Methods for analyzing membrane proteins

Abstract

This disclosure relates to a method for analyzing a sample containing intact membrane proteins in a sample containing non-synthetic biomembrane material. The method involves irradiating a gas-phase proto-membrane protein ion with infrared radiation from a laser source.

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing a sample containing non-synthetic biomembrane material, specifically a sample containing intact membranes or membrane-associated proteins, or aggregates thereof, by mass spectrometry. [Background technology]

[0002] Membrane proteins are involved in a wide range of cellular processes, including cell-cell adhesion, signal transduction, and solute flux. It is well established that the structure and function of membrane proteins can be regulated by binding to other molecules such as extracellular ligands, intramembrane lipids, and cytosolic proteins. Native mass spectrometry (nMS) provides a powerful tool for studying the relationships between these binding events, as they result in distinct changes in the composition of protein complexes that can be elucidated using mass spectrometry.

[0003] Numerous studies have been conducted to understand the structure, function, and activity of isolated proteins and model systems such as idealized model membrane proteins. For example, to study membrane protein-ligand interactions by nMS, membrane proteins or complexes are typically solubilized in membrane mimics before purification. In this specification, the term “protein” (e.g., “membrane protein”) includes protein complexes, such as protein complexes containing multiple associated polypeptide chains, or proteins that form complexes with one or more covalent and / or non-covalent ligands. Membrane proteins (or protein complexes) can be encapsulated in detergent micelles (or other suitable mimics such as lipid nanodiscs) and then delivered into the gas phase via nanoelectrospray ionization. After entering the gas phase, numerous nonspecifically bound detergent molecules must be removed (referred to as micelle removal or demicellement) to facilitate mass spectrometry of the membrane protein or complex under study.

[0004] The demicellation process is typically carried out by using energy collisions with neutral gas molecules to sufficiently increase the vibrational energy of the membrane protein-micelle complex, thereby cleaving the non-covalent bonds between the nonspecifically bound detergent molecules and the membrane protein under study. Generally, ion-neutral collision-based ion dissociation methods are commonly referred to in the art as collision-induced dissociation (CID) and, less frequently, collision-activated dissociation (CAD). In the art, specific forms and implementations of CID are often referred to by a variety of other names and acronyms.

[0005] However, such CID-based approaches to demicellement can be problematic because (i) detergent molecules optimized for membrane solubilization are not always suitable for mass spectrometry due to their relatively high binding affinity to proteins (which inhibits their gas phase removal); (ii) detergent solubilization itself can disrupt the structure, function, and activity of proteins and destabilize membrane protein complexes; (iii) weakly bound ligands can be removed along with the detergent during the micelle removal process; and (iv) sufficient vibrational excitation to induce the removal of detergent molecules by CID can also sufficiently activate target membrane proteins and alter the composition of membrane protein complexes.

[0006] The need to avoid the use of detergents in membrane protein mass spectrometry studies is clear, and in principle, this can be avoided by analyzing membrane proteins directly from the membrane environment. However, the limitations listed above regarding demicellement by collision-based approaches are common to the removal of nonspecifically bound membrane (bulk) lipid molecules in the gas phase from membrane proteins or protein complex ions. Furthermore, the removal of lipid molecules is related to its own problems, as will be discussed below.

[0007] Membrane proteins in the biological environment are associated with the cell membrane, solvent, and several other related molecules. Membrane proteins associated with numerous molecules (resulting in many combinations of molecular weights), such as nonspecifically attached lipids (bulk lipids) and residual solvent molecules, are difficult to analyze. Consider a sample containing multiple proteins, each having the same amino acid sequence with identical (covalent) post-translational modifications (i.e., they are all the same proteoform), but each associated with various combinations of nonspecifically attached adducts (bulk lipids, residual solvent molecules, etc.) (n=0, 1, 2...). Each protein with different combinations of adducts has a different total mass. Therefore, when subjected to analysis by mass spectrometry, such samples usually do not yield distinct m / z peaks in the resulting mass spectrum. Instead, highly heterogeneous mixtures of ions with the same proteoform's m / z appear in the mass spectrum as a broad continuum of unresolved m / z peaks that often cannot be masked by noise. However, different peaks are desirable because they can be used to determine protein molecular weight through the matching of a specific series of m / z peaks of mass-to-charge (m / z) ratios, which are related to the differences in the degree of ionization of proteoforms of a particular molecular weight. Such molecular weight information can provide useful insights into the structure, interactions, and function of membrane proteins.

[0008] MS analysis of free proteins, which are proteins from which all or almost all nonspecifically associated molecules, including those in a complex cellular environment, have been removed, can provide rich information. Molecular weight information provided by the mass-to-charge ratio (m / z) of free, intact proteins or protein complex ions is not always sufficient for molecular identification. If the protein under study is enriched in a cellular environment, it can be examined by tandem mass spectrometry techniques involving multiple steps of m / z selection and dissociation. This allows for the determination of protein composition and subunit structure. Furthermore, the determination of absolute molecular composition (i.e., amino acid sequence, as well as the type and location of post-translational modifications) becomes potentially possible. Such tandem mass spectrometry studies of intact proteins and protein complexes also have the potential to provide sequence tags for identifying proteins or constituent proteins, the location and type of post-translational modifications of constituent proteins, as well as molecular weight, and potentially, information on the compound class and structure of non-covalent partners. For example, Skinner et al. ("Top-down characterization of endogenous protein complexes with native proteomics," Nature Chemical Biology, Vol. 14, 2018, pp. 36-43) describe the detailed and complex compositional and structural information that can be obtained by analyzing soluble proteins and complexes in this manner.

[0009] However, it is not common to gather such detailed information from membrane proteins present among many other biomolecules (including native membranes in which they are immersed or associated). This is due to the difficulty in analyzing membrane proteins that require interactions with non-covalent lipids to maintain stability in the solution phase. Numerous membrane protein-lipid interactions interfere with the ability to observe discrete m / z peaks of proteins from m / z spectra, which hinders the usefulness of any downstream tandem mass spectrometry. Therefore, it is desirable that mass spectrometry allow for the direct analysis of membrane proteins from their biological (membrane) environment in a manner that releases proteins from any nonspecifically bound lipid molecules while preserving the integrity of the protein complex in other respects.

[0010] Therefore, researchers in this field have made considerable efforts to provide a simplified system that enables the analysis of proteins in their native environment. Researchers have attempted to achieve the removal of bulk lipids and residual solvent molecules by subjecting protein-containing gas-phase ions to various forms of CID. Most commonly, this has been done either by biasing the electrode potential so that the ions undergo sufficient energy collisions with atmospheric gas molecules as they pass through the inlet region of the mass spectrometer in one of the intermediate pressure steps of the inlet, in order to achieve CID, or by delivering the ions to a conventional RF multipole collision cell after they have passed through the inlet where they undergo energy collisions with the collision gas. Such approaches have also been used to liberate membrane proteins and protein complex ions from detergents (depending on the detergent used) and membrane mimics such as lipid nanodiscs, and have been somewhat successful. One exemplary study in this field is conducted by Gault et al. ("Combining native and 'omics' mass spectrometry to identify endogenous ligands bound to membrane proteins," Nature Methods, Vol. 17, 2020, pp. 505-508).

[0011] However, the inventors have found that these purely collisional approaches to the liberation of protein ions for analysis are very unsuccessful in the analysis of protein samples extracted directly from biological environments in association with vesicles. This is because it is difficult to remove nonspecifically bound lipids (also referred to herein as "delipidization"). The inventors have found that (1) collisional activation of the membrane protein-bulk lipid system for delipidization is insufficient, resulting in an insufficient yield of completely liberated "free" protein ions; (2) the accelerating potential used to subject vesicle ions to energy collisions can often adversely affect ion permeability in the mass spectrometer in an m / z-dependent manner; and (3) the kinetic energy threshold for collisional activation required for delipidization is generally much higher than that required for demicellement, thereby limiting the range of protein-specific noncovalent ligand interactions that can be conserved in the liberated protein ions to only those that are extremely strong. In other words, non-covalently bound protein-specific ligands that bind to the periphery of membrane proteins are almost certainly lost during delipidation, hindering the complete observation, identification, and characterization of such membrane protein-ligand complexes.

[0012] Tandem mass spectrometry (MS / MS or MS) nTo successfully perform "top-down" sequence analysis of intact protein ions via (also known as) sequencing, intact protein precursor ions must be generated effectively and efficiently, they must be m / z isolated (or m / z selected), and then, ideally, dissociated to convert them into sequence-determining product ions. Various suitable dissociation techniques are known in the art. These product ions can optionally be subjected to further operations, such as m / z isolation (m / z selection) and further charge reduction, via methods known in the art, to generate the final generation product ions. The final generation product ions are subjected to m / z analysis and detection to generate a product ion m / z spectrum. This product ion m / z spectrum is then interpreted to provide amino acid sequence information or protein identification. For the product ion spectrum to be suitable for sequence analysis, it is necessary to dissociate a large number of precursor ions. This allows for the detection of a sufficient number of final generation product ions to generate interpretable isotopic m / z peak envelopes in the m / z spectrum corresponding to a large number of different sequence product ions.

[0013] For example, if the product ion is a 10-100 amino acid fragment derived from skeletal cleavage along the primary structure of a high molecular weight precursor (such as a membrane protein or membrane protein complex), there are numerous different dissociation pathways that produce a very large number of corresponding product ion types (due to a very large number of possible binding cleavage sites). Therefore, the precursor ion signal will be split among numerous product ion types (C and N-terminal sequence ions, internal fragment ions, side-chain loss ions, etc.). Thus, tandem mass spectrometry of the starting ion will not be successful unless a sufficient yield of initial starting ions (such as bulk lipids and membrane proteins with the solvent removed) is obtained. Any resulting mass spectrum will have a signal-to-noise ratio so low that the sequence ion m / z peak in the mass spectrum cannot be distinguished. Therefore, the success of any tandem mass spectrometry experiment is determined by the amount / flux of precursor ions converted to product ions.

[0014] Therefore, due to the low yield of free protein ions, the inventors have found that conventional collision-based techniques for achieving delipidation are generally unsuitable for providing any type of free, intact membrane protein ions sufficient to perform any type of tandem mass spectrometry experiment.

[0015] Assuming that a sufficient amount of free protein precursor ions can be generated, many fragmentation methods are available to induce non-covalent and covalent cleavage to produce product ions suitable for analyzing the composition of the precursor protein. For example, along with various forms of CID, electron capture dissociation (ECD) and electron transfer dissociation (ECD) can be used. Ion-electron and ion-ion methods such as transfer dissociation and electron transfer dissociation (ETD) are used for the dissociation of intact proteins. Other known fragmentation techniques utilize light from a laser source to irradiate precursor ions with either ultraviolet (UV) or infrared (IR) light, inducing dissociation electrons or vibrational transitions respectively to induce bond cleavage. These methods are generally referred to in the art as ultraviolet photodissociation (UVPD) and infrared multi-photon photodissociation (IRMPD). All of these techniques have the ability to cleave bonds along the peptide backbone of protein ions to generate amino acid fragment ions that are suitable for the compositional analysis of precursors. For example, IRMPD has been used to analyze the amino acid sequences of purified intact soluble proteins (see, for example, Li, Nguyen, Ogorzalek Loo, Campuzano, & Loo, "An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes", Nat. Chem., vol. 12, 2018, pp. 139-148). IRMPD has also been used to cleave peptide bonds and fragment the underlying enzymatically generated peptides (see, for example, Crowe & Brodbelt, "Infrared multiphoton dissociation (IRMPD) and collisionally activated dissociation of peptides in a quadrupole ion trap with selective IRMPD of phosphopeptides", J. Am. Soc. Mass Spectrom., vol. 15, 2004, pp. 1581-1592).

[0016] Therefore, from the above perspectives, the inventors recognized the need to provide a charge-independent method for selectively removing non-specifically bound molecules such as bulk membrane lipids from membrane proteins and their complexes. Such a method is required to generate intact, free membrane protein ions in high yield, which in turn renders them suitable for top-down analysis by tandem mass spectrometry.

[0017] There is a further need for alternative and / or improved methods for characterizing intact membrane proteins in native or native-like environments. For example, there is a need for methods to characterize intact membrane proteins without relying on (or in the absence of) detergents and other non-native surfactants. As mentioned above, while detergents can assist in solubilizing membrane proteins, they typically disrupt native protein structures and destabilize native protein complexes, such that characterization or analytical data obtained from such samples may not accurately reflect the underlying native protein or protein complex. The presence of non-native detergent or surfactant molecules can further impede accurate data collection as the resulting data can be complicated by the presence of residual detergent or surfactant molecules in the sample, which can be particularly problematic for synthetic detergent molecules that may bind relatively strongly to membrane proteins. Thus, the resulting data may be characteristic of an adduct of the native protein or protein complex with the detergent. Such data can be time-consuming or complex to deconvolute. Conversely, attempts to remove detergents can disrupt the structure of the underlying native protein or protein complex. SUMMARY OF THE INVENTION

[0018] The inventors discovered that vacuum infrared (IR) activation of gas-phase ions containing membrane proteins within lipid membrane fragments can be highly effective in removing bulk lipid molecules and residual solvent molecules to provide intact membrane protein ions in the free gas phase. Furthermore, the inventors found that bulk lipid molecules can be preferentially removed from the gas-phase proto-membrane protein ions, while allowing the intact membrane protein ions in the free gas phase to retain many of their other non-covalent partners (including, for example, peptides, drugs, and other small molecules specific to the function of the protein). In the test, medium-resolution m / z analysis of the thus freed intact membrane protein ions in the gas phase produced an m / z spectrum with discrete representative m / z peaks having assignable mean m / z values. The m / z peaks in the resulting m / z spectrum corresponding to the free protein ions of such IR activation were found to have a significantly better signal-to-noise ratio (and intensity) compared to the resulting m / z peaks achievable when the instrument is configured to utilize known collision activation approaches.

[0019] While we do not wish to be bound by theory, these excellent results can be presumed to be observed for the following reasons:

[0020] The infrared wavelength photons used in the attached examples can be absorbed more readily by bulk lipid molecules than by proteins because their wavelengths excite specific vibrational modes of binding within the bulk lipids. Therefore, infrared wavelengths can be selected to resonate with, or come sufficiently close to resonate with, the vibrational modes of certain nonspecifically attached molecules (such as bulk lipid molecules).

[0021] The method of the present invention first involves generating gas-phase protoprotein ions from a sample. In a typical experiment, many such gas-phase protoprotein ions are generated. The gas-phase protoprotein ions are confined (e.g., in an ion trap). The confined gas-phase protoprotein ions are exposed to infrared (IR) radiation from a laser source. However, in addition to exposure to infrared radiation, the confined ions are typically subjected to numerous near-heat energy collisions with gases such as helium, argon, air, nitrogen, or hydrogen (among many others). These collisions have a cooling effect on the captured ions, counteracting the heating caused by the absorption of infrared laser radiation. This combined effect is thought to rapidly establish an equilibrium level of vibrational activation of the confined gas-phase protoprotein ions. Therefore, the IR-driven delipidization process can be controlled not only by adjusting the duration and intensity of the infrared beam passing through the captured ion cloud, but also by the selection of gases and gas pressures, and thus their collisional cooling effect.

[0022] The small molecule protein-specific binding partners of membrane proteins may have a somewhat higher binding affinity to the protein than nonspecifically bound solvent and bulk lipid molecules. Therefore, these combined embodiments may provide precision in process control. Such control may allow for the selection of parameters for IR activation so that the gas-phase proto-membrane protein ions can be heated precisely enough to "evaporate" the relatively weak non-covalent solvent and bulk lipid molecules (thus producing a free, intact membrane protein ion in the gas phase, typically consisting of the protein and its protein-specific binding partners), while maintaining non-covalent interactions that bind the membrane protein internally and to other protein-specific small molecule binding partners.

[0023] Furthermore, while lipid molecules have significantly smaller masses and, consequently, fewer vibrational degrees of freedom than proteins, the inventors believe that membrane lipids have a higher binding density that absorbs relatively more efficiently in IR (particularly in the wavelength range of the IR laser light used in the accompanying examples). Therefore, lipid molecules are likely to be heated to a greater extent by infrared radiation than the proteins they partially encapsulate. When such differential heating occurs, it is expected that this will promote the more efficient release of protein ions from the membrane bulk lipids.

[0024] The method described herein is particularly suitable for the analysis of membrane proteins contained in non-synthetic biomembrane materials because it can selectively remove complex mixtures of membrane bulk lipid molecules attached to membrane proteins while substantially preserving the composition of the underlying membrane proteins (complexes).

[0025] Therefore, the present invention relates to a method for analyzing a sample containing intact membrane proteins, (i) To provide a sample containing intact membrane proteins, a non-synthetic biomembrane material, a solvent, and bulk lipid molecules, (ii) Ionizing the sample to generate a gas-phase proto-membrane protein ion, wherein the gas-phase proto-membrane protein ion contains intact membrane proteins nonspecifically bound to one or more bulk lipid molecules and one or more residual solvent molecules, (iii) Confinement of gas-phase proto-membrane protein ions, (iv) By irradiating the confined gas-phase proto-membrane protein ions with infrared radiation from a laser source, one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules are separated from the intact membrane protein to generate intact gas-phase membrane protein ions, (v) A method is provided which includes m / z analysis and detection of intact membrane protein ions and / or ions derived therefrom in the gas phase.

[0026] m / z analysis may precede m / z detection, or they may be performed simultaneously.

[0027] Step (iv) may be referred to as "delipidation" or "delipidation step."

[0028] Particularly preferred examples of non-synthetic biomembrane materials include biomembrane materials extracted from or directly secreted by cells, organelles, viral envelopes, or vesicles.

[0029] A key advantage of the method of the present invention is that membrane proteins can be completely liberated from residual solvents and bulk lipid molecules. This can be achieved with a higher probability than in prior art CID-based methods, and therefore, a larger quantity of intact membrane protein ions in the gas phase can be generated from a population of proto-membrane protein ions. Consequently, in analyses in which many bulk-lipidized protein ions are subjected to the method of the present invention, the yield of completely liberated protein ions is usually higher compared to CID delipidation methods. In preferred embodiments of this method, intact membrane protein ions in the gas phase do not contain nonspecifically bound bulk lipid molecules. Similarly, intact membrane protein ions in the gas phase preferably do not contain residual solvent molecules. Intact membrane protein ions in the gas phase may further contain nonspecifically bound molecules of other non-lipids.

[0030] A relevant advantage of the method of the present invention is that it enables the characterization of intact membrane proteins in native or native-like environments, for example, in the substantial or complete absence of unnaturally occurring detergents and other non-natural surfactants. While the use of detergents is included within the present invention, the ability to evaluate samples that are at least substantially free of detergents is an advantage of this method. Specifically, it has been previously understood that endogenous membrane proteins cannot be readily released from vesicles derived from their native lipid bilayers for mass spectrometry characterization without the use of detergents and / or other non-natural surfactants, with the disadvantage of incorporating substances such as those discussed above. The ability to release and probe intact membrane proteins without the need for detergents is a remarkable advantage of the present invention.

[0031] A key advantage of the method of the present invention is the provision of ions in high yield that can be subjected to further analysis. Since intact membrane protein ions in the gas phase are produced in sufficient quantities by this method, they can be manipulated and analyzed like any other native protein (e.g., protein complex). Specifically, these intact membrane protein ions in the gas phase can be subjected to analysis by tandem mass spectrometry. For example, intact membrane protein ions in the gas phase can be subjected to an ion conversion process to produce one or more product ions, one or more product ions can be detected and m / z analyzed (optionally after being subjected to further conversion processes such as charge reduction). The first generation product ions themselves can be subjected to m / z selection before further ion conversion processes, and so on. Therefore, step (v) is, (va) To subject intact membrane protein ions in the gas phase to an ion conversion process to generate first-generation product ions, (vb) This may include selecting the first generation product ions by m / z and detecting them arbitrarily.

[0032] This method, (vc) The first generation product ions are subjected to a further ion conversion process to generate the second generation product ions, (vd) The method may further include selecting the second generation product ions by m / z and optionally performing m / z analysis and detection of the second generation product ions.

[0033] Each ion conversion process may produce one or more product ions. Therefore, in practice, by subjecting intact membrane protein ions in the gas phase to an ion conversion process, one or more product ions may be produced. If multiple such ions are produced, each ion is a first-generation product ion. If multiple such first-generation product ions are produced, one or more of them may be m / z selected (and optionally detected) in step (vb). Each first-generation product ion produced may be the same or different. If step (vc) is performed, step (vc) may be performed on one or more of the first-generation product ions. Similarly, in practice, by subjecting first-generation product ions to an ion conversion process, one or more (second-generation) product ions may be produced. If multiple such ions are produced, each ion is a second-generation product ion. Each second-generation product ion may be the same or different. If multiple such second-generation product ions are generated, one or more of them may be m / z selected (and optionally analyzed and detected) in step (vd).

[0034] For example, in a "top-down" tandem MS experiment, a specific charge state of free ions from a membrane protein is m / z selected (e.g., all other ions outside the relatively narrow m / z band corresponding to the specific charge state of the protein may be released from the relevant part of the instrument), and then subjected to further activation to dissociate the covalent bonds of the protein, generating ions characteristic of the amino acid sequence of the intact protein at the time of analysis.

[0035] A wide variety of options are available for the ion conversion process, also referred to herein as the activation process or ion conversion process. These methods include infrared multiphoton dissociation, electron transfer dissociation, activated ion electron transfer dissociation, electron capture dissociation, UV photodissociation, and collision-induced dissociation (CID). Specific examples of CID include collision cell type collision activation and RF ion trap type resonance collision activation.

[0036] Of these ion conversion processes, infrared multiphoton dissociation (IRMPD) is preferred because it is typically more reliable and efficient in achieving the dissociation of large, free membrane protein and protein complex ions. Characteristic of such free ions initially generated as proto-membrane protein ions via nESI (nanoelectrospray ionization) are their high charge (typically having more than 10 charge states, z) and their relatively low charge density. Therefore, their mass-to-charge ratio typically exceeds 3,000 daltons / elemental charge (Thompson, Th) and is typically in the m / z range of 4,000 Th to 12,000 Th. Protein and protein complex ions of such mass and charge are generally not efficiently dissociated into sequence information product ions by electron-based methods such as ETD. Furthermore, due to the very large mass difference between the collision gas molecules (typically having a mass of 40 Da or less) and the free protein or protein complex precursor ions (generally having a mass greater than 25 kDa, often in the range of 50 kDa to 150 kDa, and even greater than 200 kDa), the most common methods and practices for collision-induced dissociation, collision cell type CID, and ion trap type resonance CID may also fail to conjure sufficient vibrational excitation to the free precursor ions to yield a rich range of fragment ions resulting from the cleavage of the protein backbone, which can often be used for sequence analysis during m / z analysis and detection.

[0037] In contrast, the absorption of IR photons by protein ions appears, as expected, to be approximately proportional to molecular weight, roughly corresponding to how the number of vibrational states in the protein increases with molecular weight. Furthermore, the extraction of vibrational energy from ions through collisions with the collision gas at near-thermal kinetic energy is expected to be proportional to the surface area or cross-sectional area of ​​the ion, which is expected to be on a somewhat smaller scale than being proportional to molecular weight. Therefore, the net heating (effective temperature) of proto-membrane protein ions and intact membrane protein ions resulting from exposure of ions to IR photons is not expected to be strongly dependent on ion molecular weight. In fact, we have not observed a strong molecular weight dependence on the IR laser output power required to induce IRMPD in the nESI-generated membrane protein ions (including intact proteins and protein complexes) studied, and these protein ions were well above the molecular weight range of approximately 30 kDa to 150 kDa. The inventors also observed that IR activation can induce non-covalent cleavage, releasing small molecule binding partners from intact membrane protein ions in the gas phase as either ions or neutral substances. Furthermore, in some cases, IR activation can interfere with non-covalent interactions that bind proteins in multiprotein complex ions, observed as m / z peaks in the associated tandem m / z spectra, and can generate ions corresponding to individual protein subunits or various incomplete combinations of protein subunits.

[0038] IR activation at irradiation levels moderately higher than suitable for achieving the removal of bulk lipids from protein complexes containing nESI-generated proteins and ions (proto-membrane protein ions) generally also achieves cleavage of both the protein backbone (typically the primary dissociation pathway) and side-chain covalent bonds. Variations in the activation IR photon flux to which the membrane protein ions are exposed (generally controlled by adjusting the laser power output) and the duration of that exposure provide control over the degree of dissociation. Extending the IR photon exposure time at a fixed photon flux level sufficient to induce cleavage of covalent bonds on the protein backbone results in continuous generation of product ions, because, in the absence of means to remove the first generation of IRMPD product ions from further exposure to IR photons, each generation of product ions is subjected to continuous IR emission and activation, resulting in further dissociation as the exposure time is extended. The final product ion population includes a mixture of N-terminal and C-terminal containing sequence ions, as well as numerous other internal fragment ions. Internal fragment ions refer to fragment ions that are product ions that do not contain either the N-terminus or C-terminus of the protein.

[0039] The number of possible internal fragments is approximately (N-1), where N is the number of amino acid residues in the protein. 2The number of possible C and N-terminal ions (sequence ions) is scaled as 2 / 2, and the number of possible C and N-terminal ions (sequence ions) is scaled as 2(N-1). Each intact gas-phase membrane protein ion is expected to produce a pair of sequence ions and a certain variable number of internal fragment ions as product ions. The number of internal fragment ions produced for each intact gas-phase membrane protein ion increases with increasing activation time. Similarly, with longer activation times, the mass of the sequence ion pairs produced decreases (shorter amino acid residue length). Each sequential dissociation cleaves the original intact gas-phase membrane protein ion into more and smaller product ions. While there is some amino acid specificity for where cleavage occurs due to vibrational activation, this evolution in the composition of product ion types (also referred to in this context as fragment ions) means that the abundance of individual sequence ion types produced from an initial population of intact gas-phase membrane protein ions is generally greater than any one particular type of internal fragment ion produced.

[0040] Methods for inhibiting the generation of multiple generations of photodissociation product ions are known in the art and are referred to as product ion parking or product ion protection. Such methods may be incorporated into the methods of the present invention to improve the final abundance of higher molecular weight product ions (final product ions).

[0041] Without methods to improve the abundance of high molecular weight product ions, they may be difficult to detect above the electron noise baseline signal. However, these large and highly charged product ions are well-suited for m / z analysis and detection by charge detection mass spectrometry (CDMS), a method known in the art. Specifically, CDMS methods, such as those described by JOKafader and colleagues [Kafader, Jared O., et al. "Measurement of individual ions sharply increases the resolution of orbitrap mass spectra of proteins." Analytical Chemistry 91.4(2019):2776-2783, and Kafader, Jared O., et al. "Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes." Nature Methods 17.4(2020):391-394], would be advantageous for obtaining mass spectra and tandem mass spectra of free membrane proteins and protein complexes by the method of the present invention as described herein. Therefore, step (v) may include detection and m / z analysis of intact membrane protein ions and / or ions derived therefrom in the gas phase by CDMS.

[0042] A further important advantage of this method is its ability to optimize the rate of energy supplied to the gas-phase proto-membrane protein ions in order to control the process by which residual solvent molecules and nonspecifically bound bulk lipid molecules are separated. By using an infrared laser, the irradiation time, laser power level, wavelength, photon beam profile, and (if using a pulsed laser rather than a continuous-wave laser) the laser pulse width can all be easily adjusted. Thus, the irradiation conditions can be adjusted to selectively accumulate enough energy to break the non-covalent bonds between the protein and the nonspecifically bound molecules. Therefore, the process may involve determining the irradiation parameters used in step (iii) by repeating the process until intact gas-phase membrane protein ions can be detected after step (iv), and gradually increasing the energy of the laser radiation and / or the duration of irradiation of the gas-phase proto-membrane protein ions.

[0043] Naturally, in any subsequent IRMPD fragmentation step, these conditions can also be adjusted to produce observable series of N and C-terminal sequence ions, as well as (sequence) interpretable series of internal fragment ions.

[0044] The present invention is described in more detail below. [Brief explanation of the drawing]

[0045] [Figure 1] This is a schematic diagram of an exemplary apparatus used when carrying out an exemplary method of the present invention. [Figure 2]This figure includes a series of representative mass-to-charge ratio (m / z) spectra illustrating the use of the present invention for nonspecifically binding protein ions from lipids in E coli membrane fragments containing MacAB, among other membrane proteins and complexes. The symbols label the m / z peaks corresponding to the charge state series corresponding to common molecular weights. The value in the upper right corner of each spectrum is the intensity value of the strongest peak. An asterisk (*) is used to label m / z peaks that do not collectively correspond to the charge state sequence of proteins or other high molecular weight compounds. The infrared irradiation time was fixed at 10 ms. The IR laser power output setting (nominal, % of total power, uncalibrated) is shown for each spectrum. This figure shows the effect of laser intensity on generating intact membrane protein ions in the gas phase from a biological matrix derived from lipid membrane fragments containing intact membrane protein ions. This figure shows that optimized parameters suitable for releasing membrane proteins and complexes can be identified by positioning a set of parameters that yield the best diversity of m / z peaks in the gas phase, corresponding to intact membranes and membrane-associated proteins, as well as m / z peaks characterized by ligand binding and / or post-translational modification (PTM) retention, among the highest yield of released ions. [Figure 3] Among other molecules, representative m / z spectra are shown illustrating the use of collision activation (accelerated potentials of 50V, 100V, 150V, 200V, and 250V) in ion inlet optics (often referred to as in-source CID in this art) for releasing membrane protein and membrane protein complex ions from nonspecifically bound lipid vesicles (membrane fragments) containing the membrane protein MacAB. The values ​​in the upper right corner of each spectrum represent the intensity of the strongest peak. An asterisk (*) is used to label m / z peaks that do not correspond to any continuous series of charged states and, therefore, probably not to ions of protein origin. [Figure 4]This study includes a comparison of protein m / z peaks obtained via MacAB release, among other membrane proteins, from E coli membrane vesicles (membrane fragments) under optimized collision activation voltage (inlet CID using a 100V acceleration potential) and IR irradiation (5% of the total output laser intensity for 10 ms, uncalibrated). For the peak corresponding to the 102,775 Da protein released by in-source CID, insufficient signal intensity is observed. When using infrared irradiation, a clearly observed m / z peak intensity corresponding to the MW 102,755 Da protein (in this case, a protein complex) is high. In addition, a series of satellite m / z peaks with higher m / z values, characteristic of ligand binding and / or PTM retention, is observed. The molecular weight of this satellite distribution of m / z peaks is consistent with ligation or modification at 129 Da. [Figure 5] This figure shows the optimization of IR parameters for releasing intact BamABCDE complex membrane protein ions from lipid vesicle ions, among other membrane proteins (including the complex). The figure includes MS spectra obtained using three IR laser power settings (7%, 10%, and 15% of total power, uncalibrated), demonstrating the release of intact BamABCDE complex protein ions from ionized vesicles (proto-membrane protein ions) formed from the E. coli outer membrane, among other membrane proteins. The IR laser irradiation time was fixed at 10 ms. [Figure 6]Figure 6(A) (upper spectrum) shows the m / z spectra collected with optimized IR emission parameters to release the BamABCDE complex along with other proteins, and subsequent IRMPD (covalent fragmentation) following m / z isolation of the m / z peak matching the intact BamABCDE complex to generate product ions for sequence identification. Figure 6(A) (upper spectrum) is the mass spectrum from the ionization of E. coli lipid vesicles containing the BamABCDE complex among other membrane proteins, generated by releasing the intact membrane protein ions of the membrane protein complex using optimized IR emission parameters (determined by experiments shown in Figure 5). In Figure 6(A) (lower spectrum), the intact membrane protein ions corresponding to the m / z peak indicated by an asterisk in the upper spectrum (corresponding to the 28+ charged state of BamABCDE) were isolated and subjected to ion activation with a large number of IR photons (i.e., IRMPD) to induce peptide bond cleavage, and the new distribution of product ions was then analyzed m / z (MS2). Figure 6(B) includes detail diagrams of two m / z ranges in the lower spectrum (MS2 spectrum) of Figure 6A. These were examined for the presence of m / z peaks indicating sequence fragment product ions from the BamE subunit (i.e., b20 and y27). The bold letters above the m / z peaks in each detail diagram indicate the monoisotopic m / z peaks (upper and lower, respectively) corresponding to the b20 and y27 fragment ions. In Figure 6(C), the sequence information on the left shows the sequence of the BamE subunit in which the b20 and y27 fragment ions represent the elongation of the corresponding amino acids. The table on the right shows the experimental and theoretical molecular weights of these fragment ions, demonstrating that they are well within an acceptable threshold for sequence identification (i.e., less than 15 ppm). [Figure 7] This includes exemplary mass spectra showing the release of intact membrane protein ions of membrane proteins / complexes having MW 39kDa, 148kDa, and 271kDa, as well as other molecules, from ionized proto-membrane protein ions of extracellular vesicles (exosomes) spontaneously secreted from human body fluids using IR irradiation. [Figure 8]This figure shows the effect of IR laser power on the release of membrane proteins from the native lipid bilayer for detection by mass spectrometry. Figure 8(A) was obtained with a low laser power of 3% (corresponding to 1.8 W) and an irradiation time of 25 ms. Only soluble proteins are observed in the mass spectrum. Adjacent charge state series are indicated by circles. Figure 8(B) was obtained with a high laser power of 9% (corresponding to 5.4 W) and the same irradiation time of 25 ms. Rhodopsin (a 7-transmembrane protein) is released from the native lipid bilayer and represents the major charge state distribution in the mass spectrum (indicated by asterisks). [Figure 9-1] The use of the present invention method in sequencing rhodopsin after release from the lipid bilayer is demonstrated. A single charged state of rhodopsin was isolated and subjected to infrared multiphoton dissociation (15% (9W) with an irradiation time of 5 ms) to induce protein backbone cleavage and generate tandem mass spectrometry (MS2) spectra of panel (A). b / y type fragment ions were matched using spectral matching to the predicted MS2 spectra. Regions of interest corresponding to “proteoforms,” which are protein forms modified from the canonical sequence via the presence of post-translational modifications, are shown: (B-C) glycosylation, (D) phosphorylation, and (E) palmitoylation. Figure 9(F) is a map of the entire repertoire of fragment ions that matched those predicted for unmodified and modified rhodopsin. Approximately 14% sequence coverage was obtained, providing complete confidence in the assignment of peaks in the mass spectra (Figure 8) to rhodopsin. [Figure 9-2]The use of the present invention method in sequencing rhodopsin after release from the lipid bilayer is demonstrated. A single charged state of rhodopsin was isolated and subjected to infrared multiphoton dissociation (15% (9W) with an irradiation time of 5 ms) to induce protein backbone cleavage and generate tandem mass spectrometry (MS2) spectra of panel (A). b / y type fragment ions were matched using spectral matching to the predicted MS2 spectra. Regions of interest corresponding to “proteoforms,” which are protein forms modified from the canonical sequence via the presence of post-translational modifications, are shown: (B-C) glycosylation, (D) phosphorylation, and (E) palmitoylation. Figure 9(F) is a map of the entire repertoire of fragment ions that matched those predicted for unmodified and modified rhodopsin. Approximately 14% sequence coverage was obtained, providing complete confidence in the assignment of peaks in the mass spectra (Figure 8) to rhodopsin. [Modes for carrying out the invention]

[0046] The present invention relates to a method for analyzing intact membrane proteins by mass spectrometry, wherein intact membrane proteins can be transferred directly from their native membrane environment as ions into the gas phase, bulk lipids and residual solvent molecules can be removed, and the intact membrane protein ions in the gas phase can then be analyzed and detected by m / z analysis, or converted to other ions derived from them through further transformation processes (such as fragmentation or charge reduction) before m / z analysis and detection. Specifically, the present invention relates to a method for analyzing a sample containing intact membrane proteins, (i) To provide a sample containing intact membrane proteins, a non-synthetic biomembrane material, a solvent, and bulk lipid molecules, (ii) Ionizing the sample to generate a gas-phase proto-membrane protein ion, wherein the gas-phase proto-membrane protein ion contains intact membrane proteins nonspecifically bound to one or more bulk lipid molecules and one or more residual solvent molecules, (iii) Confinement of gas-phase proto-membrane protein ions, (iv) By irradiating the confined gas-phase proto-membrane protein ions with infrared radiation from a laser source, one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules are separated from the intact membrane protein to generate intact gas-phase membrane protein ions, (v) A method is provided which includes m / z analysis and detection of intact membrane protein ions and / or ions derived therefrom in the gas phase.

[0047] sample The present invention relates to the analysis of a sample containing a non-synthetic biomembrane material. The non-synthetic biomembrane material comprises at least intact membrane proteins, a solvent, and bulk lipid molecules. The sample is generally a liquid sample.

[0048] The sample preferably contains intact membrane proteins in the state generally found in their natural biological environment. That is, the sample preferably contains intact membrane proteins in the naturally occurring membrane environment. Therefore, the intact membrane proteins are preferably in a "wild" state.

[0049] Non-synthetic biomembrane materials may also be referred to as naturally occurring biomembrane materials. That is, non-synthetic biomembrane materials are species that can be obtained or obtained from living cells or viruses. Examples of biomembrane materials include membrane materials, bacterial cells, or viruses that can be obtained or obtained from plant or animal cells (preferably animal cells, particularly preferably human cells). Therefore, non-synthetic biomembrane materials are distinct from synthetic membranes and synthetic membrane mimics such as detergent micelles, lipid nanodiscs, and synthetic lipid monolayers or bilayers. Similar to non-synthetic biomembrane materials, synthetic membrane materials typically contain intact membrane proteins and solvents, and also contain bulk lipid molecules and / or detergent molecules. However, in synthetic membranes, bulk lipid molecules and / or detergent molecules are artificially assembled in vitro to form synthetic monolayers or bilayers. Non-synthetic biomembrane materials as described herein exclude synthetic membrane materials such as synthetic membrane mimics.

[0050] Typically, non-synthetic biomembrane materials consist of membranes (and membrane proteins) collected or released from cells (including bacterial cells) or viruses. In other words, non-synthetic biomembrane materials are typically obtained or can be obtained from cells or viruses.

[0051] If non-synthetic biomembrane materials can be obtained from or are obtainable from cells, the cells may be cultured cells or harvested cells (i.e., harvested from an organism). In such cases, the non-synthetic biomembrane material may include the cell membrane of the cell. For example, the non-synthetic biomembrane material may be obtained by lysing the outer plasma membrane of a cell. The outer plasma membrane contains a membrane (generally a lipid bilayer) that defines the cell boundary, along with membrane proteins embedded within it. The non-synthetic biomembrane material can be obtained by directly extracting the outer plasma membrane after lysis.

[0052] Non-synthetic biomembrane materials obtainable from cells are not limited to the outer plasma membrane. Free organelles or vesicles can also be extracted to obtain non-synthetic biomembrane materials.

[0053] Organelles are generally defined as specialized subunits within a cell that have specialized functions. Examples of organelles include, but are not limited to, mitochondria and the endoplasmic reticulum; examples of vesicles include the polyendoplasmic reticulum, endosomes, and phagosomes. Organelles can be released from cells (and therefore include released proteins) by lysing the cell and then extracting the organelles.

[0054] Vesicles are generally defined as membrane-bound, fluid-filled sacs. Vesicles can be found inside or outside cells, and they are often secreted from the cell surface. Examples of vesicles found outside cells include, but are not limited to, exosomes and extracellular vesicles. Extracellular vesicles can be collected directly from biological fluids or aerosols such as blood and saliva (i.e., they may be free and therefore contain free proteins). Intracellular vesicles are obtained by lysing the surrounding cells and then isolating the vesicles.

[0055] Vesicles and organelles have a membrane (generally a lipid bilayer) around their outer edge, and the membrane generally contains one or more membrane proteins embedded within it.

[0056] Non-synthetic biomembrane materials can be obtained from vesicles or organelles by lysing them.

[0057] Therefore, in preferred embodiments, non-synthetic biomembrane materials are materials extracted from or directly secreted by cells, organelles, viral envelopes, or vesicles. For example, non-synthetic biomembrane materials may be obtained by lysing the outer membrane of a cell, organelle, or vesicle, or by extracting from and isolating the viral envelope. Preferably, non-synthetic biomembrane materials may be obtained by lysing the outer membrane of a vesicle and isolating the membrane.

[0058] The non-synthetic biomembrane materials described herein may be more simply referred to as non-synthetic membrane materials or biomembrane materials.

[0059] Bulk lipid molecules are lipid molecules present in membranes from which non-synthetic biomembrane materials are obtained (such as the outer membranes of cells, vesicles or organelles, or viral envelopes). As a result, at least some of the bulk lipid molecules are generally arranged in the form of membranes. For example, some or all of the bulk lipid molecules may be arranged as lipid monolayers or lipid bilayers. In some cases (for example, when non-synthetic biomembrane materials are obtained from multilamellar vesicles), some or all of the bulk lipid molecules may be arranged in the form of multiple bilayers.

[0060] In the sample provided in step (i) of this method, the non-synthetic biomembrane material may contain intact vesicles or intact viruses or viral lipid envelopes. For example, bulk lipid molecules may form a lipid bilayer(s) containing intact membrane proteins, and the bilayer(s) may form intact single-lamellar or multi-lamellar vesicles. If such intact vesicles are present, they are typically submicron-sized. "Submicron-sized" means that the maximum diameter of the vesicle is less than 1 μm.

[0061] However, more typically, samples do not generally contain intact cells, vesicles, organelles, or viral envelopes. Rather, samples containing non-synthetic biomembrane material are generally obtained by lysing cells, organelles, vesicles, or viral envelopes and extracting membrane material from them. In addition, the extracted membrane material may have been subjected to further processes to provide the sample, such as sonication, which can destroy membrane structures such as lipid monolayers or bilayers originally present in the cells / vesicles / organelles / viral envelopes. Therefore, non-synthetic biomembrane material contains bulk lipid molecules derived from the membrane, but the entire membrane structure may not be preserved. Rather, samples typically contain membrane fragments (such as fragments of lipid bilayers) associated with intact membrane proteins. Specifically, samples may contain submicron-sized membrane fragments associated with intact membrane proteins. Submicron size means that the maximum dimension of the fragment is less than 1 μm.

[0062] Some bulk lipid molecules directly bind to intact membrane proteins. Bulk lipid molecules are said to bind nonspecifically. This means that the lipid molecules do not regulate the structure, function, assembly, or activity of membrane proteins. Therefore, lipid molecules bind to intact membrane proteins in sites that do not contribute to or regulate the structure, function, assembly, or activity of membrane proteins. For example, lipid molecules do not bind to protein receptor sites. Bulk lipid molecules bound to intact membrane proteins are bound by non-covalent interactions with the protein. While most bulk lipid molecules do not directly bind to proteins, they typically bind to other bulk lipid molecules within a monolayer or bilayer configuration.

[0063] Therefore, non-synthetic biological membranes generally contain intact membrane proteins that are present in or attached to a membrane containing bulk lipid molecules. For example, non-synthetic biological membranes typically contain intact membrane proteins nonspecifically bound to a lipid bilayer or lipid monolayer (preferably a lipid bilayer), and the lipid bilayer contains bulk lipid molecules. The lipid bilayer can form vesicles, but more typically they are small fragments (generally submicron size) of the lipid bilayer.

[0064] Bulk lipid molecules can be of one or more different types. Bulk lipid molecules are generally typical in non-synthetic biological membranes (also called native membranes). Therefore, bulk lipid molecules generally include glycerophospholipids. Bulk lipid molecules may also include phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, phosphatidylcholine, phosphatidylserine, cholesterol, diacylglycerol, fatty acids, long-chain fatty acids, sphingoglycolipids, sphingomelin, and lipopolysaccharides.

[0065] An intact membrane protein means that the protein exists in its native state, as it would in its native membrane environment. That is, any specifically bound covalent partners that were attached to the membrane protein in its native state remain attached to the membrane protein. Intact means that the protein is not fragmented. For example, if a membrane protein contains multiple subunits or monomers, an intact membrane protein typically contains all of these subunits in their native configuration, rather than just one or more fragments. However, slight modifications from the native configuration may be acceptable. For example, a partial complex of a membrane protein, which is a high molecular weight protein assembly, can be considered "intact" or "native" even if some of its subunits are not assembled in a biologically active state.

[0066] Membrane proteins can be classified into endogenous membrane proteins and superficial membrane proteins. Endogenous membrane proteins may have one or more amino acid segments embedded within the membrane and may be non-covalently bonded to bulk lipid molecules of the membrane. Superficial membrane proteins may transiently associate with bulk lipid molecules and / or endogenous membrane proteins. In one embodiment, the intact membrane protein is an endogenous membrane protein. In another embodiment, the intact membrane protein is a superficial membrane protein.

[0067] Membrane proteins can be composed of one (mono) or multiple (multi) associated polypeptide chains. Therefore, intact membrane proteins may be monomeric or polymeric membrane proteins, such as oligomeric membrane proteins. Oligomerous membrane proteins include both homooligomeric (identical polypeptide chains) and heterooligomeric (different polypeptide chains) proteins. Therefore, in one embodiment, an intact membrane protein may contain two or more non-covalent monomers, which may be the same or different.

[0068] Undamaged membrane proteins are approximately 10 3 Dalton ~ about 10 12 Dalton, for example, about 5 x 10 3Dalton ~ about 10 6 It may have a molecular weight in Dalton.

[0069] A key advantage of the method of the present invention is that it can be used to decipher the structure and / or chemical composition of unknown proteins. Therefore, the structure and / or chemical composition of intact membrane proteins may be unknown.

[0070] Non-limiting examples of the membrane protein class include G protein-coupled receptors (opsins, e.g., GPCRs such as rhodopsin), membrane transporters, membrane channels, ATP-binding cassette transporters (ABC transporters), proton-driven transporters, solute carriers, outer membrane proteins (OMPs), ATP synthases, and protein and ligand translocases. Specific examples of membrane proteins include anchor cell fusion failure protein 1 (AFF-1), p-barrel assembly machinery (BAM), bacterial molecular chaperone DnaK, cytochrome bo3, CydAB cytochrome bd oxidase complex, energy conversion Ton complex, multidrug efflux pumps such as AcrABZ-TolC and MdtABCTolC, as well as ATP synthase, membrane protein complexes in the respiratory chain (e.g., complexes I-V), adenine nucleotide translocase 1 (ANT-1), and their subunits.

[0071] An intact membrane protein may exist in the form of a complex with a ligand. Indeed, an advantage of the method of the present invention is that the structural information that can be obtained with respect to an intact membrane protein may include information about the stoichiometry of one or more ligands binding to the membrane protein, the ligand binding site, and the conformational changes that occur when ligands bind to the membrane protein.

[0072] The binding of a ligand to an intact membrane protein can be via non-covalent or covalent interactions. Typically, it is via non-covalent interactions. Specifically, the binding of a ligand to an intact membrane protein can be via ionic bonds, hydrogen bonds, and intermolecular forces such as van der Waals forces. The binding of a ligand to an intact membrane protein can be reversible or irreversible. In one embodiment, the ligand binds to the intact membrane protein via a reversible bond. Generally, ligands bind to intact membrane proteins with some degree of specificity.

[0073] Examples of ligands include, but are not limited to, RNA chains, metabolites, drugs (which may be therapeutic or diagnostic agents), metal cofactors, lipids, nucleotides, and nucleosides. Preferred examples of ligands include RNA chains, metabolites, drugs, metal cofactors, and lipids.

[0074] When the ligand is a lipid, this lipid is part of a protein (in this case, a complex) and not a membrane, and therefore differs from a bulk lipid molecule. Examples of lipids that can be ligands include fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterollipids, prenolipids, glycolipids, and polyketides.

[0075] When an intact membrane protein exists in the form of a complex with a ligand, the complex may contain one or more such complexing ligands. Each such ligand may be independently selected from the exemplary ligands described above, including RNA chains, metabolites, drugs (which may be therapeutic or diagnostic agents), metal cofactors, lipids, nucleotides, and nucleosides.

[0076] An intact membrane protein may contain one or more post-translational modifications. Some forms of post-translational modifications may involve the formation of a complex containing the membrane protein and one or more ligands. Other forms of post-translational modifications may involve the addition or modification of functional groups within the protein. Specific examples of post-translational modifications include glycosylation and phosphorylation.

[0077] The sample further contains a solvent. The solvent is typically water, but other solvents (e.g., alcohols such as ethanol or methanol) may be present. Two or more solvents may be present.

[0078] The sample may contain the above-mentioned components in addition to other constituent elements.

[0079] For example, the sample may further contain a mass spectrometry-compatible buffer. Preferably, the mass spectrometry-compatible buffer is a buffer that maintains the structural integrity of intact membrane proteins. Specifically, the mass spectrometry-compatible buffer is a buffer that can maintain the structural integrity of intact membrane proteins when the protein is associated with submicron-scale membrane fragments or vesicles and solvent molecules. Suitable mass spectrometry-compatible buffers can be easily selected by those skilled in the art. They include ammonium buffers such as ammonium acetate or ammonium bicarbonate. Ammonium acetate is particularly preferred.

[0080] If the sample contains a mass spectrometry-compatible buffer, the buffer is typically present at a concentration of at least 150 mM, for example, 250–1000 mM, or for example, 400–600 mM.

[0081] The sample may, alternatively or additionally, contain one or more of the above species as ligands. Often, these species may be added to the sample to produce intact membrane proteins in which one or more ligands form complexes with membrane proteins. Thus, the sample may contain RNA chains, metabolites, drugs (which may be therapeutic or diagnostic agents), metal cofactors, lipids, nucleotides, and nucleosides. When such ligands are added to the sample to promote the formation of ligand-containing complexes, the ligands are generally present in a molar excess compared to the membrane proteins. For example, ligands may be present in the sample in a molar ratio of 2:1 or greater compared to the membrane proteins.

[0082] In addition, the sample may contain two or more types of intact membrane proteins. For example, the sample may contain two, three, four or more different intact membrane proteins. Intact membrane proteins may include the same underlying membrane protein complexed with multiple different ligands. Alternatively or additionally, multiple different membrane proteins may be present in the sample.

[0083] The pH of a sample is typically its native pH. Native pH is the pH typically found in the biological cellular environment. Therefore, its pH is typically between pH 5 and pH 8.

[0084] Typically, membrane proteins are present in the (optionally sonicated) solution at concentrations of at least 0.5 mg / mL, for example, 0.5–20 mg / mL, for example, 1–10 mg / mL.

[0085] The sample preferably contains little to no detergent. For example, the sample may contain detergent at a concentration of less than 100 μM, for example, less than 1 μM. Preferably, the solution contains substantially no detergent or no detergent at all. The sample may contain at least substantially no detergent. The sample or solution may contain at least substantially no non-naturally occurring surfactant molecules. Examples of non-natural detergents include DDM (n-dodecyl-β-D-maltoside), n-nonyl-β-D-glucopyranoside (NG), and octyltetraglycol (C8E4). Therefore, in some embodiments, the sample or solution may not contain, at least substantially, or completely contain n-dodecyl-β-D-maltoside, n-nonyl-β-D-glucopyranoside (NG), and / or octyltetraglycol (C8E4). The sample or solution may contain less than about 100 μM of detergent, for example, less than about 50 μM, for example, less than about 20 μM, for example, less than about 10 μM, for example, less than about 1 μM, for example, less than about 0.1 μM, or less than 0.01 μM. Similarly, non-synthetic biomembrane materials typically do not contain, or at least substantially, detergent.

[0086] gas-phase proto-membrane protein ions The method described herein involves generating gas-phase proto-membrane protein ions from a sample, and then selectively removing solvent and bulk lipid molecules from the gas-phase proto-membrane protein ions to generate intact gas-phase membrane protein ions.

[0087] The gas-phase proto-membrane protein ion contains intact membrane proteins nonspecifically bound to one or more bulk lipid molecules and one or more residual solvent molecules. The gas-phase proto-membrane protein ion is generated by ionizing the sample. As described above, the sample typically contains intact membrane proteins associated with bulk lipid molecules in the form of a membrane (typically a lipid bilayer). Generally, the membrane is simply a submicron-sized fragment, rather than an entire vesicle or other object. Therefore, the gas-phase proto-membrane protein ion typically contains intact membrane proteins associated with bulk lipid molecules in the form of a membrane (typically a lipid bilayer, but monolayers are also possible), and residual solvent molecules.

[0088] Proto-membrane protein ions can be charged ions directly generated by the ionization of a sample. However, more generally, ions generated by the ionization of a sample travel through the inlet to the mass spectrometer, and after passing through various stages of vacuum, they release several nonspecifically bound molecules when they reach the position where they are confined and subsequently irradiated. Thus, proto-membrane protein ions also refer to ions derived from ions directly generated by the ionization of a sample, with the loss of one or more neutral or ionic bulk lipid molecules and / or one or more solvent molecules.

[0089] Gas-phase proto-membrane protein ions may more concisely be referred to herein as “gas-phase PMP ions” or “initial gas-phase MP ions.” Alternatively, since ions must be understood to be in the gas phase once they enter through the inlet of the mass spectrometer, gas-phase proto-membrane protein ions may simply be referred to herein as proto-membrane protein ions.

[0090] Residual solvent molecules are molecules of the solvent present in the sample. Therefore, while residual solvent molecules are typically water molecules, they can also include other solvent molecules such as alcohol molecules.

[0091] While some lipid and solvent molecules may be lost during the ionization process, generally speaking, gas-phase PMP ions contain multiple bulk lipid molecules and multiple residual solvent molecules.

[0092] Generally, PMP ions contain at least five bulk lipid molecules. For example, gas-phase PMP ions may contain 20 or more, 50 or more, or even 100 or more bulk lipid molecules. Naturally, in practice, this process results in a large distribution of gas-phase PMP ions, with each gas-phase PMP ion having a different number of bulk lipid molecules.

[0093] Similarly, gas-phase PMP ions generally contain at least one residual solvent molecule. For example, gas-phase PMP ions may contain 50 or more, 100 or more, or even 1000 or more residual solvent molecules. In this case as well, the process actually results in a large distribution of PMP ions where the number of residual solvent and bulk lipid molecules differs for each PMP ion.

[0094] Typically, gas-phase PMPs contain at least substantially no detergent. Typically, gas-phase PMPs contain fewer than 1000 detergent molecules, e.g., fewer than 100, e.g., fewer than 50, e.g., fewer than 20, e.g., fewer than 10, e.g., fewer than 5, 4, 3, 2, or 1. In some embodiments, gas-phase PMPs contain no detergent at all.

[0095] This process further includes irradiating with gas-phase PMP ions to separate residual solvent molecules and nonspecifically bound bulk ligand molecules. This process generates intact membrane protein ions in the gas phase. Since this involves the liberation of intact membrane protein ions from residual solvent molecules and nonspecifically bound bulk ligand molecules, the intact membrane protein ions in the gas phase may also be referred to as "liberated intact membrane protein ions," "gas-phase liberated intact membrane protein ions," "gas-phase intact membrane protein ions, or intact membrane protein ions." More concisely, the intact membrane protein ions in the gas phase may be referred to as "gas-phase IMP ions" or IMP ions.

[0096] Because solvent molecules are small and often easily separated, it is generally possible to remove all solvent molecules from intact membrane proteins during the ionization and irradiation process. Therefore, intact membrane protein ions in the gas phase do not contain residual solvent molecules.

[0097] The irradiation process can completely "evaporate" bulk lipid molecules and solvent molecules from the gas-phase PMP ions. However, a key advantage of this irradiation process is that the degree of evaporation can be finely controlled to limit or completely avoid the simultaneous loss of other non-covalently bound protein-specific ligands from the membrane protein, thus avoiding the dissociation of protein substructures. This means that the degree to which bulk lipid molecules are removed can be finely controlled compared to the control given by CID-based methods of the art. The method of the present invention makes it possible to remove all bulk lipid molecules and all solvent molecules. The intact membrane protein ions in the gas phase generally consist of intact membrane proteins (including any ligands complexed with them). The intact membrane protein ions in the gas phase consist only of intact membrane proteins (along with all of the ligands complexed with them at the time of the generation of the initial PMP ions from which they were induced). For example, the intact membrane protein ions in the gas phase may consist only of intact membrane proteins in their ionized form. That is, the intact membrane protein ions in the gas phase are completely free from bulk lipids and solvent molecules. Therefore, intact membrane protein ions in the gas phase do not contain bulk lipid molecules or solvent molecules (i.e., they contain 0 bulk lipid molecules and 0 solvent molecules).

[0098] When an intact membrane protein ion in the gas phase is bound to one or more (protein-specific) ligands, the intact membrane protein exists within that intact membrane protein ion in the form of a complex with one or more ligands. As described above, ligands, if present, are usually non-covalently bound to the membrane protein. For example, if it is desirable to study the binding between ligands and membrane proteins, it may be advantageous for the intact membrane protein ion in the gas phase to contain ligands. Therefore, an intact membrane protein ion in the gas phase may contain one or more non-covalently bound ligands (ligands bound to the membrane protein).

[0099] The ligands are as described herein. Therefore, the ligands are preferably selected from lipids, RNA chains, metabolites, drugs, and metal cofactors. If the ligand is a lipid, the lipid is different from the bulk lipid molecule; that is, the chemical structure of this lipid is different from the chemical structure of the bulk lipid molecule.

[0100] In other embodiments, the structure of membrane proteins may be studied in the absence of ligands. Therefore, in some cases, the intact membrane protein ions in the gas phase do not contain nonspecifically bound molecules of other non-lipids. (i.e., the intact membrane protein ions in the gas phase do not contain either bulk lipid molecules or other nonspecifically bound molecules.) Specifically, in some cases, the intact membrane protein ions in the gas phase do not contain ligands.

[0101] In intact membrane protein ions in the gas phase, it is preferable for the membrane protein to maintain its native conformation.

[0102] The intact membrane protein ions in the gas phase themselves can be directly analyzed and detected by m / z analysis. Advantageously, one or more ions derived from the intact membrane protein ions in the gas phase can be analyzed and detected by m / z analysis. These downstream products of the intact membrane protein ions in the gas phase can be generated in a variety of different ways, as described in more detail below. Generally, references to ions derived from intact membrane protein ions in the gas phase should be understood as referring to product ions generated from the intact membrane protein ions in the gas phase. Product ions directly generated from intact membrane protein ions in the gas phase are referred to as first-generation product ions. Product ions generated from first-generation product ions may be referred to as second-generation product ions. Second and subsequent-generation product ions may be referred to as next-generation product ions.

[0103] Therefore, ions derived from intact membrane proteins in the gas phase are product ions of any generation produced by one or more ion-to-ion, ion-neutral, ion-surface, photon-ion, and electron-ion interactions. There are many different such ion-to-ion processes known in the art. Intact membrane protein ions in the gas phase, or first-generation or next-generation product ions, can be converted into one or more product ions by such processes. Some are applicable to cations, some to anions, and some to both. These one or more processes may be simultaneous or sequential and may involve the generation of any number of intermediate derived product ions, including zero, which are further subjected to such processes.

[0104] Therefore, ions derived from intact membrane protein ions in the gas phase can be defined as product ions generated from intact membrane proteins in the gas phase by an ion conversion process.

[0105] The ion conversion process and the interactions may be dissociative, such as ion-neutral collisions used to achieve various forms of CID, single-photon-ion interactions used to achieve ultraviolet photodissociation (UVPD), or multiphoton interactions used to achieve IRMPD. The ion conversion process may involve multiple types of interactions occurring simultaneously, such as in activated-ion electron transfer dissociation (AI-ETD), in which a polyvalent analyte polypeptide cation undergoes a dissociative electron extraction reaction with an electron transfer reagent anion while also being irradiated with IR photons.

[0106] The ion conversion process and the interaction are reactive and may involve the formation and breakdown of both covalent and non-covalent chemical bonds. For example, an intact membrane protein ion or an induced ion may be subjected to ion-ion interactions with a reagent ion, to which the reagent ion may attach by either covalent or non-covalent bonds.

[0107] The ion conversion process and interaction described may be charge reduction. An example of such a charge reduction interaction is a non-dissociative ion-electron type reaction (ECnoD), in which a free electron is captured by an intact membrane protein cation or cationic intermediate inducer ion in the multivalent precursor gas phase, and the resulting charge reduction radical ion remains stable and does not dissociate. A further example of a charge reduction interaction is an ion-ion-proton transfer reaction. For an intact membrane protein cation in the gas phase or an induced cationic ion ionized through multiple protonations, the reaction with a proton transfer reagent anion removes a proton from the cation without inducing dissociation, resulting in a 1-charge reduction product ion.

[0108] Product ions can, in some cases, be fragments of intact membrane protein ions in the gas phase. Fragments are ions produced by the dissociation of another ion (e.g., intact membrane protein ions in the gas phase). Fragments are typically produced by the dissociation of covalent bonds. Products resulting from the cleavage of one or more non-covalent subunits or ligands from intact membrane protein in the gas phase are also considered fragments. Fragments can be obtained directly by dissociating intact membrane protein ions in the gas phase, or by dissociating fragmentation products of intact membrane protein ions in the gas phase (e.g., by fragmenting first-generation or next-generation product ions).

[0109] It should be understood that product ions derived from intact membrane protein ions in the gas phase may include ions that have been charge-modified (generally via ion-ionic or ion-electron reactions) to increase or decrease their charge, in order to aid in m / z analysis and detection, and the interpretation of the corresponding m / z spectra.

[0110] Step (i) - Provision of samples A preferred process for providing samples containing non-synthetic biomembrane materials is described in International Application No. PCT / GB2019 / 052421, which is incorporated herein by reference in its entirety.

[0111] Generally, step (i) involves isolating a non-synthetic biomembrane material from a biological sample. The biological sample is not particularly limited. For example, it may be a blood sample, a saliva sample, a bacterial culture sample, for example, an aggregate of cells from a biopsy, an entire organ composed of multiple cell types, for example, the brain or pancreas, or a tissue culture, for example, cells grown outside of a living organism.

[0112] Non-synthetic biomembrane materials may include proteins directly released from a biological sample. In other cases, some initial processing of the sample may be required to access the membrane material of interest. For example, if the membrane protein of interest is contained in a membrane inside a cell, step (i) may involve releasing the membrane from the cell by lysing the cell. For example, if the non-synthetic biomembrane of interest is the membrane of an organelle or intracellular vesicle, the process may include providing a sample containing a living cell and lysing this cell to release the organelle and / or intracellular membrane.

[0113] Furthermore, in a preferred embodiment, step (i) includes disrupting a non-synthetic biological membrane to generate a non-synthetic biological membrane material. It has been previously found that disrupting the cell membrane before ionization generates gas-phase proto-membrane protein ions that are more readily analyzable. Furthermore, the inventors have previously found that this membrane disruption, which generates intact membrane proteins associated with membrane fragments (typically submicron-sized membrane fragments), can be efficiently and conveniently achieved by sonication. Therefore, in one embodiment, step (i) includes sonicating the sample.

[0114] Suitable ultrasonic treatment conditions are discussed in international application PCT / GB2019 / 052421.

[0115] The sample may be sonicated for more than 1 minute. The sample may be sonicated for less than 5 minutes. For example, the sample may be sonicated for 2 to 4 minutes, for example, 2 to 3 minutes, for example, 2.5 minutes.

[0116] The sample may be intermittently sonicated, for example, by periodically applying and removing ultrasound. For example, ultrasound may be applied in cycles of 1-5 seconds "on" and 3-10 seconds "off," or for example, 2-4 seconds "on" and 5-7 seconds "off." When the sample is intermittently sonicated, the "off" period is still taken into consideration when calculating the duration for which sonication is applied.

[0117] Ultrasonic treatment can be applied using probe sonicators such as the Vibra-Cell VCX-500 Watt or Sonics.

[0118] Ultrasonic treatment can be carried out at temperatures below 20°C, for example, below 15°C, or for example, below 10°C. In some embodiments, ultrasonic treatment may be carried out on ice. This is to prevent the sample from heating as a result of ultrasonic treatment.

[0119] Therefore, in a particularly preferred embodiment, step (i) includes isolating a non-synthetic biomembrane from a biological sample and disrupting the non-synthetic biomembrane to produce a non-synthetic biomembrane material. Typically, the disruption process produces submicron-sized membrane fragments associated with intact membrane proteins. Preferably, the disruption is carried out by sonication.

[0120] If the sample does not contain a mass spectrometry-compatible buffer, step (i) typically also includes suspending the non-synthetic biomembrane material in an aqueous solution containing a mass spectrometry-compatible buffer. This step may be performed before or after the sonication step, if one is performed. Examples of mass spectrometry-compatible buffers include ammonium buffers such as ammonium acetate or ammonium bicarbonate. Ammonium acetate is particularly preferred.

[0121] Furthermore, aqueous solutions containing mass spectrometry-compatible buffers typically have ionic strengths similar to those under physiological conditions, as well as a pH of approximately 5–8. These conditions are considered to best preserve the confirmation of the native nature of intact membrane proteins, including non-covalent interactions with any ligand, through the process of ionization to the gas phase.

[0122] In practice, a sample generally contains multiple membrane proteins. A sample may contain multiple chemically identical membrane proteins (i.e., proteins having the same amino acid sequence, the same post-translational modifications, etc.). Typically, a sample contains multiple different membrane proteins. The membrane proteins present may or may not have the same amino acid sequence. If a sample contains multiple membrane proteins with the same amino acid sequence, the present membrane proteins may nevertheless be chemically different from one another (e.g., by the number and / or position of post-translational modifications). Therefore, a sample typically contains multiple membrane proteins with different chemical structures. For example, a sample may contain two or more membrane proteins, each having a different chemical structure.

[0123] Process (ii) - Ionization In step (ii), the sample from step (i) is ionized to generate proto-membrane protein ions as defined above. The ionization process is generally carried out by some form of electrospray ionization, preferably nano-electrospray ionization or static electrospray ionization. Any ionization method capable of generating gas-phase proto-membrane protein ions (as defined above) would be suitable for carrying out the present invention.

[0124] In the nanoelectrospray ionization process, nano-sized droplets are generated. This can be achieved, for example, by routine adjustment of electrospray conditions using an electrospray capillary emitter (particularly one with a tip having a diameter of less than 10 μm, preferably less than 5 μm, most preferably 0.5 to 2 μm).

[0125] In a preferred embodiment, the ionization process of step (ii) is a native electrospray ionization process. In a native electrospray ionization process, the conditions during the electrospray procedure are kept close to physiological conditions. Specifically, the pH of the sample during the electrospray process is maintained at a physiological pH, typically between 5 and 8. Under such conditions, the native conformation of intact membrane proteins is optimally preserved.

[0126] Electrospray ionization is typically carried out at or near atmospheric pressure. The resulting gas-phase proto-membrane protein (PMP) ions, as described herein, contain bulk lipid molecules (in some cases, even entire vesicles) along with embedded intact membrane proteins. The intact membrane proteins (in the sample, as well as in the gas-phase PMP ions and gas-phase intact membrane protein ions) may exist, for example, in the form of complexes with one or more ligands. The gas-phase PMP ions also contain residual solvent molecules.

[0127] Due to the transport of gas-phase PMP ions generated by the electrospray process from the near-atmospheric pressure region where they were generated into the vacuum of the mass spectrometer, some degree of desolvation and separation of bulk lipid molecules typically occurs immediately after the ionization process.

[0128] Following ionization, the proto-membrane protein ions are transported to the vacuum of the mass spectrometer by passing the sample through a heated capillary tube or preferably a small pore into a region of pressure considerably lower than atmospheric pressure. In the case of static electrospray ionization and nano-electrospray ionization, the emitter tip is located within a few millimeters of the capillary inlet or pore. For example, this process may involve passing the proto-membrane protein ions through a capillary tube heated to a temperature above 250°C, e.g., 250–450°C, e.g., 300–400°C. These high temperatures help remove residual solvent molecules without denaturing the intact membrane proteins.

[0129] The ionization process can be carried out using a mass spectrometer equipped with a appropriately configured nanoelectrospray ion source. A preferred example is a Thermo Scientific® Orbitrap Eclipse® Tribrid® mass spectrometer equipped with a Thermo Scientific® NanoSpray Flex NG® ion source configured for static native electrospray ionization. This instrument utilizes a heated metal capillary atmosphere against a vacuum interface. Modified versions of the instrument having its ion source and ion source configuration have been used to carry out the exemplary methods described herein.

[0130] In practice, as described above, the sample generally contains multiple membrane proteins. Therefore, ionizing the sample as in step (ii) typically generates multiple gas-phase protomembrane protein ions (also referred to as multiple gas-phase PMP ions or a group of gas-phase PMP ions). Each gas-phase protomembrane protein ion is as described herein.

[0131] Each gas-phase proto-membrane protein ion contains intact membrane proteins. The intact membrane proteins present in each of the gas-phase PMP ions within a group of gas-phase PMP ions may be the same or different. In some cases, each gas-phase PMP ion within a group of gas-phase PMP ions contains intact membrane proteins having the same chemical structure. Alternatively, the group of gas-phase PMP ions may contain intact membrane proteins having different chemical structures. Each gas-phase PMP ion within a group of gas-phase PMP ions may contain intact membrane proteins having different chemical structures. For example, a group of gas-phase proto-membrane protein ions may contain two or more gas-phase proto-membrane protein ions, each containing intact membrane proteins having different chemical structures.

[0132] During the ionization of a sample, the resulting gas-phase PMP ions have a distribution of charge states. Each gas-phase PMP ion within this group can be ionized to one of several charge states (even if each gas-phase PMP ion has the same amino acid sequence or is composed of intact membrane proteins that are chemically identical). For example, the group of gas-phase PMP ions may contain two or more gas-phase PMP ions, each having a different charge state.

[0133] Furthermore, each gas-phase PMP ion contains one or more bulk lipid molecules and one or more residual solvent molecules. The number of bulk lipid molecules present in each gas-phase PMP ion within a group of gas-phase PMP ions may be the same or different. Similarly, the number of residual solvent molecules present in each gas-phase PMP ion within a group of gas-phase PMP ions may be the same or different. Generally, for a large number of gas-phase PMP ions, it includes gas-phase PMP ions having a broad distribution of the number of bulk lipid molecules.

[0134] The ionization step generates gas-phase PMP ions as described above. However, this is typically not suitable for m / z analysis or m / z selection for tandem m / z spectroscopic experiments (m / z is the mass-to-charge ratio expressed as m, where m is the number of elementary charge elements and m is the mass per z). This is because, in practice, each gas-phase PMP ion in the gas-phase PMP ion population contains a variable number of bulk lipid molecules and a variable number of residual solvent molecules, so the population of gas-phase PMP ions typically produced in step (ii) generally has a very broad m / z distribution. Furthermore, even within multiple gas-phase PMP ions, including individual gas-phase PMP ions containing the same membrane protein, as well as the type and number of bulk lipid and solvent molecules (same chemical composition), the gas-phase PMP ions will have a variety of charge states. Therefore, the distribution of the generated gas-phase PMP ions will appear in the m / z spectrum only as a continuity of unresolved ion signals that produce a somewhat elevated baseline, essentially indistinguishable from noise. To produce an m / z spectrum with discrete, representative m / z peaks having assignable average m / z values, the substantial fraction of gas-phase PMP ions would need to be freed of all of their bulk lipid molecules and residual solvent molecules.

[0135] Step (ii) moves the membrane protein (in the form of PMP ions) to the gas phase. Therefore, all PMP ions and all ions derived therefrom (intact membrane protein ions in the gas phase, product ions, etc.) are gas-phase ions. Such ions can be interpreted as being in the gas phase even if there is no explicit reference to the gas phase.

[0136] Process (iii) - Confinement Once gas-phase PMP ions are generated and transported into the vacuum of a mass spectrometer, they are then confined. "Confined" means that the ions are localized within an electromagnetic field. This localization may be around a point, along an axis, or along a curved path. Confinement indicates the boundary of the ion's motion and position in either two or three dimensions. A confined gas-phase proto-membrane protein ion may be referred to as a "confined gas-phase proto-membrane protein ion," while the gas-phase PMP ion itself is defined as described above.

[0137] The gas-phase protomembrane protein ions, and the intact gas-phase membrane proteins that are converted in step (iv), are confined throughout step (iv) and generally throughout at least a portion of step (v) of the Method (i.e., their motion is bounded in at least two dimensions in an electromagnetic field). If one or more further steps are performed after step (iv), any ions generated therein may also be confined during these steps.

[0138] In practice, multiple gas-phase proto-membrane protein ions are generally confined in step (iii). Multiple gas-phase proto-membrane protein ions are as defined above. The group of gas-phase PMP ions is typically confined or localized within a certain volume of space. The localized group of gas-phase PMP ions may be referred to as an ion cloud. Thus, the following optional method steps may be similarly carried out for multiple intact gas-phase membrane protein ions generated in step (iv). Alternatively or additionally, such processes may be carried out for multiple ions derived from intact gas-phase membrane protein ions, or for multiple intact gas-phase membrane protein ions generated in step (iv).

[0139] Using any suitable apparatus, gas-phase proto-membrane protein ions (and / or intact gas-phase membrane protein ions and / or ions derived therefrom) can be confined. Suitable examples of apparatus that can be used to confine these species include mass filters (such as quadrupole mass filters), radio frequency ion guides (such as radio frequency two-dimensional multipole ion guides, radio frequency stacked ring ion guides, or radio frequency stacked ring ion funnels), or ion traps (a typical example being a double-pressure radio frequency quadrupole linear ion trap). One example is a radio frequency multipole ion guide (ion routing multipole), which can operate as an ion guide or ion trap by adjusting the magnitude of the radio frequency (RF) and direct current (DC) potentials applied to its electrodes.

[0140] The confinement of proto-membrane protein ions, intact membrane protein ions, or ions derived from membrane protein ions can be carried out using one or more such devices. Using two or more components of such devices, any such type of ion can be confined during each of steps (iii), (iv), and (v).

[0141] For example, the exemplary experiments described herein are performed with an improved Thermo Scientific® Orbitrap Eclipse® Tribrid® mass spectrometer, where gas-phase proto-membrane protein ions, generated by a source and passing through a heated metal inlet capillary, are immediately confined by an RF stacked ring ion funnel and then permeated through a further pair of RF multipole ion guides, which move the gas-phase PMP ions to a quadrupole mass filter, and from there to the high-pressure region of a dual-pressure RF quadrupole linear ion trap analyzer via further RF ion guides, including an RF C-trap, an ion-routing multipole (collision cell), and another RF multipole ion guide. Thus, the confinement of gas-phase proto-membrane protein ions is achieved by the RF ion funnel, a pair of RF multipole ion guides, an RF quadrupole mass filter, a third RF multipole ion guide, an RF C-trap, an ion-routing multipole / trapping collision cell, a fourth RF multipole ion guide, and an RF quadrupole linear ion trap. Intact membrane protein ions in the gas phase are confined within an RF quadrupole ion trap during step (iv). To enable and achieve various ion transformation processes that may optionally occur during step (v), intact membrane protein ions and ions derived from intact membrane protein ions can move to, be confined in, and move out of both ion-routing multipole / trapping collision cells (collision cell type CID) and the high-pressure region of the RF quadrupole linear ion trap (ion trap type resonant CID, IRMPD, ETD, and proton-transfer charge reduction ion-ion reactions). In step (v), to achieve an optional sub-step of m / z isolation of intact membrane protein ions or ions derived therefrom, each ion is confined in the high-pressure region of the RF quadrupole linear ion trap.

[0142] In step (v), to perform m / z analysis of intact membrane protein ions or ions derived therefrom using an Orbitrap m / z analyzer, each ion moves to an RF C-Trap, is confined therein, and is then radially released into a high orbitrap m / z analyzer. In step (v), to perform m / z analysis of intact membrane protein ions or ions derived from intact membrane protein ions using an RF linear ion trap m / z analyzer, each ion moves to a low-pressure region of an RF quadrupole linear ion trap, is confined therein, and is then m / z selectively and continuously released into an electron multiplier tube-based ion detector.

[0143] This configuration is described only to illustrate, and not to limit, various parts of the apparatus that may be used to contain the ions generated during the method of the present invention. Other preferred arrangements of one or more RF ion guides, RF quadrupole mass filters, RF quadrupole ion traps, and m / z analyzers can be readily conceived by those skilled in the art.

[0144] The confinement of gas-phase PMP ions (and other ions generated during this method) is, of course, essential to spatially localize the ions so that they can be exposed to infrared radiation of sufficient intensity and duration during step (iii), and so that the intact membrane protein ions thus generated in the gas phase can be optionally exposed to further processes to ultimately produce induced ions that are ultimately directed to and detected by an m / z analyzer. However, additional advantageous processes may be carried out during the confinement of ions (such as gas-phase PMP ions).

[0145] The confined ions can be exposed to the collision gas. Exposure to the collision gas can be used at different time points in this method to achieve many different results.

[0146] For example, exposure of gas-phase proto-membrane protein ions to a collision gas can remove one or more residual solvent molecules and bulk lipid molecules in the collision-induced dissociation process. Therefore, exposure to the collision gas can help remove these undesirable residual solvent and bulk lipid molecules. Exposure of gas-phase PMP ions to the collision gas for the removal of residual solvent and bulk lipid molecules may occur before and / or during step (iv). Preferably, exposure of gas-phase PMP ions to the collision gas for the removal of residual solvent and bulk lipid molecules occurs before step (iv) and during step (iii).

[0147] Exposure to the collision gas may also be used to aid in the fragmentation of intact membrane protein ions in the gas phase generated in step (iv), and / or ions derived therefrom. Thus, the process may, after step (iv), include exposing the intact membrane protein ions in the gas phase in the CID process to the collision gas to dissociate the intact membrane protein ions in the gas phase and generate two or more fragments. Alternatively or additionally, the process may include exposing product ions derived from the intact membrane protein ions in the gas phase to the collision gas to further dissociate the product ions and generate two or more fragments. The fragments thus generated may be, for example, ligands dissociated from the membrane protein (such as nonspecifically bound ligands), subunits of the membrane protein (if the membrane protein is a multimeric complex), or fragments generated by the cleavage of covalent bonds within the membrane protein.

[0148] In such cases, if exposure to a collision gas is used to aid in the fragmentation of intact membrane protein ions and / or derived product ions in the gas phase generated in step (iv), the ions to be fragmented must generally be confined in an RF ion trap or multipole collision cell at relatively high pressure (generally greater than 1 mTorr). Thus, in such cases, the process may include, after step (iv), transferring the intact membrane protein ions or derived product ions in the gas phase to a high-pressure ion trap or collision cell.

[0149] As described above, the collision gas used to dissociate intact membrane protein ions or derived product ions in the gas phase of an RF ion trap (typically an RF quadrupole linear ion trap) via a resonance trap type CID is typically a very light gas such as hydrogen or helium, preferably helium, because the use of high molecular weight gases is likely to hinder the device's ability to be used for m / z isolation and m / z analysis. In such cases, the pressure of the collision gas in the ion trap (e.g., an RF quadrupole linear ion trap) is typically in the range of 1 to 10 mTorr. The collision gas used to dissociate intact membrane protein ions or derived product ions in the gas phase of an impact cell is generally a noble gas such as neon, argon, or xenon, or nitrogen (N2) or sulfur hexafluoride (sulfur The impacting gas is a relatively unreactive molecular gas such as hexafluoride (SF6), and generally nitrogen or argon is preferred. In such cases, the pressure of the impacting gas in the impacting cell may be, for example, 1 to 20 mTorr or more.

[0150] Collision-induced dissociation (CID) fragmentation is a less preferred method of fragmentation according to this method. When applied to intact membrane protein ions, it tends to result in lower yields of sequence fragment ions, as well as relatively higher yields of side chain and loss fragment ions. Nevertheless, fragmentation by CID can be a useful technique and is an example of an ion conversion process as described herein. Other more preferred ion conversion processes (in particular IRMPD) are described below, and these methods are also carried out while the ions are confined, for example, in an RF quadrupole linear ion trap or collision cell.

[0151] However, of particular importance, the effect of the presence of the collision gas in the method of the present invention is to provide a cooling effect during step (iv). During step (iv), when gas-phase PMP ions are conserved in the presence of the collision gas, near-thermal energy collisions with gas atoms or molecules extract vibrational energy from the gas-phase PMP ions, counteracting the heating effect of infrared irradiation. This allows the gas-phase PMP ions to reach a certain level of vibrational excitation equilibrium when exposed to infrared irradiation. Therefore, by controlling the gas pressure and properties of the gas present during step (iv), further control over the heating of the gas-phase proto-membrane protein ions is possible and can be used to optimize the production of intact membrane protein ions in the gas phase. Thus, step (iv) can preferably be carried out while the gas-phase proto-membrane protein ions are confined in the presence of the collision gas (e.g., in an RF quadrupole linear ion trap).

[0152] As described above, suitable collision gases that can be used to cool the gas-phase proto-membrane protein ions include nitrogen, hydrogen, or a noble gas such as helium, argon, or neon, preferably hydrogen, or more preferably helium, when the gas-phase PMP ions are confined within an RF quadrupole linear ion trap analyzer. In such cases, the pressure of the collision gas may be, for example, less than 10 mTorr, e.g., 5 to 10 mTorr, typically about 7 mTorr.

[0153] For any specific ion confinement device used to confine gas-phase proto-membrane protein ions, the characteristics (wavelength, beam power, beam diameter, etc.) and optimal settings of the infrared photon beam irradiated in step (iv) of the method according to the present invention should be readily determined experimentally.

[0154] The advantage of confining ions generated during this method arises not only from the ability to expose such confined ions to the collision gas, but also from the ability to select a specific ion m / z range, such that ions with m / z outside the selected range are often not confined and therefore excluded. All RF ion confinement devices have m / z dependence in ion confinement. RF quadrupole field devices, such as RF quadrupole m / z filters and RF quadrupole linear ion traps, can operate to have very sharp transitions between m / z confinement and m / z emission or non-confinement ranges. Numerous techniques for achieving selection of a specific m / z range using RF quadrupole field devices are well known in the art. Depending on the specific device, the m / z selection range may be narrower than 0.1% of m / z at the midpoint of the selected m / z range. When performed on an ion beam, the selection of a range of mass-to-charge ratios is often referred to in the art as "mass selection," and somewhat more precisely, as "m / z selection." The selection of a specific mass-to-charge ratio or range of mass-to-charge ratios, and the exclusion of ions with m / z values ​​outside that selected range when performed on a captured ion population, is often referred to in the art as "mass isolation," and somewhat more precisely as "m / z isolation." For the purposes of this specification, m / z isolation and m / z selection may be considered synonymous.

[0155] m / z isolation may be performed in relation to gas-phase proto-membrane protein ions, gas-phase intact membrane protein ions, or ions derived from gas-phase intact membrane protein ions. This allows for the analysis of gas-phase proto-membrane protein ions or gas-phase intact membrane protein ions having a specific m / z ratio or being within a specific m / z range. This ability to m / z select ions or groups of ions to be subjected to further transformation processes allows for a more detailed analysis of the structure and composition of the membrane proteins therein. After the generation of any ions, the process may include a step of performing m / z isolation to isolate ions having a specific m / z or m / z value range. This may be referred to as m / z isolation or m / z selection.

[0156] The m / z selection of gas-phase proto-membrane protein ions, gas-phase intact membrane protein ions, or ions derived from gas-phase intact membrane protein ions can be performed rather coarsely in, for example, an RF ion guide and much more precisely in an RF quadrupole linear ion trap analyzer or a quadrupole m / z filter. Ions with undesirable m / z ratios may be emitted from the RF ion guide, RF quadrupole linear ion trap, or RF quadrupole m / z filter. This may involve actively adjusting the magnitude and frequency components, or in some examples applying an additional type of voltage to the RF ion guide, RF quadrupole linear ion trap, or RF quadrupole m / z filter electrodes to emit ions in a specific m / z range, or simply allowing ions with undesirable m / z ratios to leak out.

[0157] In practice, the process involves pre-determining a desired range of m / z ratios to be retained, and then releasing ions with m / z ratios outside that range from an RF ion guide, RF quadrupole linear ion trap, RF quadrupole m / z filter, or other device in which the ions are confined.

[0158] For example, in some embodiments of the method described herein, during step (iii) and / or step (iv), gas-phase PMP ions and / or intact membrane protein ions in the gas phase are retained in an ion trap or permeated through an ion guide or mass filter, and the method is The method includes isolating gas-phase PMP ions and / or intact membrane protein ions in the gas phase by releasing one or more ions outside a predetermined m / z window from an RF ion guide, an RF quadrupole linear ion trap, and an RF quadrupole m / z filter.

[0159] This m / z selection step can, of course, be repeated before and / or after an ion conversion process performed on intact membrane protein ions in the gas phase and ions derived therefrom. For example, in some embodiments of the method described herein, after step (iv), intact membrane protein ions in the gas phase or ions derived therefrom are held in an ion trap or permeated through an ion guide or mass filter, and the method is performed The method includes m / z isolation or m / z selection of intact membrane protein ions in the gas phase or ions derived therefrom by releasing one or more m / z ions outside a predetermined m / z range from an ion trap, ion guide, or mass filter (generally, an ion trap, ion guide, or mass filter is an RF ion guide, an RF quadrupole linear ion trap, or an RF quadrupole m / z filter).

[0160] Step (iv) - Irradiation Step (iv) of this method involves irradiating the confined gas-phase proto-membrane protein ions with infrared radiation from a laser source to separate one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules from the intact membrane protein, thereby generating intact gas-phase membrane protein ions. The intact gas-phase membrane protein ions are as described above.

[0161] The inventors have found that by using irradiation with infrared radiation from a laser source, solvent molecules and, surprisingly, bulk lipid molecules can be selectively and completely "evaporated" from gas-phase proto-membrane protein ions to produce intact gas-phase membrane protein ions. The inventors have found that the use of such IR activation generally produces membrane-integrated membrane protein ions from multiple gas-phase PMP ions in higher yields than by using well-optimized conventional inlet CIDs.

[0162] While we do not wish to be bound by theory, it is hypothesized that infrared radiation is in near-resonance with one or more vibrational modes of bulk lipid molecules. Therefore, infrared radiation may be absorbed more efficiently by those molecules, leading to the dissociation of those molecules from gas-phase proto-membrane ions, in preference to the absorption of radiation by the membrane proteins themselves and the resulting dissociation. This resonance or near-resonance overlap of photon wavelengths and covalent absorption bands in bulk lipids is hypothesized to allow for higher absorption of IR radiation per vibrational degree of freedom for bulk lipids than for membrane proteins. Thus, with appropriate selection of laser power settings and exposure time, this differential heating separates relatively low-affinity bulk lipid molecules from membrane proteins without dissociating the membrane proteins themselves during step (iv).

[0163] It can be further inferred that solvent molecules have a lower binding affinity than bulk lipids, and even if solvent molecules have roughly the same IR energy absorption per vibrational degree of freedom as membrane proteins, the IR radiative flux sufficient to separate bulk lipids is more than sufficient to separate solvent molecules.

[0164] Therefore, infrared irradiation selectively separates bulk lipid molecules and residual solvent molecules, resulting in a high yield of intact membrane protein ions in the gas phase. As described above, intact membrane protein ions in the gas phase do not contain bulk lipid molecules or residual solvent molecules.

[0165] Both fixed-wavelength and tunable-wavelength infrared laser sources are known. Fixed-wavelength laser sources generally offer advantages such as high power output, ease of use, high commercial availability, and significantly lower cost. Tunable-wavelength IR laser sources allow for the optimization of the IR wavelength used in readily available methods. For example, it is expected to be particularly advantageous to be able to select the output wavelength of infrared radiation from an IR laser source that has the best resonance overlap with bulk lipid molecules present in the gas-phase proto-membrane protein ions generated in a particular analysis. This may allow for the optimization of the selective loss of bulk lipid molecules from gas-phase PMP ions and the maximization of the probability of generating intact gas-phase membrane protein ions from each gas-phase proto-membrane protein ion.

[0166] Therefore, in a preferred embodiment of the present method, the wavelength of the infrared radiation output from the laser source is selected to preferentially excite one or more vibrational modes of one or more bonds in nonspecifically bound bulk lipid molecules. A suitable wavelength may be selected by varying the wavelength of the infrared radiation and repeating the disclosed method until intact membrane protein ions can be detected after step (iv).

[0167] The laser source may be a continuous-wave laser source or a pulsed laser source. Pulsed laser sources generally have the characteristic that the output pulse length, pulse frequency, and potentially pulse output power can be adjusted, and therefore, in connection with carrying out the disclosed method, the yield of intact membrane protein ions from irradiated gas-phase proto-membrane protein ions can be optimized through iteration of the method and variation of laser parameters. Pulsed laser sources may be advantageous in that they may be able to accumulate a larger amount of vibrational energy in gas-phase proto-membrane protein ions, in particular to the preferentially absorbing bonds of attached bulk lipid molecules, at very short time intervals. This may promote rapid heating of bulk lipids and their separation while limiting the transfer of vibrational energy from non-covalent bulk lipids to membrane proteins, thereby reducing membrane dissociation during bulk lipid removal.

[0168] The experimental studies illustrated herein utilized a continuous fixed-wavelength infrared laser. This laser was a CO2 gas laser designed for industrial marking and engraving applications, having a nominal output wavelength of 10.6 μm and a specific nominal maximum output power (uncalibrated) of 60 watts. The laser output power could be controlled by an analog voltage that allowed the output power to be adjusted from 0% to 100% of the maximum output. A pair of adjustable mirrors were used to align the laser beam with the central axis of the high-pressure region of the three segments of an RF quadrupole linear ion trap. During these early pioneering experiments, the RF quadrupole field generation potential and the axial confinement segment DC bias potential were applied to the electrodes of the high-pressure region of the RF quadrupole linear ion trap during irradiation step (iv) of the disclosed method, such that the cloud of gas-phase PMP ions should be radially confined within a radius of less than 1 mm of the device axis and confined in a region along its axis that roughly corresponds to the central segment of the device, which is approximately 37 mm long. The IR laser beam was focused to position the beam waist within a high-pressure quadrupole linear ion trap, and the beam diameter was fairly uniform, with a diameter radius of 2–3 mm along the axial portion of the device where the gas-phase PMP ions were confined, according to a relatively coarse method available for measurement. The laser output power control signal was under the complete control of the instrument's built-in control system, which could operate the laser output to the desired output power to initiate irradiation of gas-phase PMP ions and return it to zero at the end of the irradiation period. According to the laser specifications, the response time of the laser output transitioning in either direction between virtually no output power and stable output power was well less than 1 millisecond. Fixed-wavelength IR lasers with similar specifications are readily available commercially. Common nominal output wavelengths for continuous-wave CO2 gas lasers in the same output power range are 9.3 μm, 10.2 μm, and 10.6 μm.

[0169] Therefore, in some cases, the infrared radiation from the laser source may be pulsed radiation, and in other cases, the infrared radiation from the laser source may be continuous wave radiation. Preferably, the infrared radiation from the laser is continuous wave radiation.

[0170] The wavelength of radiation from a laser source lies in the infrared region of the electromagnetic spectrum. Therefore, the wavelength of infrared radiation is generally 780 nm to 1 mm. Considering that the present invention relies on the absorption of infrared radiation by the vibrational modes of lipid molecules, the wavelength of infrared radiation is preferably a wavelength that overlaps with one or more vibrational modes of bulk lipid molecules. The vibrational modes of bulk lipid molecules tend to be in the micron range. Therefore, preferably, the wavelength of infrared radiation is in the range of 1.4 μm to 1 mm. More preferably, the wavelength is 3 μm to 100 μm, for example, about 5 μm to about 50 μm, for example, about 8 to about 15 μm, and most preferably, the wavelength is 9 to 11 μm.

[0171] Conveniently, the laser source may be a CO2 laser. Therefore, the wavelength of infrared radiation from the laser source may have one or more dominant wavelengths from the following: 10.6 μm, 10.2 μm, 9.6 μm, and 9.3 μm. Particularly preferably, the wavelength of infrared radiation from the laser source has a dominant wavelength of 10.6 μm.

[0172] The irradiation step of the method of the present invention is intended to dissociate all bulk lipid molecules (either neutral or ionic) from the gas-phase proto-membrane protein ions and, if necessary, remove residual solvent molecules. Furthermore, the irradiation step is also intended not to dissociate the membrane proteins. Rather, step (iv) generates intact gas-phase membrane protein ions. The generated intact gas-phase membrane protein ions can hold any, and preferably all, complexed, specifically bound ligands, such as drugs, RNA chains, protein-specific lipids, and post-translational modifications.

[0173] Therefore, the degree of vibrational activation of gas-phase PMP ions during step (iv), which is controlled by factors including irradiation power, is generally significantly lower than the level of vibrational activation required to achieve covalent dissociation within membrane proteins via conventional IRMPD processes.

[0174] It should be understood that, for a given time interval and a given level of vibrational activation of gas-phase PMP ions with respect to the collision gas and collision gas pressure, both non-covalent and covalent dissociation have a certain probability of occurring. As discussed above, the binding energies of non-covalent bonds that hold bulk lipids and solvent molecules within gas-phase PMP ions are generally expected to be lower than the binding energies of non-covalent bonds that bind specifically bound ligand molecules to membrane proteins. They are almost certainly lower than the binding energies of covalent bonds within membrane proteins. Consequently, at preferably low levels of vibrational activation, the dissociation of non-covalent bonds binding solvent molecules and bulk lipids to membrane proteins is far more likely to occur within the activation time interval than the cleavage of any single non-covalent or covalent bond in the membrane protein.

[0175] The preferential absorption of IR radiation by bulk lipids increases the relative vibrational activation per vibrational degree of freedom (temperature) of bulk lipids in gas-phase proto-membrane protein ions to such an extent during the irradiation period that it further biases the probability of various dissociation pathways toward achieving separation of bulk lipids without dissociation of membrane proteins.

[0176] Therefore, the probability of generating intact membrane protein ions in the gas phase from confined gas-phase proto-membrane protein ions can be optimized by using a relatively low laser power compared to the preferred laser power for achieving a combination of covalent and non-covalent dissociation of membrane proteins.

[0177] When multiple gas-phase proto-membrane protein ions are confined in step (iii), these ions are typically irradiated during step (iv) to generate multiple gas-phase intact membrane protein ions, which may also be referred to as a collection of gas-phase intact membrane protein ions. Each gas-phase intact membrane protein ion within the collection of multiple gas-phase intact membrane protein ions is as described herein.

[0178] Each intact membrane protein ion in a set of intact gas phases may be the same or different. In some cases, each intact gas phase membrane protein ion has the same chemical structure. Alternatively, each intact gas phase membrane protein ion in a set of intact gas phases may have different chemical structures. For example, a set of intact gas phases may contain two or more intact gas phase membrane protein ions, each having a different chemical structure.

[0179] The intact membrane protein ions present may also have different charge states. In some cases, each intact membrane protein ion in multiple gas phases may have the same charge. Alternatively, each intact membrane protein ion in multiple gas phases may have different charge states (even if each intact membrane protein has the same amino acid sequence or is chemically identical). For example, multiple intact membrane protein ions in multiple gas phases may contain two or more intact membrane protein ions, each of which may have a different charge state.

[0180] If a population of confined gas-phase proto-membrane protein ions is irradiated during step (iv), ideally, all of the confined gas-phase PMP ions should be exposed to light of the same intensity during irradiation step (iv). In this case, the probability of generating intact gas-phase membrane protein ions is the same as if all of the confined gas-phase proto-membrane protein ions were chemically and structurally identical. The optical system delivering the IR light is preferably configured to provide a high degree of uniformity of the photon flux within the volume in which the gas-phase PMP ions / gas-phase IMP ions are confined. Furthermore, in order to minimize the maximum power output and therefore the size and cost of the IR laser, it is preferable to maximize the photon flux over the entire region in which gas-phase PMP ions are localized during the irradiation period of step (iv) for any given laser power setting. Optimal parameters can be determined by routine trial and error. For example, parameters can be determined by performing repeated experiments and adjusting the laser power output, irradiation time, and cooling impaction gas pressure (within the capabilities of the equipment used) to optimize the yield of intact gas-phase membrane protein ions.

[0181] The gas-phase proto-membrane protein ions (or, if present, multiple gas-phase proto-membrane protein ions) are confined in step (iii). They are typically localized within a defined volume in space. Thus, step (iii) may include confining the gas-phase PMP ions (or multiple gas-phase PMP ions) within a volume. As described above, during step (iv), it is preferable that the infrared radiation from the laser source has a uniform photon beam within the volume. Thus, the laser source may be configured to provide a uniform photon beam within the volume.

[0182] For example, this method typically involves performing irradiation for a period of up to 1 second, preferably less than 50 ms. This method may also involve performing irradiation for a period ranging from 1 μs to 50 ms. The period during which irradiation using infrared radiation from a laser source is performed may be referred to as the "irradiation period." For example, a particular device operating according to this method may be configured to perform irradiation for a duration that may range from 100 μs to 20 ms.

[0183] It should be understood that intact membrane protein ions in the gas phase can form during or immediately after irradiation (while the gas-phase proto-membrane protein ions remain sufficiently vibrationally activated after energy absorption). That is, one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules can separate from the intact membrane protein in the gas-phase proto-membrane protein ions during irradiation. Alternatively, they can separate from the intact membrane protein after irradiation. Therefore, while irradiation is applied to confined gas-phase proto-membrane protein ions, the composition of the gas-phase proto-membrane protein ions can change during irradiation. For example, the gas-phase proto-membrane protein ions can change due to the separation of one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules. Intact membrane protein ions in the gas phase can form from the gas-phase proto-membrane protein ions during irradiation.

[0184] When a population of gas-phase proto-membrane protein ions is irradiated, the rate at which residual solvent molecules and nonspecifically bound bulk lipid molecules separate from the intact membrane protein may differ among the gas-phase proto-membrane protein ions. As an example, consider two populations of gas-phase proto-membrane protein ions, each containing the same intact membrane protein, the same number and type of residual solvent molecules, and the same number and type of nonspecifically bound bulk lipid molecules. Even if the two gas-phase proto-membrane protein ions are subjected to the same irradiation conditions during step (iv), the separation of the residual solvent molecules and the nonspecifically bound bulk lipid molecules is likely to occur at different times.

[0185] Therefore, when irradiation is performed on a population of gas-phase proto-membrane protein ions, the irradiation parameters (such as irradiation duration and laser power) are typically selected to maximize the number of intact gas-phase membrane ions generated from the population of gas-phase proto-membrane protein ions. In other words, these parameters are typically selected to maximize the size of the final population of intact gas-phase membrane protein ions.

[0186] For example, the method may include operating the laser at 12% or less of its available power output during irradiation step (iv), for example, 10% or less of its available power output. In contrast, to achieve efficient IRMPD of identically confined ions under identical conditions using irradiation events of identical duration, it would typically be necessary to operate the laser at more than 10% of its available laser power output, for example, 15% or more of its available laser power output.

[0187] The specific irradiation parameters to be used during irradiation in step (iv) are generally best determined through trial and error. Important irradiation parameters include the length of time the irradiation is performed ("irradiation period"), the power of the laser source (e.g., 3–12% of the available power, equivalent to approximately 1.8–7.2W for a 60W continuous-wave infrared CO2 laser), and the wavelength of the laser source.

[0188] In some embodiments, the laser power is about 0.1 to about 10% (e.g., about 0.06 W to about 6 W) of the power of a 60 W continuous-wave infrared CO2 laser, for example, about 1% to about 8% (e.g., about 0.6 W to about 4.8 W), for example, about 2% to about 6% (1.2 W to about 3.6 W). For example, such power levels are useful in some embodiments for releasing intact membrane proteins from vesicles induced from their native lipid layers. In some embodiments, the laser power is about 10% to about 30% (e.g., about 6 W to about 18 W) of the power of a 60 W continuous-wave infrared CO2 laser, for example, about 12% to about 20% (e.g., about 7.2 W to about 12 W), for example, about 15% to about 18% (e.g., about 9 W to about 10.8 W). For example, such power levels are useful in some embodiments for releasing intact polypeptide chains of membrane proteins or fragments thereof.

[0189] Therefore, in some embodiments, the irradiation in step (iv) is, for example, at wavelengths of about 3 μm to 100 μm, for example, about 5 μm to 50 μm, for example, about 8 to 15 μm, for example, about 9 to 11 μm, at about 0.1% to 30% (e.g., about 0.06 to 18 W), for example, about 1% to 8% (e.g., about 0.6 W to 4.8 W) of a 60 W continuous-wave infrared CO2 laser. This may include irradiating at a power corresponding to, for example, about 2% to about 6% (1.2W to about 3.6W), or about 12% to about 20% (e.g., about 7.2W to about 12W), or for example, about 15% to about 18% (e.g., about 9W to about 10.8W), for a maximum period of about 1 second, preferably about 1 μs to about 50 ms, for example, about 100 μs to 25 ms, or for example, about 5 ms to about 25 ms.

[0190] Further irradiation parameters that may be varied include the size and position of the beam waist, as well as the divergence of the IR photon beam with respect to the volume to which gas-phase proto-membrane protein ions (or clusters of gas-phase proto-membrane protein ions) are localized when confined during irradiation step (iv). When a pulsed laser is used, the laser's output pulse power, pulse length, and repetition rate are all parameters that can potentially be adjusted to promote the formation of intact gas-phase membrane protein ions.

[0191] If a population of gas-phase proto-membrane protein ions is confined during step (iii), the parameter can be optimized to maximize the yield of intact gas-phase membrane protein ions from the confined gas-phase proto-membrane protein ion population during step (iii).

[0192] In some embodiments, the method includes determining the irradiation parameters used in step (iv) by repeating the process until intact membrane protein ions in the gas phase can be detected after step (iv), and by varying the power of the laser radiation and / or the duration of irradiation. When irradiation parameters far from optimal are used, it is generally impossible to observe m / z peaks corresponding to a particular population of intact membrane protein ions in the gas phase above the baseline of the m / z spectrum (which includes signals from a broad m / z distribution of residual gas-phase proto-membrane protein ions, various gas-phase matrix ions that do not contain membrane proteins, and electronic noise). When m / z peaks corresponding to gas-phase IMP ions can be distinguished from the spectral baseline, suitable irradiation parameters are obtained.

[0193] Determining these irradiation parameters is a routine optimization problem that can be readily performed by those skilled in the art. Such optimization would typically involve multiple trial experiments using various combinations of different irradiation parameters (including irradiation time and laser power settings) to determine the irradiation parameter settings that optimize the generation of intact membrane protein ions in the gas phase. Specifically, optimization may be performed to determine the combination of irradiation parameters that maximizes the population of intact membrane protein ions in the gas phase generated in step (iv).

[0194] One or more merit indices may be used to evaluate the m / z spectra generated in these trial experiments. One such merit index may be the abundance of m / z peaks in the m / z spectrum corresponding to the detected intact membrane protein ions. Another such merit index may be the number of clearly observable m / z peaks in the m / z spectrum corresponding to the desired, different intact membrane protein ions. Optimization experiments may be performed manually by selecting irradiation parameter settings for each trial experiment.

[0195] Many methods for the complete or partial automation of this type of optimization are known in the art. The parameters of the functions of modern, highly computerized mass spectrometers can be optimized based on the detected ion signal and the attributes of the m / z spectrum produced by the instrument using automated procedures to "tune" or "calibrate" the instrument. Similarly, the optimization experiments and determination of the optimal parameter settings for process (iv) can be partially or completely automated.

[0196] Process (v) - m / z analysis and detection Step (v) of this method includes m / z analysis and detection of intact membrane protein ions and / or ions derived therefrom in the gas phase.

[0197] The method described herein is a mass spectrometry method. Therefore, step (v) includes determining the m / z of intact membrane protein ions and / or ions derived therefrom in the gas phase.

[0198] In practice, the method typically involves measuring the signal intensity as a function of the mass-to-charge (m / z) ratio by any suitable measuring means. Thus, step (v) generally involves transferring intact membrane protein ions and / or ions derived therefrom in the gas phase to an m / z analyzer and detector, and m / z analysis and detection of the intact membrane protein ions and / or ions derived therefrom in the gas phase. Any suitable combination of m / z analyzer and detector may be used. For example, a high-field orbittrap analyzer was used in the exemplary method described herein. In other examples, an RF quadrupole linear ion trap analyzer and its associated transform dynode-electron multiplier tube-based ion detector in the same instrument may be used. Other examples of m / z analyzer-detector combinations that would be suitable for use with the method described herein include time-of-flight analyzers and associated ion detectors, as well as Fourier transform ion cyclotron resonance (FT-ICR) analyzers.

[0199] By performing step (v), an m / z spectrum, or m / z spectral data, is typically generated. The m / z spectrum can be analyzed to determine the m / z of any ion produced by the processes described herein.

[0200] To aid in analysis and characterization, after the generation of any ion, the process may include a step of performing m / z isolation to isolate ions having a specific m / z or m / z value range. The isolated ions can be subjected to an ion conversion process or can be subjected to m / z analysis and detection.

[0201] Ion conversion process It is possible to perform m / z analysis and detection on the intact membrane protein ions in the gas phase generated in step (iv), and thus determine their m / z ratio. However, if the identity of a particular observed intact membrane protein ion in the gas phase is unknown, it is advantageous to subject the intact membrane ion in the gas phase and / or one or more product ions derived therefrom to one or more ion transformation processes (e.g., fragmentation or charge reduction). Then, it is possible to perform m / z analysis and detection on the various product ions thus derived from the intact membrane protein ion in the gas phase. If such processes are carried out on a population of chemically identical intact membrane protein ions in the gas phase having the same structure and molecular composition, information about the structure and chemical identity of the intact membrane protein ions in the gas phase can be obtained.

[0202] Therefore, in a preferred embodiment of step (v), intact membrane protein ions in the gas phase are subjected to one or more ion conversion processes to produce at least one product ion, which is then m / z analyzed and detected. "Product ion" refers to an ion produced from another ion. Product ions are produced by ion conversion processes performed on another ion. In this context, the first-generation product ion is an ion produced directly from intact membrane protein ions in the gas phase.

[0203] In another preferred embodiment of step (v), the product ions may be subjected to one or more ion conversion processes to generate one or more next-generation product ions. Any product ions (i.e., any product ions derived from previous-generation product ions) can be detected by m / z analysis. For example, a gas-phase membrane protein ion may be fragmented to generate a product ion, and this first-generation product ion may be subjected to further fragmentation through one or more ion conversion processes. Any next-generation product ions formed through this series of conversion processes can be detected by m / z analysis.

[0204] Therefore, step (v) may include m / z analysis and detection of intact membrane protein ions and / or ions derived therefrom in the gas phase by tandem mass spectrometry.

[0205] Therefore, as an example, step (v) may include fragmenting intact membrane protein ions in the gas phase to produce product ions which are fragment ions. These fragment ions can then be directly analyzed and detected by m / z analysis. Alternatively, these fragment ions can be charge-modified before m / z analysis and detection. For example, the charge of the fragment ions can be reduced to increase their m / z ratio, which can increase the separation at m / z between product ions that may be produced when a population of chemically identical intact membrane protein ions in the gas phase is simultaneously transformed according to step (v) of the Method.

[0206] In yet another alternative, the fragment ions may be fragmented themselves to generate further fragment ions of the next generation. These further fragment ions may be subjected to m / z analysis and detection, or to one or more further ion conversion processes. The further fragment ions may be charge-modified and then subjected to m / z analysis and detection (for example, their charge may be reduced before m / z analysis and detection), or the further fragment ions may be further fragmented. That is, three or more fragmentation processes may be carried out during process (v). For example, 3, 4, 5, 6, 7, 8, 9, or 10 fragmentation processes may be carried out during process (v) to yield multiple fragment ions.

[0207] Multiple fragmentation processes result in a large number of fragmented ions. Therefore, when fragmentation processes are carried out, m / z selection of a desired ion or some ions is generally performed during the fragmentation process, and optionally before m / z analysis and detection. The m / z selection process is discussed above in "Step (iii) - Confinement".

[0208] However, the ion conversion process does not necessarily have to be a fragmentation process. Step (v) may include subjecting intact membrane protein ions in the gas phase to one or more ion conversion processes to produce product ions (specifically, first-generation product ions). These first-generation product ions can then be detected by direct m / z analysis. Alternatively, the first-generation product ions can be subjected to one or more further ion conversion processes to produce second-generation product ions. The second-generation product ions can be detected or subjected to further ion conversion processes. That is, three or more ion conversion processes may be carried out in step (v). For example, 3, 4, 5, 6, 7, 8, 9, or 10 ion conversion processes may be carried out in step (v).

[0209] Therefore, process (v) is, (va) To subject intact membrane protein ions in the gas phase to a conversion ion process to generate first-generation product ions, (vb) This may include selecting the first generation product ions by m / z and performing m / z analysis and detection as optional.

[0210] "M / Z analysis" means that ions are subjected to an m / z-dependent dispersion process. This process makes it possible to determine the mass-to-charge ratio of detected ions. Generally, the process disperses ions in space, time, or motion frequency, depending on the ion's m / z. This means that when an ion is detected, its m / z can be determined by referring to its detection location, the time it takes to reach the detector, the path stability determined by the voltage applied to the m / z analyzer electrodes, or the frequency of its motion indicated by the frequency of the detection signal it induces. M / Z-dependent dispersion processes typically involve interaction with an electromagnetic field. One example involves the interaction between an ion and an electric field in a time-of-flight mass spectrometer. In this case, the ion is given kinetic energy by the electric field and is therefore accelerated to a velocity dependent on its m / z. Thus, the time of flight required to reach the detector after interaction with the electric field depends on the ion's m / z, making it possible to determine its m / z.

[0211] In some cases, two or more first-generation product ions are generated. In such cases, step (vb) includes m / z selection of at least one of the first-generation product ions. For example, step (vb) may include m / z selection of all first-generation product ions generated in step (vb), and detection and m / z analysis of those first-generation product ions. Alternatively, one or more of the first-generation product ions may be subjected to one or more further ion conversion processes before m / z analysis and detection.

[0212] Therefore, process (v) follows process (vb), (vc) The first generation product ions are subjected to an ion conversion process to generate the second generation product ions, (vd) The method may further include selecting the second generation product ions by m / z and optionally performing m / z analysis and detection of the second generation product ions.

[0213] In some cases, two or more second-generation product ions are generated. In such cases, step (vd) includes m / z selection of at least one of the second-generation product ions (and optionally, detection and m / z analysis). For example, step (vd) may include detection and m / z analysis of all second-generation product ions generated in step (vc). Alternatively, one or more of the second-generation product ions may be m / z selected and then subjected to one or more further ion conversion processes before m / z analysis and detection.

[0214] Further ion conversion processes and optional detection processes may be carried out to generate additional product ions, and these processes may be designated as steps (ve), (vf), etc.

[0215] If the ion conversion process is a fragmentation process, the ions generated by the fragmentation process during step (v) may be, for example, ligands dissociated from membrane proteins (such as nonspecifically bound ligands), subunits of membrane proteins (membrane proteins forming a multimeric complex), or fragments generated by the cleavage of covalent bonds within membrane proteins. The fragmentation process carried out during step (v) may further generate uncharged fragments.

[0216] In practice, this process is typically carried out for multiple gas-phase IMP ions, and typically multiple first-generation product ions are generated. Similarly, subsequent ion conversion processes typically generate multiple next-generation product ions. Each product ion in the multiple next-generation product ions may be the same or different.

[0217] A wide variety of ion conversion processes (particularly fragmentation processes) are conceivable for any fragmentation process carried out during step (v). For example, dissociation ion conversion can be achieved by one or more of the following methods: infrared multiphoton dissociation (IRMPD), electron transfer dissociation, activated ion electron transfer dissociation, electron capture dissociation, UV photodissociation, and collision-induced dissociation. Generally, among these, IRMPD is preferred because the inventors have found that it generally provides sequence information ions in high yield from membrane gas phase protein ions (regardless of their molecular weight).

[0218] Therefore, in some embodiments, step (v) is, (va)IRMPD fragments intact membrane protein ions in the gas phase and generates fragmented ions, (vb) This may include selecting fragment ions by m / z.

[0219] In a particular embodiment, step (v) is: (va)IRMPD fragments intact membrane protein ions in the gas phase and generates fragmented ions, (vb) Selecting fragment ions by m / z using the CDMS method, and performing optional m / z analysis and detection.

[0220] The selection of the most appropriate ion conversion process in step (v) may depend on the type of desired product (e.g., fragmentation product). For example, some fragmentation techniques are better suited to breaking non-covalent bonds (as may be desired when the intact membrane protein ion itself in the gas phase is fragmented by removing any ligand within the multimeric membrane protein and / or dissociating any associated subunits). Such techniques include IRMPD, collision-induced dissociation (including RF ion trap type resonance collision-induced dissociation, and beam type / collision cell type low-energy collision-induced dissociation). Surface-induced dissociation is also a less preferred alternative method.

[0221] In contrast, when the intention is to dissociate covalent bonds, preferred techniques include IRMPD, collision-induced dissociation (including trap-type CID and low-energy beam-type CID), ultraviolet photodissociation (UVPD), and electron transfer dissociation. When IRMPD, UVPD, or ETD are used, it may be desirable to utilize some form of fragment ion protection to limit the extent of internal (non-C and N-terminus, including the product) formation (see, for example, product ion parking, U.S. Patent Application Publication 2010 / 0084548 and Ugrin et al., Journal of the American Society of Mass Spectrometry, 2019, vol. 30 pp. 2163-2173 (these are incorporated herein by reference)).

[0222] Therefore, in some embodiments, step (v) is, (va) Collision-induced dissociation fragments intact membrane protein ions in the gas phase, generating fragmented ions, (vb) Selecting fragment ions by m / z, (vc)IRMPD fragments the fragment ions to generate further fragment ions, (vd) This may include m / z analysis and detection of the further fragment ions.

[0223] As described above, this method may include modifying the charge of an ion (which may be called a precursor ion) to generate the ion to be detected or fragmented, prior to any step in which the ion is detected or fragmented.

[0224] For example, process (v) is, (va) Fragmenting intact membrane protein ions in the gas phase (preferably by IRMPD) to generate fragment ions, (vb) Select the m / z range that includes the m / z of the fragment ion, thereby selecting the fragment ion by m / z. (vc) Reducing the charge of the fragment ion to generate a charge-reduced fragment ion, (vd) This may include detecting and m / z analyzing charge-reduced fragment ions.

[0225] Charge reduction is generally achieved by proton-transfer ion-ion reactions, but it can also be achieved by other means, such as (but not limited to) electron-transfer ion-ion reactions or electron-capture (ion-electron) reactions (though less preferably).

[0226] Further analysis Previously, this method has been described and defined in relation to a single m / z analysis and detected system of ions derived from a single proto-membrane ion generated in step (ii) of this method. In practice, this method is performed to simultaneously generate and analyze a population of ions. Depending on the m / z analyzer used and various other instrument considerations, the number of ion m / z values ​​collectively analyzed and detected in a single repeat of this method can range from one order of magnitude to over 100,000. Furthermore, this method generally involves multiple repetitions of generating and analyzing multiple ion populations from the same sample. Generally, the aggregated data from all these applications of this method, when digitized through the m / z analysis and detection performed in step (v) of this method, becomes a set of m / z ion signal abundance versus data, which can be plotted as an m / z spectrum. When presented as an m / z spectrum, this data shows m / z peaks indicating the detection of intact membrane protein ions and / or derived product ions of various types in the gas phase. A key objective of the present invention is that structural and chemical information about one or more membrane proteins in a given sample can be determined using their m / z spectra.

[0227] For example, at a simple level, analysis of the obtained m / z spectrum may allow for the chemical identification of any ligand bound to a membrane protein in intact membrane protein ions in the gas phase. It may also provide information about the relative abundance of different proteoforms of the same protein in the provided sample, or the relative abundance of different proteins in the provided sample. Therefore, generally, this method allows for the identification of any ligand bound to a membrane protein in a given sample. (vi) Using the m / z spectral data obtained in step (v) (for example, by analyzing the m / z spectra resulting from the m / z analysis and detection of fragment ions of a specific membrane protein ion type generated by multiple applications of the Method to the provided sample), information regarding the structure and / or identity, and / or abundance of intact membrane proteins in the provided sample may further be provided.

[0228] Specifically, step (vi) may involve utilizing the m / z ion signal abundance data obtained in step (v) to identify membrane proteins, obtain information regarding the amino acid sequence of membrane proteins, or identify binding partners of membrane proteins. Here, membrane proteins are membrane proteins present in the method of the present invention, and as described above, the process can be repeated to identify multiple different membrane proteins present in the sample.

[0229] Automated software tools are known in the art to assist analysts in extracting such information from aggregated m / z ion abundance signal data (m / z spectral data). These data analysis tools are highly sophisticated and can enable the extraction of very detailed information about intact membrane proteins (multiple copies) in the sample from which the m / z analysis and detected ions were derived, which generated the m / z spectrum. Depending on the specific experiment (details of the ion transformation performed in step (v) of the method that generated the aggregated m / z detected ion abundance signal to generate the m / z spectral data), such information may include the amino acid sequence of all or part of the intact membrane protein, and / or the location and / or type of post-translational modifications of the intact membrane protein.

[0230] Suitable software for extracting information such as m / z spectral data includes Protein Prospector (UCSF), Byos / Byonic (Protein Metrics), Proteome Discoverer (Thermo Scientific), TD Validator, Prosight Native, Prosite Lite, and all other software packages from Proteinaceous Inc., as well as MASH Explorer (University of Wisconsin-Madison).

[0231] Exemplary Embodiments By combining specific features of each of the above steps (i) to (v), a particularly preferred embodiment of the method can be obtained. Examples of such combinations are as follows:

[0232] A method for analyzing a sample containing multiple copies of intact membrane proteins, wherein the method is (i) To provide a sample containing a non-synthetic biomembrane material containing multiple copies of intact membrane proteins, a solvent, and bulk lipid molecules, (ii) Ionizing the sample to generate multiple gas-phase proto-membrane protein ions, each of which contains intact membrane proteins nonspecifically bound to one or more bulk lipid molecules and one or more residual solvent molecules, (iii) Confinement of multiple gas-phase proto-membrane protein ions, (iv) By irradiating multiple confined gas-phase proto-membrane protein ions with infrared radiation from a laser source, one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules are separated from the intact membrane protein to generate multiple gas-phase intact membrane protein ions, Selectively isolating intact membrane protein ions from multiple gas phases at m / z, (va) Selectively subjecting intact membrane protein ions in the gas phase to an ion conversion process to generate multiple first-generation product ions, The first generation product ion is selected by arbitrary m / z, (vb) Intact membrane protein ions in the gas phase, or if step (va) has been performed, first-generation product ions are to be analyzed and detected by m / z analysis. A method comprising (vi) using the m / z-ion signal intensity data obtained in step (v) (for example, when such data is aggregated with data from multiple applications of the Method to multiple copies of the intact protein to provide the m / z spectrum of the product ions of a particular intact membrane protein), to obtain information regarding the structure and / or identity of the intact membrane protein.

[0233] It should be noted that when multiple types of first-generation product ions are generated, selectively selecting the first-generation product ions by m / z may involve selecting one or more types of first-generation product ions by m / z (which can be achieved through appropriate m / z selection).

[0234] A method for analyzing a sample containing multiple copies of intact membrane proteins, wherein the method is (i) To provide a sample comprising a non-synthetic biomembrane material containing multiple copies of intact membrane proteins, a solvent, and bulk lipid molecules, wherein the non-synthetic biomembrane material is extracted from or directly secreted by cells, organelles, viral envelopes, or vesicles. The sample is subjected to sonication, (ii) Ionizing the sample to generate multiple gas-phase proto-membrane protein ions, each of which contains intact membrane proteins nonspecifically bound to one or more bulk lipid molecules and one or more residual solvent molecules, (iii) Confinement of multiple gas-phase proto-membrane protein ions, (iv) By irradiating multiple confined gas-phase proto-membrane protein ions with infrared radiation from a laser source, one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules are separated from the intact membrane protein to generate multiple intact membrane protein ions, and multiple intact gas-phase membrane protein ions are selectively isolated in m / z. (v) A method comprising m / z analysis and detection of intact membrane protein ions and / or ions derived therefrom in multiple gas phases.

[0235] A method for analyzing a sample containing intact membrane proteins (particularly multiple copies of intact membrane proteins), wherein the method is (i) To provide a sample comprising a non-synthetic biomembrane material containing intact membrane proteins, a solvent, and bulk lipid molecules, wherein the non-synthetic biomembrane material is extracted from or directly secreted by cells, organelles, viral envelopes, or vesicles. The sample is subjected to sonication, (ii) Ionizing a sample by electrospray ionization to generate gas-phase proto-membrane protein ions, wherein the generated gas-phase proto-membrane protein ions include intact membrane proteins associated with submicron-scale membrane fragments or vesicles and one or more residual solvent molecules. (iii) Confinement of gas-phase proto-membrane protein ions, (iv) By irradiating confined gas-phase proto-membrane protein ions with infrared radiation from a laser source, one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules are separated from the intact membrane protein to generate intact gas-phase membrane protein ions, and the intact gas-phase membrane protein ions are selectively isolated in m / z. (v) A method comprising m / z analysis and detection of intact membrane protein ions and / or ions derived therefrom in the gas phase.

[0236] A method for analyzing a sample containing multiple copies of intact membrane proteins, wherein the method is (i) To provide a sample comprising a non-synthetic biomembrane material containing multiple copies of intact membrane proteins, a solvent, and bulk lipid molecules, wherein the non-synthetic biomembrane material is extracted from or directly secreted by cells, organelles, viral envelopes, or vesicles. The sample is subjected to sonication, (ii) Ionizing a sample by electrospray ionization to generate multiple gas-phase proto-membrane protein ions, wherein each gas-phase proto-membrane protein ion contains intact membrane proteins associated with one or more submicron-scale membrane fragments or vesicles and one or more residual solvent molecules. (iii) Confining multiple gas-phase proto-membrane protein ions and exposing the multiple gas-phase proto-membrane protein ions to a collision gas to remove one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules by collision-induced dissociation, (iv) By irradiating multiple confined gas-phase proto-membrane protein ions with infrared radiation from a laser source, one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules are separated from the intact membrane protein to generate multiple gas-phase intact membrane protein ions, and the multiple gas-phase intact membrane protein ions are selectively isolated in m / z. (v) A method comprising m / z analysis and detection of intact membrane protein ions and / or ions derived therefrom in multiple gas phases.

[0237] A method for analyzing a sample containing multiple copies of intact membrane proteins, wherein the method is (i) To provide a sample comprising a non-synthetic biomembrane material containing multiple copies of intact membrane proteins, a solvent, and bulk lipid molecules, wherein the non-synthetic biomembrane material is extracted from or directly secreted by cells, organelles, viral envelopes, or vesicles. The sample is subjected to sonication, (ii) Ionizing a sample by electrospray ionization to generate multiple gas-phase proto-membrane protein ions, wherein each gas-phase proto-membrane protein ion contains intact membrane proteins associated with one or more submicron-scale membrane fragments or vesicles and one or more residual solvent molecules. (iii) Confining multiple gas-phase proto-membrane protein ions and exposing the multiple gas-phase proto-membrane protein ions to a collision gas to remove one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules by collision-induced dissociation, (iv) Generating multiple intact gas-phase membrane protein ions by irradiating multiple confined gas-phase proto-membrane protein ions with infrared radiation from a laser source, thereby separating one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules from the intact membrane protein, wherein the laser source has a wavelength of 1.4 μm to 1 mm, preferably 9 to 11 μm, and preferably the irradiation period is less than 50 ms. Selectively isolating intact membrane protein ions from multiple gas phases at m / z, (v) A method comprising m / z analysis and detection of intact membrane protein ions and / or ions from multiple gas phases.

[0238] A method for analyzing a sample containing multiple copies of intact membrane proteins, wherein the method is (i) To provide a sample comprising a non-synthetic biomembrane material containing multiple copies of intact membrane proteins, a solvent, and bulk lipid molecules, wherein the non-synthetic biomembrane material is extracted from or directly secreted by cells, organelles, viral envelopes, or vesicles. The sample is subjected to sonication, (ii) Ionizing a sample by electrospray ionization to generate multiple gas-phase proto-membrane protein ions, wherein each gas-phase proto-membrane protein ion contains intact membrane proteins associated with one or more submicron-scale membrane fragments or vesicles and one or more residual solvent molecules. (iii) Confining multiple gas-phase proto-membrane protein ions and exposing the multiple gas-phase proto-membrane protein ions to a collision gas to remove one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules by collision-induced dissociation, (iv) Generating multiple intact gas-phase membrane protein ions by irradiating multiple confined gas-phase proto-membrane protein ions with infrared radiation from a laser source, thereby separating one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules from the intact membrane protein, wherein the laser source has a wavelength of 1.4 μm to 1 mm, preferably 9 to 11 μm, and preferably the irradiation period is less than 50 ms. Selectively isolating intact membrane protein ions from multiple gas phases at m / z, (va) Subjecting multiple intact membrane protein ions in the gas phase to an ion conversion process to generate multiple first-generation product ions, typically by collision-induced dissociation or IRMPD, Selectively isolating multiple first-generation product ions at m / z, (vb) Detection and m / z analysis of multiple first-generation product ions, or (vc) Typically, this involves either collision-induced dissociation or IRMPD, which fragments multiple first-generation product ions to generate multiple second-generation product ions. Selectively isolating multiple second-generation product ions at m / z, (vd) A method comprising m / z analysis and detection of multiple second-generation product ions.

[0239] It should be noted that when multiple types of first-generation or second-generation product ions are generated, selective m / z selection of the first or second-generation product ions may involve m / z selection of one or more types of first-generation or second-generation product ions (which can be achieved through appropriate m / z selection).

[0240] Any of the methods described herein, The method may further include obtaining information about the structure and / or identity of the intact membrane protein by utilizing the m / z-ion signal intensity data obtained in step (v) (for example, when such data is aggregated with data from multiple applications of the method to multiple copies of the intact protein in order to provide the m / z spectrum of the product ions of a particular intact membrane protein ion).

[0241] While specific embodiments and configurations and materials have been discussed herein, it should be understood that various changes or modifications in form and detail can be made without departing from the scope and spirit of the invention. The following examples are provided to illustrate the invention and should not be considered limiting. [Examples]

[0242] Example 1 This method has been scaled down to be carried out using an improved Thermo Fisher® Orbitrap Eclipse® Tribrid® mass spectrometer. However, any suitable mass spectrometer instrument with an infrared laser source (10.6 μm) and a suitable m / z range, configured so that the infrared light beam of sufficient intensity can interact with the confined proto-membrane ions generated from the sample (as described above) by native electrospray ionization for a duration sufficient to yield completely free, intact membrane protein ions. Now referring to Figure 1, a simplified schematic diagram of the improved Orbitrap Eclipse Tribrid MS instrument, the following describes how various embodiments of the method of the present invention are carried out using this instrument.

[0243] Samples in native pH solutions, generally as described above and for the specific examples described below, are ionized by electrospray ionization (static native electrospray source) 1 to generate proto-membrane protein ions containing bulk lipids, solvent molecules, small molecules, and membrane proteins and complexes, other lipids and lipophilic compounds that specifically associate with membrane-associated proteins, along with other biomolecules that associate with the membrane bilayer. These proto-membrane protein ions travel through a heated metal capillary inlet 2 representing atmospheric pressure relative to the vacuum interface of the mass spectrometer. Exiting inlet 2, the proto-membrane proteins enter the RF ion funnel ion guide 3, permeate through a first vacuum region 21, then traverse a second vacuum region 22 via a first (linear) RF multipole ion guide 4, and then traverse a third vacuum region 23 via a second (curved) RF multipole ion guide device 5. Proto-membrane protein ions can be optionally subjected to CID within the first RF multipole ion guide 4 by adjusting the voltage gradient along and / or between devices 2, 3, 4, and / or 5 to accelerate the proto-membrane protein ion velocity. As a result, collisions with background (primarily atmospheric) gas molecules within the RF multipole ion guide 4 can lead to an increase in ionic vibrational energy, inducing (unfavorable) dissociation of non-covalent and covalent bonds of the proto-membrane ions (inlet CID). At a suitably selected accelerating multipole device bias potential, covalent cleavage from inlet CID can result in partial desolvation and delipidation of proto-membrane protein ions with limited or no loss of non-covalent partners or covalent cleavage, and therefore may be advantageous. Proto-membrane protein ions exiting the second RF multipole 5 enter and traverse the vacuum region 24 via the quadrupole m / z analyzer 6, electrostatic ion gating lens 8, RF multipole ion guide device 7, and RF C-trap ion transfer device 9, and then enter the RF multipole ion trap / ion guiding device and ion routing multipole (IRM) 11. The potential of the electrostatic ion gating lens 8 can be switched to allow or deflect and exclude ions further downstream in the device.The IRM has its own enclosure 28, which allows a flow of nitrogen to be introduced into it to function as an attenuation / impacting gas without increasing the pressure in the quadrupole m / z filter 6, and as a result, ion permeation through the m / z filter is not impaired when operating in m / z selective mode. From the IRM, protomembrane protein ions permeate through the RF multipole ion guide 12 to a fourth vacuum region 28, and are then stored in the high-pressure trap region of the dual-pressure RF quadrupole linear ion trap linear ion trap (HPT) 13.

[0244] After a sufficient population of proto-membrane protein ions has accumulated in the HPT13, the potential of the electrostatic ion gating lens 8 is switched to block further transmission and accumulation of ions, activating the IR laser light source 20 and directing its output IR light beam 16 along the axis of the HPT13 to irradiate the proto-membrane protein ions confined along the device axis in the central part 13.2 of the device and activate them by vibration. As is well known in the art, the DC bias potentials of the front part 13.1, central part 13.2, and rear part 13.3 of the HPT generate an axial confinement potential, and radial confinement is provided by an RF quadrupole field established by the RF potential applied to the device electrodes. The high pressure of helium in the HPT (approximately 6 mTorr) allows for collision stabilization and accumulation of the proto-membrane protein ions injected into the HPT, as well as localization of the proto-membrane protein ions along the device axis. The IR transmission optical window 19 allows the laser beam to enter the vacuum chamber at the rear end of the ion trap vacuum region 27. The beam from the IR laser source is already aligned with the axis of the HPT using mirror 17, and the IR lens 18 creates an elongated waist of the IR light beam along the HPT 13 axis in the central part 13.1 of the HPT. Residual solvent and bulk lipid molecules non-covalently bound to the proto-membrane protein ions absorb photons from the IR beam 16 that excite them by vibration. This dissociates all the solvent and bulk lipids from a significant portion of the confined proto-membrane protein ions, resulting in a population of intact membrane protein ions that remain trapped within the HPT 13. In some examples, it is advantageous to apply a broad m / z isolation band auxiliary voltage waveform to the HPT 13 to eliminate various background ions that are co-generated and transmitted with the proto-membrane ions before irradiation of the proto-membrane protein ions with the IR beam, and to exclude ions with m / z values ​​outside the m / z range of most of the accumulated proto-membrane ions from the device.

[0245] After the cessation of IR activation of the ions confined in the HPT (e.g., protonated membrane protein ions, free intact membrane protein ions, residual background ions, etc.), the population of these ions can be analyzed according to methods established in the art for the Orbitrap Eclipse Tribrid instrument. This ion population can be processed by native electrospray ionization generated from a "normal" sample composed of soluble proteins and protein complexes, sent to the instrument, desolvated by inlet CID, delivered to and confined in the HPT 13, and analyzed as such. For example, m / z analysis of the ion population within the HPT after IR irradiation can be achieved by moving the ion population to the C-Trap9 and radially emitting the ion population from there through optical elements within the vacuum region 26 into the Orbitrap m / z analyzer 10 within the ultra-high vacuum region 25. The Orbitrap m / z spectrum of this ion population (generally, detected ion signal transient data from many repetitions of the experiment, and thus obtained by averaging m / z analyses of multiple ion populations) includes m / z peaks corresponding to various charge states of free intact membrane protein ions from multiple specific membrane protein isoforms in the sample, as well as m / z peaks corresponding to various other background ion types including residual PMP ions and an elevated baseline. Alternatively, the ion population within the HPT 13 can be moved to the low-pressure trap region of the dual-pressure region RF quadrupole linear ion trap linear ion trap (LPT) 14, m / z analyzed by continuous radial emission, and detected by an electron multiplier tube-based ion detector 15. The ion trap m / z spectrum of this ion population (generally, m / z and detected ion signal data from many repetitions of the experiment, and thus the result of averaging m / z analyses of multiple ion populations) includes m / z peaks corresponding to various charge states of free intact membrane protein ions from multiple specific membrane protein isoforms in the sample, as well as m / z peaks from various other background ion types including residual PMP ions and an elevated baseline. <0,000,899><0,000,900><0,000,901>Tandem mass spectrometry (MS / MS, or MS) is used to analyze the free, intact membrane protein ions released from PMP ions by IR irradiation of ions within HPT13. 2To perform the analysis, ions with m / z values ​​outside the narrow range corresponding to the specific charge state of the membrane protein ion of interest, as determined by the m / z peak observed in the previously acquired m / z spectrum, are isolated in the ion trap by applying a narrow m / z isolation band auxiliary voltage waveform to the HPT13. After the application of this m / z isolation waveform, the population of ions remaining in the HPT consists of multiple intact membrane protein ions of the specific charge state of interest of the selected intact membrane protein ions, as well as a smaller number of various types of background ions, as well as residual PMP ions, and potentially intact membrane protein ions from other protein isoforms present in the sample at much lower abundances than the protein isoforms investigated by MS / MS. The m / z isolated ions are then subjected to one or more of the various ion conversion processes available on the instrument. The m / z isolated ions can be moved to the IRM7 at relatively high energies to perform beam-type HCD. Alternatively, the m / z isolated ions can be retained in HPT 13 and subjected to ion trap type (resonance) CID, IRMPD, ETD, AI-ETD, or PTCR. [Note: Trap type (resonance)] CID, ETD, and PTCR are standard ion conversion processes available in commercially available configurations of Thermo Fisher® Orbitrap Eclipse® Tribrid® mass spectrometers, and the manner in which these processes are achieved in the instrument is well known in the art. However, the most important modification of the instrument, and the modification required to demonstrate the method of the present invention, was the adaptation of an IR laser light source 20 to the instrument to enable IR irradiation of ions confined in HPT 13 (and LPT 14). This naturally allows, as described above, irradiation of PMP ions to obtain IMP ions by achieving complete "evaporation" of the solvent and bulk lipids from these ions. Advantageously, this modification also enables IRMPD and AI-ETD on the instrument.To achieve IRMPD for the m / z isolated population ions within HPT13 that substantially contain IMP ions, IR laser 20 operates at an output power above that appropriate to efficiently convert PMP ions to IMP ions without inducing covalent bond cleavage, over a certain time interval. To achieve AI-ETD, while the IR laser light source is operated, the isolated ions are subjected to an ion-ion reaction with ETD reagent ions in HPT13. The IR laser light source output power level for AI-ETD is the same as that used for the conversion of PMP ions to IMP ions according to the present invention. Product ions from any of these conversion processes can then be delivered to Orbitrap analyzer 25 or LPT14 for m / z analysis and detection, as described above in the first example that explains the MS analysis of IMP ions generated through IR irradiation of PMP ions within HPT13.

[0247] In the following discussion of FIGS. 2-7 demonstrating the usefulness of the method according to the present invention, the process of complete removal of solvent molecules and bulk lipid molecules from PMP ions by IR irradiation-induced vibrational activation to generate IMP ions is referred to, for simplicity, as IRMPD-L. IRMPD-L represents InfraRed Multi Photon De-Lipidisation.

[0248] Referring now to FIG. 2, the utility of IRMPD-L was demonstrated using vesicles derived from E. coli cells engineered to contain the MacAB efflux pump among many proteins endogenous to the inner and outer membranes. Membrane fragments were generated using gentle sonication, and the resulting membrane fragments were analyzed as described above (e.g., transported via nanoelectrospray ionization to generate PMP ions to an improved Thermo Scientific Orbitrap Eclipse mass spectrometer and delivered to HPT 13 to generate intact membrane protein and complex ions, IMP ions, by IRMPD-L). At low IR energy (1.0% of 60 W, i.e., 0.6 W) and low irradiation time (10 ms), in the m / z spectrum (average) generated by m / z analysis using the Orbitrap analyzer of ions retained in HPT after the IRMPD-L process, a series of low-resolution charge state m / z peaks (FIG. 2) were generated. By increasing the laser intensity to 5.0% (about 3.0 W), in the corresponding (averaged) m / z spectrum of ions retained in HPT13 after IRMPD, three well-resolved series of m / z peaks corresponding to consecutive charge states of IMP ions of a common membrane protein isoform (charge state envelope) were generated. The L process. The most abundant one is centered at about 6500 Th), has a charge state of 25+, and corresponds to an endogenous membrane protein with a mass of 149,577 Da in terms of mass. Another charge state envelope centered at the 19+ charge state (about 5500 Th) corresponds to a protein with a mass of 104,775 Da. Furthermore, a series of low-abundance m / z peaks centered at the 25+ charge state near about 9000 Th were observed, corresponding to a protein with a mass of 218,312 Da. The strongest peak has a total ion intensity of 2.28×10 3 and increasing the IRMPD-L irradiation energy (7%, about 4.2 W), the abundance of this peak was 1.66×10 3The charge state distribution corresponding to the 218,312 Da protein decreased to a certain point, and disappeared, presumably due to the dissociation of the complex into partial complexes or covalently dissociated fragment ions. Increasing the laser power to 9% (approximately 5.4 W) resulted in a further decrease in the IMP m / z peak intensity, presumably due to the absorption of IR beam energy by complex ions emitted from membrane proteins and PMP ions (which dissociates them via the IRMPD process). Therefore, when using the IRMPD-L process, which is the method of the present invention, the intensity of IR irradiation of PMP ions must be adjusted to maximize the yield of IMP ions.

[0249] For comparison, the yield of IMP ions using an inlet CID (see Figure 1) that delivers PMP ions to the CID in a first RF multipole ion guide 4 was characterized using the same MacAB sample as above, by adjusting the voltage gradients along and / or between RF ion guiding devices 2, 3, 4, and / or 5, which are generally preferred prior art methods for liberating IMP ions from PMP ions, using an inlet CID. Figure 3 shows the m / z spectra of ions generated via the inlet CID for several collision (acceleration) voltages with potentials applied to adjacent optical elements tuned to optimize ion transmission. At a collision voltage of 50 V, very low ion signal (m / z peak) intensities were observed, but several m / z peaks corresponding to the protein charge state of 149,577 Da were observed. At an increased accelerating voltage of 100V, two m / z peak envelopes of charge state series were observed in the m / z spectrum, one of which corresponded to a protein of 149,577 Da (1.02 × 10⁻¹⁵ ions). 3(Intensity of ). The other charge state distribution corresponded to a trace amount of 102,755 Da protein (19+ central charge state around 5000 Th). However, no other m / z peaks were observed, and none of these corresponded to a continuous series of charge states (e.g., peaks with m / z intervals consistent with integer differences in charge states for a common neutral mass). Increasing the accelerating potential to 150 V resulted in a slight increase in ionic intensity (m / z peak of 25+ charged state ions of 149,5777 Da protein, 1.26 × 10⁻⁶). 3 The intensity increased to 102,755 Da, but the m / z peak from the protein disappeared. Further increases in acceleration energy were investigated, and all of them resulted in a decrease in the ion signal in the m / z spectrum. Therefore, for MacAB samples, the IRMPD-L technique was clearly superior to collision-based techniques in removing lipids from PMP ions, as it yielded higher m / z peak intensity for the m / z peak corresponding to intact membrane protein ions (IMP ions) and produced a clear m / z spectrum suitable for peak assignment.

[0250] In Figure 4, the enhanced ability of IRMPD-L to completely remove nonspecifically associated lipid molecules from proto-membrane protein ions compared to collision-based approaches is further demonstrated by comparing the m / z peaks observed for 102,775 Da proteins (20+ and 10+ charged states) in m / z spectra obtained using optimized delipidation parameters for both in-source CID and IRMPD-L. Using IRMPD-L, the higher signal-to-noise ratio of m / z peaks in the m / z spectra allows for a clear assignment of the charge states of these peaks and observation of "satellite" m / z peaks, one of which corresponds to the addition of a 129 Da molecule (metabolites, post-translational modifications, or other small molecule ligands to the protein). (These m / z peaks are not observed by removing nonspecifically bound bulk lipids from PMP ions using in-source CID). Therefore, Figure 4 illustrates the advantages of IRMPD-L over conventional collision-based approaches in its ability to generate m / z spectra of membrane protein ions corresponding to the retention of non-covalent bonds (e.g., ligand binding such as metabolites and small molecules) and / or covalent adducts (e.g., retention of PTMs, which are unstable and known to dissociate during CID processes).

[0251] The usefulness of the IRMPD-L method of the present invention for obtaining intact membrane protein ions by removing solvents and bulk lipid molecules from proto-membrane protein ions (PMP ions) containing multi-subunit membrane protein complexes originally embedded in vesicles. The heteropentamerial β-barrel assembly mechanism complex was expressed in E. coli cells, and after harvesting the cell membrane, the membrane was fragmented into smaller membrane vesicles using gentle sonication in ammonium acetate buffer. Referring here to Figure 5, PMP ions were generated by nanoelectrospray ionization, and membrane protein and complex ions (IMP ions) were generated by delipidation of bulk carrier lipid molecules using the IRMPD-L method of the present invention. Using a 10 ms IR irradiation interval at 7% of the available laser power setting in IRMPD-L, m / z spectra (average) were obtained for the ions retained in the HPT and are shown in the upper m / z spectra shown in Figure 5. We identified several m / z peak series corresponding to the continuous charge states of membrane protein isoforms. One of these m / z peak series (charge state envelope) corresponds to the 199,200 Da membrane protein complex (centered around the 28+ charge state m / z peak near 7000 Th in Figure 5), which, when converted to a neutral mass, exhibits a deviation of approximately 0.5% from the mass (200,035 Da) calculated from the known sequence of the overexpressed, fully assembled Bam complex. The normalized intensities of the m / z peaks for various charge states of the IMP ion for this protein (circles in Figure 5) decreased during IRMPD-L as the laser power setting increased (from 7% to 10% and 15% of maximum power). Simultaneously with this decrease, the abundance of m / z peaks corresponding to different ionization states of the intact Bam membrane complex increased, as did the intensity of lower m / z features (m / z peaks), which likely corresponds to the increased generation of fragment ions, including partial complex ions, generated by IRMPD of the intact membrane protein (complex) ions. As can be seen from the figure, the upper m / z spectrum contains clear and well-defined m / z peaks, generated at laser power levels well below the threshold for standard IRMPD. The m / z peaks have satisfactory intensity.

[0252] To demonstrate that sequence information can be obtained from IMP ions of a fully assembled Bam complex, IMP ions at 7378Th (27+ charged state in the signal distribution at 199kDa) were isolated m / z using a selection window of approximately 10Th. Subsequently, this population of IMP ions was subjected to IR multiphoton dissociation (IRMPD). This involved high laser energy (over 15% of available energy) to induce cleavage of peptide bonds in each protein subunit, generating a product ion distribution consisting of peptide and protein fragment ions. The resulting spectrum showed many low-charged state ions (1+~7+), indicating that the majority of the protein complex was fragmented into amino acid sequence ions. By manually searching this spectrum for expected fragments of all five subunits, many potential matches were generated, and therefore the experiment focused on characterizing two expected ions corresponding to cleavage along the peptide backbone of the small His6-tagged BamE subunit. 20 The m / z peak series with 1Da intervals corresponding to ions, monovalent y 27 The ions were observed with a series of well-resolved peaks corresponding to them (Figure 6B). Importantly, the matched fragment ions contained an engineered C-terminal His6 tag (Figure 6C) and spanned a region expected to be completely devoid of post-translational modifications. Furthermore, comparison of the observed mass of the fragments with their theoretical masses showed that the mass deviation was well within an acceptable threshold for reliable molecular identification (less than 10 ppm), providing enhanced confidence in the assignment.

[0253] Therefore, it was shown that it is possible to release IMP ions from PMP ions with a laser power of 7%, and then dissociate the intact membrane protein complex using a higher laser power of over 15%, thereby obtaining sequence ions that define the subunits directly released from this complex.

[0254] As a final, brief example of the usefulness of the IRMPD-L method, Figure 7 demonstrates that, once optimized parameters are obtained, intact membrane proteins and membrane-associated proteins can be released from PMP ions containing entire secretory extracellular vesicles (exosomes) obtained from human body fluids.

[0255] Example 2 Experiments were conducted to demonstrate the effect of IR laser power on the release of membrane proteins from the native lipid bilayer for detection by mass spectrometry, and to show the applicability of the present invention's method to sequencing of rhodopsin after release from the lipid bilayer.

[0256] (a) Method (i) Preparation of rod disk film Bovine eyes were obtained from commercial slaughterhouses. Rod exosegmental membranes were obtained from batches of 50–100 eyes with dark-adapted retinas and isolated as previously described. [1,2] The isolated membranes were suspended in 200 mM ammonium acetate and homogenized using a probe sonicator equipped with a stepped-tip microtip (2 mm, Vibra-Cell VCX-500 Watt, Sonics) and at maximum amplitude (40%) (1 second on, 2 seconds off) with 2 J per cycle for 1.5 minutes. This yielded disk membrane vesicles containing approximately 9 μM of rhodopsin, which were diluted 2-fold with 200 mM ammonium acetate buffer at pH 7.4 immediately before native MS analysis.

[0257] (ii) Native mass spectrometry and top-down mass spectrometry The experiment was conducted using an improved Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). A Synrad Firestar Ti60 CO2 continuous-wave IR laser (10.6 μm, 60 W) was coupled to the instrument so that the beam was focused into the high-pressure cell of a dual-cell quadrupole linear ion trap (QLIT), and its timing and power output were controlled by the instrument software. These instrument improvements have been previously described. [3]The vesicles were ionized via a nanoelectrospray using a gold-plated borosilicate glass capillary (1.2 mm OD) prepared in-house. The capillary voltage was maintained at 1.0–1.2 kV relative to the instrument's pore (heated to approximately 100–200°C). The ions were activated in the source using a mild activation energy (50 V source CID) before entering the next differential pressure region containing the curved flatapole. The instrument was operated in high-pressure mode (20 mtorr in ion routing multipole), and the ions were irradiated in the ion trap for 25 ms with a laser output power in the range of 0–6 W (0–10% output power), followed by MS. 1 I switched back to orbitrap for detection. MS 1 The spectrum was collected at m / z 200 with a resolution of 15,000. 2 For the experiment, ions were isolated in an ion trap and irradiated with an IR laser at a laser power in the range of 9.0–10.8 W (15–18%) for 5 or 10 ms to induce peptide bond dissociation. The fragment ions were then returned to the Orbittrap for detection at m / z 200 with a resolution of 200,000.

[0258] (iii) Data analysis and software The proteins were initially identified using Prosight Native (Proteinaceous) through a database search of the entire bovine proteome. [4] Next, the fragments were manually validated using TDValidator(Proteinaceous). [5] We manually inspected the assigned peaks and removed any incorrect assignments.

[0259] (b) Results (i) Release of membrane and surrounding proteins from vesicles by IR light We previously described a mass spectrometer incorporating a 10.6 μm wavelength CO2 laser directed to a high-pressure cell of a linear ion trap, and demonstrated that the laser output power and irradiation time can be adjusted. [3]

[0260] Here, we sought to determine whether this method could release endogenous membrane proteins embedded in vesicles derived from native lipid bilayers. This was previously considered unattainable because such proteins are expected to have relatively low absorption cross-sections at the wavelengths of light used here.

[0261] To investigate the effect of laser power on lipid vesicles, the power was increased in increments of 0.6 W (1% of the total laser power) while maintaining a constant irradiation time of 25 milliseconds (Figure 8). The mass spectra obtained at each laser power appear different. At a low laser power of 1.8 W, very few distinguishable peaks corresponding to 44448, 45619, 47194, and 51705 Da appear from an apparent high baseline. The high baseline may be due to the inherent heterogeneity of the vesicles, and ion signals are detected at every m / z, ultimately hindering the determination of the charge state of lower-abundance protein species. The mass spectrum generated after irradiation at a laser power of 5.4 W instead reveals well-resolved peaks for numerous protein distributions, including novel charge state distributions not observed under previous conditions. The most abundant distribution above 4500 m / z is tentatively assigned to the GPCR, rhodopsin, corresponding to a molecular weight of 41714 Da with additional satellite features, indicating the presence of several proteoforms of its protein species coexisting within the disk membrane.

[0262] The significant differences in protein distribution observed at each laser power level suggest that IR lasers can influence differential emission of proteins from lipid vesicles. While we do not wish to be bound by theory, we hypothesize that at relatively low laser power levels (0.6–4.2 W), laser irradiation disrupts the lipid bilayer, releasing soluble proteins trapped within the vesicle core. At higher laser power levels, we hypothesize that the absorption cross-section of bulk phospholipids is sufficiently high, resulting in the dissipation of the lipid bilayer and thus the release of membrane proteins and associated complexes for mass spectrometry. By carefully tuning these parameters, many non-covalent protein-protein interactions remain undisturbed.

[0263] (ii) Native top-down sequencing of rhodopsin proteoform High-resolution mass spectra obtained using a 5.4W laser output power reveal heterogeneity in the rhodopsin proteoforms, as six observed distributions exist between m / z 5160–5300, corresponding to the 8+ charged state. The deconvoluted masses of the major peaks of each major proteoform correspond to 41476Da, 41714Da, 41876Da, 42038Da, 42200Da, and 42362Da. Each proteoform deviates significantly from the reported sequence mass of 39008Da, and therefore, we attribute the additional masses (2468–3354Da) to the various post-translational modifications reported. Rhodopsin is the most well-characterized GPCR, and various post-translational modifications have been reported: acetylation at M1, N-linked glycosylation at N2 and N15, palmitoylation at S322 and S323, phosphorylation at seven potential sites (S334, T335, T336, S338, T340, T342, S343), and a disulfide bond between C110 and C187 linking intradiscal loops 1 and 2. Using intact masses, we can infer which modifications may be present. For example, the mass difference between the two lowest molecular weight proteoforms (41476Da and 41714Da) is 238Da, which potentially indicates a difference in palmitoylation at one of the two lipid-promoting sites. Furthermore, the mass difference among the latter five proteoforms was consistently 162 Da, suggesting heterogeneity in glycan configuration due to the subsequent addition of a single hexose. The identity of the glycans and their occupancy were confirmed using glycoproteomics, and then the proteoforms observed in the native mass spectra were assigned using their abundances. Thus, the proteoform with the highest abundance in the native mass spectrum contains two N-linked glycans at sites N2 and N15, and the glycans at each site are both composed of Man3GlcNAc3.Regarding the remaining proteoforms, the N-linked glycan at site N2 remains static, while the glycan at N15 may differ by only a single mannose, resulting in proteoforms having Man4GlcNAc3, Man5GlcNAc3, and Man6GlcNAc3 at N15. However, the remaining mass difference (over 500 Da) between sequence mass and measured mass is not so straightforward to assign, as the mass may be achieved using combinations of several other posttranslational modifications.

[0264] To confirm the identity and localization of specific post-translational modifications, we isolated the 8+ charged state at m / z 5215 using a 35 Da m / z window. Thus, the isolation window encompassed any proteoform present in the mass range of approximately 41.5–41.9 kDa, which was then subjected to infrared multiphoton dissociation (IRMPD) using high laser power (15–18% or 9–10.8 W) with a short (5 ms) irradiation time to generate sequence information ions from intact membrane proteins. 2 The most abundant fragment in the spectrum is a y-type fragment ion (y), which originates from a highly flexible skeletal cleavage within the C-terminus. 10 1+ -y 22 1+ ) is assigned to (Figure 9). Good C-terminal sequence coverage reveals evidence of numerous important post-translational modifications, and the sequential fragmentation of adjacent amino acids (i.e., sequence tags) allows for their precise localization. 10 1+ -y 22 1+ A wealth of information is embedded within the series of consecutive fragments from which we localize the phosphate modification to S334. 15-19 1+ Evidence is found for both unmodified and phosphorylated fragment ions. The very abundant 3+ and 2+ fragment series above 2500 m / z contain even more information, and clusters of trivalent fragment ions between 2540 and 2620 m / z are y-type fragments originating from cleavage within intradiscal loop 3. 66 3+ , y 673+ , and y 68 3+ This may be due to the following: Fragments originating from the same skeletal section in intradiscal loop 3 also exist as divalent ions with m / z 3800-3900, where the relative distribution of one and two palmitic acid modifications is maintained.

[0265] Despite the widespread availability of several sequence tags in fragmentation data to aid in reliable assignment of rhodopsin, the presence of N-linked glycans at N2 and N15 hinders the analysis of N-terminal fragments using simple arithmetic to determine amino acid mass differences between fragment ions. Over 300 fragment ions are available via MS. 2This was observed spectrally, and is a significant difference from the approximately 100–150 fragment ions detected for other proteins in the vesicles. This is due to the fact that the number of fragment ions that can be generated from a single backbone cleavage is greater because the simultaneous glycan fragmentation combinatorially increases the number of observed b-type fragment ions. In the case of proton-driven fragmentation, such as in IRMPD, the glycan predictably fragments along the glycosidic bond, producing Y-type and Z-type fragments, and across the glycan ring, yielding X-type fragments. The mass of these theoretical fragments can then be added to the theoretical mass of protein fragment ions derived from amino acid cleavage involving N-linked glycans. We searched for fragment ions that would result from all possible combinations of glycan and protein cleavage and found evidence of fragments derived from backbone cleavage at the N-terminus of rhodopsin. In many cases, several assigned fragments originated from the same protein backbone cleavage but retained differences in the partial glycans covalently bonded to asparagine 2 and 15. Under the dissociation conditions used here, the remaining partial glycans typically consisted only of the N-acetylglucosamine core, with only a few fragments retaining any portion of the branched mannose moiety being assignable. This is expected, as collision-based and IR-based fragmentation typically do not preserve the fragile branching of glycan modifications. Nevertheless, the goal was not to reconstruct the complex glycan from the native fragmentation spectrum, but rather to identify patterns in the fragment ions such that heterogeneous membrane protein sequence information could be obtained without interference. In summary, we achieve 14% sequence coverage on endogenous GPCRs directly from their native lipid bilayer, including direct evidence and localization of several important PTMs such as phosphorylation, palmitoylation, and N-linked glycosylation. Further analysis would allow for even higher sequence coverage.

[0266] Therefore, the method disclosed herein provides a robust approach for accurately characterizing native membrane proteins in a native environment. This example describes the characterization of rhodopsin as an exemplary protein, but the disclosed method is suitable for a wide variety of membrane protein analytes.

[0267] References 1. Jastrzebska et al. (2006) "Functional and Structural Characterization of Rhodopsin Oligomers", Mechanisms of Signal Transduction, Vol. 281, pp. 11917 - 11922 2. Kevany et al. (2013) "Structural and Functional Analysis of the Native Peripherin-ROM1 Complex Isolated from Photoreceptor Cells", Membrane Biology, Vol. 288, pp. 36272 - 36284 3. Lutomski et al. (2023), "Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors", Angew. Chem. Int. Ed., Vol. 62, e2023056 4. Durbin et al. (2023), "ProSight Native: Defining Protein Complex Composition from Native Top-Down Mass Spectrometry Data", J. Proteome Res., Vol. 22, pp. 2660 - 2668 5.Fornelli et al.(2018),「Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques」,Anal.Chem.,Vol.90,pp.8421-8429

Claims

1. A method for analyzing a sample containing intact membrane proteins, wherein the method is (i) To provide a sample containing the intact membrane protein, a non-synthetic biomembrane material, a solvent, and bulk lipid molecules, (ii) Ionizing the sample to produce a gas-phase proto-membrane protein ion, wherein the gas-phase proto-membrane protein ion contains intact membrane protein nonspecifically bound to one or more bulk lipid molecules and one or more residual solvent molecules, (iii) Confinement of the gas phase proto-membrane protein ions, (iv) By irradiating the confined gas-phase proto-membrane protein ions with infrared radiation from a laser source, one or more residual solvent molecules and one or more nonspecifically bound bulk lipid molecules are separated from the intact membrane protein to generate intact gas-phase membrane protein ions, (v) A method comprising m / z analysis and detection of intact membrane protein ions and / or ions derived therefrom in the gas phase.

2. The method according to claim 1, wherein the sample comprises a non-synthetic biomembrane material extracted from or directly secreted by cells, organelles, viral envelopes, or vesicles.

3. The method according to claim 1 or 2, wherein the intact membrane protein ions in the gas phase do not contain nonspecifically bound bulk lipid molecules, and preferably do not contain nonspecifically bound molecules of other non-lipids.

4. The method according to claim 1 or 2, wherein in the non-synthetic biomembrane material provided in step (i), the intact membrane protein is present in or attached to the lipid bilayer containing the bulk lipid molecule.

5. The method according to claim 4, wherein the lipid bilayer forms vesicles.

6. The method according to claim 1 or 2, wherein step (i) comprises ultrasonically treating the sample.

7. The method according to claim 1 or 2, wherein the sample is ionized by electrospray ionization, preferably by nano-electrospray ionization, or by static electrospray ionization.

8. The method according to claim 1 or 2, wherein the sample is ionized by native electrospray ionization.

9. The method according to claim 1 or 2, wherein the gas-phase protomembrane protein ion of step (ii) comprises one or more submicron-scale membrane fragments or vesicles associated with the intact membrane protein and solvent molecules.

10. The method according to claim 1 or 2, further comprising exposing the gas-phase proto-membrane protein ions to a collision gas to remove one or more residual solvent molecules by collision-induced dissociation.

11. The method according to claim 1 or 2, wherein the wavelength of the infrared radiation from the laser source preferentially excites one or more vibrational modes of one or more bonds in the nonspecifically bound bulk lipid molecules.

12. The method according to claim 1 or 2, wherein the infrared radiation from the laser source is continuous wave radiation.

13. The method according to claim 11, wherein the wavelength of the infrared radiation from the laser source is 780 nm to 1 mm, preferably 1.4 μm to 1 mm, more preferably 3 μm to 100 μm, and most preferably 9 to 11 μm.

14. The method according to claim 11, wherein the wavelength of the infrared radiation from the laser source has a dominant wavelength of one or more of 10.6 μm, 10.2 μm, 9.6 μm, and 9.3 μm.

15. The method according to claim 1 or 2, wherein the irradiation by infrared radiation is carried out for a period of up to 1 second, preferably less than 50 ms.

16. Process (v) is, (va) Subjecting the intact membrane protein ions in the gas phase to an ion conversion process to generate first-generation product ions, (vb) The method according to claim 1, comprising m / z selection of the first generation product ions.

17. (vc) The first generation product ions are subjected to a further ion conversion process to generate a second generation product ions, The method according to claim 16, comprising (vd) m / z analysis and detection of the second generation product ions.

18. The method according to claim 16 or 17, wherein each ion conversion process is selected from infrared multiphoton dissociation, electron transfer dissociation, activated ion electron transfer dissociation, electron capture dissociation, UV photodissociation, and collision-induced dissociation.

19. (vi) The method according to claim 16 or 17, further comprising analyzing the m / z spectral data of the product ion to obtain information regarding the structure and / or identity of the intact membrane protein.

20. The method according to claim 19, wherein the information includes the amino acid sequence of all or part of the intact membrane protein, and / or the location and / or type of post-translational modification of the intact membrane protein.

21. The gas-phase protomembrane protein ions and / or intact membrane protein ions in the gas phase are retained in an ion trap or permeated through an ion guide or mass filter, and the method is The method according to claim 1 or 2, comprising m / z isolation of the gas-phase proto-membrane protein ions and / or intact membrane protein ions in the gas phase by releasing one or more ions outside a predetermined m / z range from the ion trap, ion guide, or mass filter.

22. The method according to claim 1 or 2, comprising reducing the charge of a precursor ion to generate the ion before the ion is detected or subjected to any step in an ion conversion process.

23. The method according to claim 1 or 2, comprising repeating the method until intact membrane protein ions in the gas phase can be detected after step (iv), and determining the irradiation parameters used in step (iii) by varying the energy of the infrared radiation from the laser source and / or the duration of the irradiation.

24. The method according to claim 1 or 2, wherein the intact membrane protein comprises two or more non-covalent monomers, and these monomers may be the same or different.

25. The method according to claim 1 or 2, wherein the intact membrane protein ions in the gas phase comprise one or more non-covalent ligands.

26. The method according to claim 25, wherein the non-covalent ligand is selected from the group consisting of lipids, RNA chains, metabolites, drugs, and metal cofactors.

27. The method according to claim 1 or 2, wherein the sample preferably comprises a mass spectrometry-compatible buffer that maintains the structural integrity of the intact membrane protein in submicron-scale membrane fragments or vesicles associated with the intact membrane protein and solvent molecules.

28. The method according to claim 1 or 2, wherein step (v) comprises determining the m / z ratio of intact membrane protein ions and / or ions derived therefrom in the gas phase.

29. The method according to claim 1 or 2, wherein the sample, the non-synthetic biomembrane material, and / or the gas-phase protomembrane protein ions are at least substantially free of detergent.

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