctional. In some embodiments, the chemical tag is selected from one or more of compounds 7-9, or a derivative thereof. In some embodiments, the chemical tag is compound 7. In some embodiments, the chemical tag is compound 8. In some embodiments, the chemical tag is compound 9.

[0097] In some embodiments, the chemical tag is heterobifunctional. In some embodiments, the chemical tag is selected from one or more of compounds 7-9, or a derivative thereof. In some embodiments, the chemical tag is compound 7. In some embodiments, the chemical tag is compound 8. In some embodiments, the chemical tag is compound 9.

[0098] In some embodiments, the chemical tagging reaction is a two-step process. In some embodiments, a diamine is used in the two-step chemical tag labeling reaction. In some embodiments, the diamine is selected from one or more of compounds 10-11, or a derivative thereof. In some embodiments, the chemical tag is compound 10. In some embodiments, the chemical tag is compound 11.

[0099] In some embodiments, the chemical tagging reaction is photoreactive process. In some embodiments, the chemical tag is selected from one or more of compounds 12-15, or a derivative thereof. In some embodiments, the chemical tag is compound 12. In some embodiments, the chemical tag is compound 13. In some embodiments, the chemical tag is compound 14. In some embodiments, the chemical tag is compound 15.

[0100] In embodiments, the structures of chemical tags, compounds 1 - 15, comprise the following structures:

[0101] In embodiments, the reactive groups can be the same or different. In embodiments, the reactive groups can be installed concurrently or sequentially. [0102] In embodiments, the first reactive groups can react with any amino acid, such as with the more reactive amino acids: lysine, arginine, aspartic acid, glutamic acid, histidine, cysteine and tyrosine. In embodiments, the first reactive groups can react with the N-terminus of a polypeptide. In embodiments, the first reactive groups can react with the C- terminus of a polypeptide. In embodiments, the reactive groups may target lysine using acylation or alkylation reactions, such as involving isothiocyanates, isocyanates, acyl azides, NHS (H-hydroxysuccinimide) esters, sulfonyl chlorides, epoxides, carbonates, fluorophenyl esters, fluorobenzene derivatives, imidoesters, carbodiimide-activated carboxylates, acid chlorides, and/or anhydrides. Any one or more of the listed groups may be expressly excluded.

[0103] In embodiments, the first reactive group can be applied in a two-step process. In embodiments, the carboxylic acid on glutamate and aspartate residues is preactivated. In some embodiments, a diazomethane or diazoacetyl reagent is used. In some embodiments, a carbonyldiimidazole (CDI) is used to conjugate a carboxylic acid to a primary amine. In some embodiments, a carbodiimide is used to crosslink a carboxylic acid to a primary amine. In some embodiments, the carbodiimide is selected from l-ethyl-3-(3- dimethylaminopropyljcarbodiimide (EDC) or dicyclohexylcarbodiimide (DCC).

[0104] In embodiments, the second reactive group can react with any amino acid (as above) and specifically also include photoactivation chemistry, such as diazirines, diazo compounds, aryl azides, and benzophenones.

[0105] In embodiments, the crosslinking reaction can be a photo-initiated chemical reactions. In some embodiments, the crosslinking reagent is a photoreactive group. In some embodiments, the photoreactive group can be selected from phenyl azide, ortho-phenyl azide, meta-hydroxyphenyl azide, tetrafluorophenyl azide, ortho-nitrophenyl azide, metanitrophenyl azide, diazirine, azido-methyl coumarin, and/or psoralen. In some embodiments, the photoreactive group is phenyl azide. In some embodiments, the photoreactive group is ortho-phenyl azide. In some embodiments, the photoreactive group is meta-hydroxyphenyl azide. In some embodiments, the photoreactive group is tetrafluorophenyl azide. In some embodiments, the photoreactive group is ortho-nitrophenyl azide. In some embodiments, the photoreactive group is meta-nitrophenyl azide. In some embodiments, the photoreactive group is diazirine. In some embodiments, the photoreactive group is azido-methylcoumarin. In some embodiments, the photoreactive group is psoralen.

[0106] In embodiments, the concentrations of the chemical tags may be between 0.01 and 100 mM, such as between 0 and 10 mM, and such as between 0.01 and 1 mM. [0107] In embodiments, the reaction time can range from 1 min to 48 hours, more preferably from 1 min to 1 hour, and still more preferably from 1 min to 10 min.

[0108] In embodiments, the accelerants can be used to increase the rate of the chemical reactions for targeted amino acids under low-temperature, nonaqueous conditions. Non-limiting examples include: organic acids, organic bases, and catalysts for “click” chemistry. Any one or more of the listed accelerants may be expressly excluded.

[0109] In embodiments, leaving groups are installed on amino acids to increase reactivity. Leaving groups include, without limitation, carbodiimides, aminium/uronium and phosphonium salts, propanephosphonic acid anhydride for the activation of carboxylic acids. Any one or more of the listed groups may be expressly excluded.

[0110] In embodiments, protection groups are used to limit reactivity of non-targeted reaction groups on amino acid side chains, such as targeted esterification of carboxylic acids to restrict their reactivity during coupling to lysines.

[OHl] In embodiments, the crosslinking reagents include, but are not limited to: MDS (m- maleimidobenzoyl-N-hydroxysuccinimide ester), GMBS (N-y- maleimidobutyryloxysuccinimide ester), EMCS (N-(s-maleimidocaproyloxy) succinimide ester), sulfo-EMCS (N-(s-aleimidocaproyloxy) sulfo succinimide ester), aryl-azides ((N-((2- pyridyldithio)ethyl)-4-azidosalicylamide), ANB-NOS (N-5-Azido-2- nitrobenzyloxysuccinimide) and sulfo- SANP AH), diazirines, disuccinimidyl suberate (DSS), disuccinimidyl tartrate (DST), dithiobis succinimidyl propionate (DSP), BMOE, DTME, isocyanate, pyridyldisulfide, thiosulfonate, vinyl sulfonate, maleimide, carbodiimide, NHS esters, imidoesters, pentafluorophenyl ester, hydroxymethyl phosphine, hydrazine, alkoxyamine, haloacetyls, pyridyl disulfides, Staudinger reagent pairs, SIA, SBAP, SIAB, Sulfo-SIAB, AMAS, BMPS, GMBS, Sulfo-GMBS, MBS, Sulfo-MBS, SMCC, Sulfo-SMCC, EMCS, SulfoEMCS, SMPB, Sulfo-SMPB, SMPN, LC-SMCC, Sulfo-KMUS, SPDP, LC- SPDP, Sulfo-LC-SPDP, SMPT, PEG4-SPDP, PEG12-SPDP, formaldehyde, FAMD-diazirine, or NHS-FAM. In embodiments, the crosslinking reagent is ethylenediaminetetraacetic dianhydride. Any one or more of the listed reagents may be expressly excluded.

[0112] In an embodiment, crosslinking is performed in a two-step reaction. In embodiments, the reaction includes pre-activation with a carboxylic acid-targeting agent (e.g., pentafluorophenyl trifluoroacetate) to create a mixed anhydride in the proteome. In embodiments, this is then reacted with simple diamines in a crosslinking step, or internally quenched with a nearby free amine somewhere else in the proteome to create a zero-length crosslink. Exemplary embodiments are shown in FIGs. 12A-12C. Compositions

[0113] In embodiments, provided is a composition of covalently labeled (e.g., crosslinked) cells, wherein the covalent labeling (e.g., crosslinking) method includes: freezing a cell by ultra-rapid freezing; contacting the frozen cell with an organic solvent and a chemical tag under conditions to remove cellular water and cause covalent labeling (e.g., crosslinking) of cellular protein by the chemical tag. In embodiments, the covalent labeling (e.g., crosslinking) method includes one or more of the methods described herein.

[0114] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

EXAMPLES

[0115] One skilled in the art would understand that descriptions of making and using the particles described herein is for the sole purpose of illustration, and that the present disclosure is not limited by this illustration.

Example 1: Embedding MS-compatible Cryotags Into Cells Using Freeze-Substitution

[0116] We tested our method of in situ labeling using crosslinker-mimicking monovalent reagents, to explore the feasibility of coupling under the conditions imposed by freezesubstitution. Initial evaluation was performed on human A549 cells frozen in liquid nitrogen and freeze-substituted with a fluorescein-conjugated diazirine compound in methanol at -80°C, followed by irradiation at 355 nm, to test for penetrance and cell structure preservation. Fluorescence imaging and immunoprecipitation of fluorescein conjugated biomolecules (followed by LC-MS/MS) indicated that the method of freeze-substitution could couple reactive compounds into the proteome in an unbiased manner and preserve cellular structure. Assessment of freeze-substituted N-hydroxysuccinimide ester reactivity under cryogenic temperatures revealed low coupling at 50 mM, around 0.4% of the proteome at -20°C for 48 hours. Anhydride reagents vastly outperformed the NHS-ester at -20°C, supporting 63% labelling of the proteome at 50 mM and 48 hours, and coupling almost exclusively to lysines. Labeling reduced to 0.9% at -80°C and 48 hours incubation, but could be driven to 40% with high reagent concentrations. Acyl chlorides could be uses as an alternative, supporting coupling at -80°C and reduced reactions times (minutes), while retaining high specificity for lysines.

[0117] A new exchanger device can be used to maintain a dry reaction environment and achieve repetitive/integrative labelling with lower crosslinker concentrations, based on anhydride and acyl chloride reagents. The data herein point to the success of this approach, indicating that highly selective crosslinking reactions can be achieved in the vitrified state with high yield.

[0118] To determine the possibility of embedding MS-compatible crosslinkers (which are larger than traditional fixatives and embedding agents) into cells using freeze- substitution, fluorescent probes were designed to contain reactive groups capable of covalently labeling proteins. In the first instance, a diazirine-substituted fluorescein was synthesized and introduced into human A549 cells that were plunge-frozen in liquid nitrogen (LN2), using the freeze-substitution process. Briefly, 0.5 mM solution of the reagent in -80°C methanol was incubated with the cells over a 24-hour period, followed by a photolysis process to convert the diazirine into a protein-reactive functional group. Fluorescence imaging demonstrated that the reagent could penetrate the cell and label contents in an unbiased manner (FIGS. 1A and IB), the latter confirmed by a proteomics analysis.

[0119] We next sought to determine if alternative solution chemistries could be used to covalently couple a fluorescent molecule into the cell. Here, we synthesized NHS-FAM to target free lysines. After freeze substitution in -80°C methanol to introduce the reagent, the samples were incubated at three different temperatures, showing virtually no reactivity at - 80°C, modest reactivity at -20°C and marginally higher reactivity at room temperature (FIGS. 2A and 2B) This result shows that, while labeling can occur in a water-free environment, the yields are modest and strongly dependent upon temperature.

[0120] Next, we explored other, more reactive, labeling chemistries in place of NHS esters, to allow for covalent labeling reactions under colder conditions. There are a range of options that can target free amines, a functional group for XL-MS. Virtually all reagents used in protein biochemistry involve either aldehydes, carbodiimides, isocyanates, imidoesters, sulfonyl chloride, fluorophenyl esters or the NHS esters, given their relative stability in water. However, we selected anhydrides and acyl chlorides, reagents that are not stable in water but much more reactive to amines than any of the common reagents.

[0121] First, we chose to freeze-substitute A549 cells with propionic anhydride at - 80°C, using acetone, and then performed the reactions under various conditions (FIG. 3). This experiment shows that extremely high labeling of the proteome can be achieved even at -80°C. It is also advantageous to first remove cellular water, and add a base to catalyze the reaction (at least at the lower reaction temperatures). Similar results were obtained with lower reagent concentrations using even more amine-reactive reagents like acyl chlorides. It is important to emphasize that neither of these reagent classes would work in conventional XL-MS experiments as water would hydrolyze the reagents before they could react with protein. We note further that the reactivity profile of both anhydrides and the acyl chlorides were very similar to the classical NHS esters (i.e. strongly favoring free amines). This makes detection in the proteome straightforward, and indicates their usefulness in crosslinking reagents.

[0122] Next, we developed a method for preparing larger volumes of cells using a modified spray freezing method, where suspensions of cells were aerosolized into a specially designed cryogenic chamber (FIG. 4A). This process is effective at generating small droplets prior to impinging on the cryogen, to generate ultra-rapid freezing rates necessary for vitrification. This procedure can be coupled with a flask of suspension cells, or mated with a specialized adapter to a fluorescent-activated cell sorter (FACS) instrument, to freeze only selected populations of cells.

[0123] The frozen cell droplets are then simply introduced into a specialized solvent exchanger (FIG. 5). We have determined that, for effective XL-MS crosslinking reactions, the reagent works well when introduced at low concentrations (<50 mM). Therefore, an exchanger is used to introduce successive solutions of fresh reagent. In some embodiments, this approach is critical, as it allow for integration of more crosslinked product to ensure that a suitable yield is obtained. That is, control over the crosslinking yield is required in order to effectively sample the interactome.

[0124] FIGS. 1A and IB show a freeze substitution to introduce FAMD-diazirine into human A549 cells. FIG. 6A shows the preservation of structure, followed by photolytic coupling using 355 nm UV light. Negative: no reagent used in freeze substitution. Photolysis generated fluorescein-protein conjugates (green) showing labeling throughout the cell. DAPI labeled DNA (blue) marks the nucleus. P-W: photolysis of entrained FAMD-diazirine, followed by wash-out of uncoupled reagent using -80°C acetone. W-P: washout followed by photolysis. W: washout. Scale bar=20 pm. FIG. 2B is the structure of FAMD-diazirine.

[0125] FIGS. 2A and 2B shows a freeze substitution at -80°C to introduce 0.5 mM NHS-FAM into human A549 cells with preservation of structure, followed by fixation in methanol to support imaging, at various temperatures. Fluorescence images (FIG. 2A) demonstrate increased labeling at higher reaction temperatures (after freeze substitution), supported by quantification of labeling (FIG. 2B). Scale bar=20 pm.

[0126] FIG. 3 shows covalent labeling of human A549 cells under freeze-substitution conditions with propionic anhydride. Three reaction temperature were profiled, and the addition of a base catalyst in freeze substitution (here triethylamine) was explored. We determined if a freeze-substitution step using neat acetone prior to introducing the propionic anhydride (“pre-desiccation”) conferred any advantage. Quantitation was by proteomics, using mass spectrometry to detect what fraction of the whole proteome was modified with the reagent or not.

[0127] FIGS. 4A and 4B show an example spray freeze apparatus for ultrafast cryogenic cooling of cells in liquid ethane (FIG. 4A) and the effect of pressure on droplet size (FIG. 4B) The device provides control over the size of the droplets containing the cells, based on gas pressure (right, scale in microns, size expressed as diameter).

[0128] FIG. 5 shows an example solvent exchanger for freeze- substitution, based on a fixed temperature design using dry ice (-80°C), allowing for both continuous and stop-flow solvent exchange in a water-free design.

[0129] FIG. 6 shows a schematic for an example freeze substitution device.

Example 2. Cryotagging process: Fast Freezing of Cells

[0130] The goal of fast freezing of cells is to achieve cooling rates approaching 10,000K/s to preserve cell and protein structure, which can be achieved by spraying droplets with sizes of approximately 50 micron in diameter. Alternatively, ultrarapid plunge freezing is another option for fast cell freezing, along with high-pressure freezing, which drops the required freezing rate, to allow regular rates of freezing ~1000K/s.

[0131] We have achieved sufficiently fast spray freezing with a simple air-brush (FIG. 8A). The air brush can be replaced with any sort of nebulizer, not just pressurized gas. For example, acoustic waves can be used to generate droplets. FIGs. 8A-8B and 9 show that gas nebulization can efficiently spray freeze type of suspended cells. We used E. coli for the cells in FIG. 9, and show that our droplet size is independent of the cell density that we spray. Droplets (containing a red dye) were sprayed into oil to prevent evaporation and allow measurement (FIG. 8B). As the graph in FIG. 9 shows, we meet our size targets for fast freezing.

Example 3. Cryotagging process: Desiccation

[0132] A solvent exchanger is used to control the whole cryotagging process. Dimensions are entirely flexible, but the solvent exchanger allows us to capture slurries up to ~2 ml in volume. This provides an adequate amount of cells for downstream analysis by proteomics. We make a slurry of the frozen cells in an ultracold solvent like acetone (-80°C) and then add this slurry to the solvent exchanger. [0133] It is critical to keep the cells frozen the whole time, from transfer to desiccation. The flow cell holds a membrane through simple compression that can tolerate cold temperatures and organic solvents, and has a porosity small enough to retain cells. Ideal membranes are nylon filters (0.2 micron porosity) or PTFE membranes (0.2-1 micron porosity). Any sort of solvent exchanger could work, including an aspiration-based device. The device can further contain a flow cell, for connecting it to an autosampler and a solvent delivery system (like an HPLC) to infuse ultracold organic solvents like acetone, ethanol, methanol, or any solvent that can (1) dissolve frozen water and (2) remain liquid at cryogenic temperatures, which is defined as anything between -20°C and -100°C. An exemplary temperature for desiccation is -80°C.

Example 4. Cryotagging process: Freeze Substitution

[0134] An HPLC system for solvent management gives us the ability to inject the cryotagging reagent. After desiccation we can introduce alternative solvents, or even blends of solvent (including small percentages of water, up to ~5%) that may be better for solvating the cryotag.

[0135] The introduction of mixed solvents is important for preserving protein structure and preparing the frozen cells for labeling. That is, if we don’t have the right formulation, then the reagents will just aggregate with the protein and not diffuse throughout the sample. The formulation may be mixed solvents such as low concentrations of water, DMF, or DMSO in acetone. One example would be 5% water in acetone, which promotes the solubilization of protein side chains at their surfaces and free diffusion of the cryotagging reagents. A less favorable solvent blend would be, for example, 50% DMF in acetone at zero degrees, as this blend may precipitate the proteins and destroy both cellular and protein structures.

[0136] The system allows for one-time injections, multiple repeat injections or even continuous infusion of the cryotag/crosslinker. Multiple or continuous injection allows us to “integrate” the cryotag/crosslinker, that is, build up more reaction products. This can be necessary if the yield is low from a single injection. For continuous injection, the cryotag would simply be added to the HPLC solvent, rather than injected in-line. The flow cell can be connected to waste, but also to any analytical device (e.g., UV-Vis or mass spectrometer) to monitor reagent introduction efficiency and reactivity.

[0137] Further, the solvents from the HPLC are dynamically cooled in a chamber that holds the flow cell.

Example 5. Cryotagging process: Chemical Reaction [0138] The flow cell can be placed in a programmable chiller, which allows control of the temperature from -80°C to RT, for periods of minutes to hours. The flow cell also optionally contains a “window” (an optically transparent port) that allows for the introduction of light. This light can be used to achieve photo-initiated chemical reactions with the right sort of reagent at any temperature regime, but especially at ultracold temperatures.

[0139] When introduced in this fashion, a variety of chemistries can be tolerated, over a range of temperatures (FIG. 10), here represented by covalent labeling with simple “monovalent” cryotags targeting lysine side chains (i.e., one half of a crosslinker cryotag). The freeze substation process also allows for reaction additives like bases to increase reactivity.

[0140] This also works for crosslinking, as demonstrated in the example of FIG. 11 using a cyclic anhydride. Any chemistry that works under cryogenic conditions can be used in cryotagging, for example, amine-directed tags target primary amines (lysine side chains and the N-terminus of a polypeptide). As a further example, a two-step reaction that preactivates carboxylic acids (on glutamic acid and aspartic acid) works as well (FIG. 12A).

Example 6. Whole cell tagging of E. coli.

[0141] The bead-bound cryotagging process was as depicted in FIG. 10. For whole-cell labeling, an apparatus was used. Briefly: E. coli DH5a (pUC19) cells were collected and washed 3X in 1XPBS at 4°C. Using optimal spray conditions (16cm distance from ethane, 25psi gas pressure on air brush), E. coli cell suspension was spray-frozen for 120 seconds into liquid ethane, in 10 second bursts.

[0142] Ethane was evaporated at -80°C and sample transferred to cold acetone, also at -80 °C, then decanted into an equally cold flow cell for desiccation and freeze substitution. Anhydrous acetone at a rate of 0.100 mL/min was infused at -80°C overnight for desiccation. 45pL of cryotagging solution (500 mM coupling reagent and 1 M triethylamine in neat acetone) was injected, for a labeling concentration of lOmM reagent and 20 mM base.

[0143] Flow was stopped and mixture incubated for Ihr at RT. After incubation, cells were washed in cold acetone at 0.5mL/min and residual reagent quenched with excess ethanolamine in methanol. Organic solvent was removed and the protein digested overnight with trypsin, for analysis by LC-MS/MS on an Orbitrap Eclipse. Labeling levels were measured by PEAKS studio using E. coli proteome as database. The results comparing cryotagging using bead-based labeling (Example 4, below) vs. whole cell labelling are shown in FIGs. 14A and 14B. The results show the differences in efficiency and labelling preference in the presence of ~5% water in acetone vs. anhydrous acetone for both whole cell and bead-based cryotagging.

Example 7. Labelling of E. coli lysate. [0144] The experiment was conducted using a simplified version of the workflow, to rapidly explore different chemistries.

[0145] E. coll cells were denatured and total cellular protein captured on cation exchange resin (SP3 beads), approximately the same size as cells, for ease of handling and to present standard reaction conditions. E. coll lysate was reduced and alkylated with lOmM DTT and 80 mM CAA, respectively. Lysate loaded onto SP3 beads and washed. Beads transferred to glass vial.

[0146] 2mL cryotagging solution was added to beads under anhydrous conditions. lOmM of cryotagging reagent: Propionyl NHS-ester, Propionyl chloride, Propionic anhydride, and Pentafluorophenyl trifluoroacetate (20mM triethylamine was added for Propionyl NHS- ester, Propionic anhydride and Pentafluorophenyl trifluoroacetate).

[0147] Samples (n=3 for each condition) were reacted at RT for Ihr, -20°C for 18 hours, or -80°C for 48 hours.

[0148] Beads were collected and washed.

[0149] Bound and labeled proteins were digested overnight with trypsin.

[0150] LC-MS/MS data were acquired on Orbitrap Eclipse Mass spectrometer. Labeled proteins were quantitated with PEAKS studio using E. coll proteome as the database. The percent labeling for four exemplary cryotags in shown in FIG. 10.

Example 8. Air brush droplet size distribution range determination using E. coli.

[0151] An experiment to determine a droplet size distribution range was performed using E. coli cells. E. coli DH5a (pUC19) grown O/N in 2YT + AMP100 at 37°C 200rpm. Cells collected and washed 3X in 1XPBS at 4°C, then centrifuged at 5000rpm for 10 minutes. [0152] Cells pooled into stock tube after final wash. An ODeoo was obtained for the following cell suspensions: Blank (no cells), Undiluted, 1/2 dilution, 1/3 dilution, 1/4 dilution, 1/5 dilution, and a 1/10 dilution.

[0153] Cell suspensions were connected to airbrush, the airbrush was set at 16cm above beaker containing neutral, optically clear mineral oil with gas pressure set at 25psi. The cell suspensions were sprayed for 10 seconds into oil. Droplets observed under dissection microscope. Droplets were analyzed in ImageJ with scale set to 5mm representing the whole field of view. Results are shown in FIG. 9.

[0154] In-focus droplets (n > 20) were measured for diameter.

Example 9. Air brush as a spray freezing apparatus for the ultra-rapid cryofixation of cells for cryotagging. [0155] An airbrush was connected to compressed nitrogen gas cylinder, pressure of gas set at 25psi. The airbrush is set 16cm above nylon mesh placed in copper flask. The copper flask contained liquid ethane, cooled with liquid nitrogen (FIG. 8A).

[0156] A solution of red food dye was prepared and connected to a small airbrush. The airbrush was set at 16cm above a beaker containing neutral, optically clear mineral oil and sprayed for 10 sec at 25 psi. Droplets observed with dissection microscope and images taken (FIG. 8B).

Claims

WHAT IS CLAIMED IS:

1. A method of analyzing cellular protein interactions by mass spectrometry, comprising: a) freezing a cell by ultra-rapid freezing; b) contacting the frozen cell with an organic solvent and a chemical tag under conditions to remove cellular water and cause crosslinking of cellular protein by the chemical tag; c) analyzing the crosslinked cellular protein by mass spectrometry to determine cellular protein interactions.

2. A method of determining protein interactions within a cell, the method comprising: a) freezing a cell by ultra-rapid freezing; b) contacting the frozen cell with an organic solvent and a chemical tag under conditions to remove cellular water and cause crosslinking of cellular protein by the chemical tag; c) analyzing the crosslinked cellular protein by mass spectrometry to determine protein interactions.

3. The method of claim 1 or 2, wherein the crosslinking reagent comprises a reagent that can be reactive at subzero (0°C) temperatures.

4. The method of claim 3, wherein the crosslinking reagent comprises a spacer.

5. The method of claim 3 or 4, wherein the crosslinking reaction is sequential or concerted.

6. The method of claim 5, wherein the crosslinking reagent is homobifunctional.

7. The method of claim 5, wherein the crosslinking reagent is heterobifunctional.

8. The method of claim 1 or 2, wherein the chemical tag is an anhydride, acyl chloride, or activated ester.

9. The method of any one of claims 1 to 3, wherein the cell is frozen using a technique selected from spray-freezing, plunge freezing, self-pressurized rapid freezing, or high pressure freezing.

10. The method of claim 9, wherein the cell is aerosolized before freezing.

11. The method of claim 10, wherein the cell is frozen in a droplet less than 100 microns in diameter.

12. The method of claim 11, wherein the cell is frozen in a droplet less than 50 microns in diameter.

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13. The method of any one of claims 1 to 12, wherein the cell is frozen using a sprayfreezing device, ultrasonic waves, nebulizer, or microfluidic device

14. The method of claim 13, wherein the cell is frozen using a spray-freezing device.

15. The method of claim 14, wherein the spray freezing device is an air sprayer.

16. The method of any one of claims 1 to 15, wherein the cell is frozen at a rate of at least 1000 K/s.

17. The method of claim 16, wherein the cell is frozen at a rate of about 10,000 K/s.

18. The method of claim 14, the spray-freezing device comprising a cell reservoir, wherein the cell reservoir contains a cell suspension for freezing; a nitrogen/air supply; and a channel between the cell reservoir and the nitrogen/air supply, wherein the cell reservoir and the nitrogen/air supply are in fluid contact via the channel; wherein the cell suspension comprises the cell.

19. The method of claim 14, wherein the spray-freezing device comprises a cryogenic reservoir; a heat sink within the cryogenic reservoir; a non-ferrous container within the heat sink, the non-ferrous container containing the cryogen; and a membrane suspended within the non-ferrous container, such that when the frozen cells are in contact with the membrane, the cells are immersed in the cryogen.

20. The method of claim 18 or 19, wherein the nitrogen/air supply is at a pressure of about 10 psi to about 30 psi.

21. The method of any one of claims 1 to 20, wherein the cell is aerosolized into droplets by contacting the cell suspension with the nitrogen/air supply.

22. The method of any one of claims 19 to 21, wherein the cryogenic reservoir contains a cryogenic material.

23. The method of any one of claims 1 to 22, wherein the cell is frozen by contacting the droplet containing one or more cells with a cryogen.

24. The method of claim 23, wherein the cryogen comprises short chain hydrocarbons precooled by a cryogenic material.

25. The method of claim 24, wherein the short chain hydrocarbon is selected from ethane or propane.

26. The method of any one of claims 18 to 25, wherein the frozen cell droplets are sprayed onto the membrane by the nitrogen/air supply.

27. The method of claim 26, wherein the membrane comprises a mesh.

28. The method of any one of claims 1 to 27, wherein the cells are sorted using fluorescent activated cell sorting (FACS) prior to freezing.

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29. The method of claim 28, wherein the FACS is performed in a FACS machine, the FACS machine comprising a nozzle, wherein the nozzle produces droplets having a diameter less than 100 microns.

30. The method of any one of the above claims, wherein the cryogen is stirred during the freezing step.

31. The method of claim 19, wherein the cryogenic reservoir is comprised of a heat sink within the cryogenic reservoir; a non-ferrous container within the heat sink, the non-ferrous container containing the cryogen immersed in liquid nitrogen with stirring of the cryogen.

32. The method of any one of claims 13 - 31, wherein the frozen cells and cryogen mix to form a cell slurry.

33. The method of claim 32, wherein the cell slurry is transferred from the cryogenic reservoir to a freeze substitution device under cryogenic temperatures.

34. The method of claim 33, wherein the cryogenic reservoir and freeze substitution device are linked.

35. The method of claim 34, wherein the cryogenic reservoir and freeze substitution device are in fluid communication within the same larger device or system.

36. The method of any one of claims 1 - 35, wherein the chemical tag is added with a freeze substitution device, the device comprising: an in-line inlet tube in fluid contact with an autosampler; an autosampler for adding a chemical tag in fluid contact with an organic solvent pump and in-line inlet tube; a downstream chiller and temperature control chamber surrounding the in-line inlet tube originating from the autosampler and in fluid contact with the organic solvent pump; wherein the in-line inlet tubing is in fluid contact with a reaction chamber located within the chiller and temperature control chamber; wherein the reaction chamber comprises a porous membrane capable of retaining frozen cells; wherein the in-line inlet becomes the outlet tube downstream from the reaction chamber; and; wherein the applied fluid from the organic solvent pump and autosampler flows in one direction from the inlet to outlet direction.

37. The method of claim 36, wherein the chemical tag is added in the reaction chamber.

38. The method of claim 37, wherein the chemical tag selected from compounds 1 - 15.

39. The method of any one of claims 1 to 38, wherein the chemical tag is at a concentration of about 50 mM or less in the organic solvent.

40. The method of any one of claims 1 to 39, wherein step b) comprises continuous solvent exchange.

41. The method of any one of claims 1 to 40, wherein step b) comprises stop-flow solvent exchange.

42. The method of claim 40 or 41, wherein a solvent exchanger is used for the solvent exchange.

43. The method of claim 42, wherein the solvent exchanger comprises a solvent reservoir containing the organic solvent and the chemical tag, and a reaction chamber, wherein the solvent reservoir and reaction chamber are in fluid communication, such that the organic solvent flows into the reaction chamber; and wherein the solvent reservoir and reaction chamber are kept below freezing.

44. The method of claim 43, wherein the reaction chamber contains the frozen cell and contains less than 5% (v/v) water to organic solvent.

45. The method of claim 44, wherein the reaction chamber contains the frozen cell and contains less than 1% (v/v) water to organic solvent

46. The method of any one of claims 43 to 45, wherein the solvent reservoir and reaction chamber are kept below freezing by immersion in a cryogenic material.

47. The method of claim 46, wherein the cryogenic material comprises solid carbon dioxide, carbon dioxide, nitrogen, oxygen, argon, helium, or hydrogen.

48. A spray freeze apparatus comprising: a cryogenic reservoir; a heat sink within the cryogenic reservoir; a copper container within the heat sink, the copper container containing a cryogen; and a membrane suspended within the copper container, such that when frozen cells are in contact with the membrane, the frozen cells are immersed in the cryogen.

49. The spray freeze apparatus of claim 48, further comprising a cell reservoir; a nitrogen/air supply; and a channel between the cell reservoir and the nitrogen/air supply, wherein the cell reservoir and the nitrogen/air supply are in fluid contact via the channel.

50. The spray freeze apparatus of claim 49, wherein the membrane is a mesh.

51. A system for freezing of cells for analysis by mass spectrometry, the system comprising a spray freeze apparatus of claim 50 having a cell solution in the cell reservoir.

52. The system of claim 51, further comprising a cryogenic material in the cryogenic reservoir.

53. The system of claim 52, wherein the cryogenic material comprises carbon dioxide, nitrogen, oxygen, argon, helium, or hydrogen.

54. The system of any one of claims 50 to 52, further comprising a solvent exchanger.