Sample digestion for mass spectrometers

Abstract

Devices and methods for analyzing organic molecules in a condensed phase sample are disclosed. The method may include: generating a plasma with an electric spark; performing plasma treatment by exposing the condensed phase sample to the plasma, thereby generating fragments of organic molecules in the condensed phase sample; and after completion of the plasma treatment, detecting these fragments using a mass spectrometer or other analysis device and characterizing the fragments, molecules or samples with the detected fragments. These fragments may in particular be cleaved from the backbone of organic molecules.

Description

[0001] This invention relates to sample digestion processes for subsequent mass spectrometry analysis, and more particularly, to such digestion processes performed within condensed-phase samples, for example, prior to the subsequent desorption of sample components for mass spectrometry analysis. For example, the digestion may be the digestion of organic molecules in such condensed-phase samples. The invention also relates to the analysis of samples in which molecular fragments are generated in the condensed-phase sample through the aforementioned digestion or cleavage, followed by mass spectrometry analysis (e.g., including desorption or ionization) of the fragments for detection.

[0002] Introduction The identification of proteins, peptides, and other organic molecules (especially organic polymers) can be achieved through top-down whole-molecule mass spectrometry, but this technique is quite challenging, especially for proteins, and requires specific mass spectrometers. As an alternative, bottom-up mass spectrometry can be used to analyze target molecules; however, this method typically requires enzymatic digestion of the molecules, which is not only time-consuming but also requires reagents in the liquid phase. This enzymatic digestion technique often leads to an imbalance in the distribution of analytes in the sample, resulting in a loss of structural and spatial resolution.

[0003] The aim is to address these and other issues present in the relevant existing technologies. Summary of the Invention

[0004] This invention provides a method for analyzing samples using mass spectrometry or other analytical techniques. Specifically, it uses a "bottom-up" approach to analyze organic molecular samples, such as peptides and proteins, without the need for enzymatic digestion, which is not only time-consuming but also requires multiple liquid chromatography reagents. Unlike traditional methods, this method involves performing plasma pretreatment or digestion on the condensed phase sample before any subsequent desorption or other processes to introduce sample debris generated during plasma digestion into the mass spectrometer or other type of analyzer. Typically, pretreatment and plasma digestion are performed and completed in a first location. After pretreatment, the sample is transferred to a second location for desorption and / or ionization and / or other processes as part of the process of introducing the generated debris into the mass spectrometer or other analyzer. For example, the introduction into the mass spectrometer or other analyzer can occur at least 1 second, at least 5 seconds, at least 10 seconds, at least 30 seconds, at least 1 minute, at least 1 hour, or at least 1 day after the completion of the plasma digestion process.

[0005] After the plasma digestion process, other analytical methods and corresponding analyzers used to analyze the samples include: nuclear magnetic resonance (NMR) or electron paramagnetic resonance (EPR) spectroscopy and analyzers; as well as various optical spectroscopy and analyzers, such as analyzers operating in the infrared, ultraviolet and / or visible light regions, and related analyzers using Raman spectroscopy, absorption spectroscopy and other techniques.

[0006] Specifically, the present invention provides a method and apparatus for analyzing condensed phase samples: a pretreatment stage is performed by using an electric spark or similar means, wherein plasma fragmentation and digestion are performed in the condensed phase sample, for example, without immediate desorption of the sample; then, after the pretreatment is completed, at least a portion of the sample containing the generated fragments is analyzed using mass spectrometry or other analytical methods.

[0007] The target molecules to be cleaved in the sample can be organic molecules, such as proteins or peptides, especially organic polymer molecules. The above technique is particularly advantageous for detecting organic molecular fragments generated from cleavage along the polymer backbone.

[0008] Desorption or similar treatment of the sample into a mass spectrometer or other analytical instrument is preferably performed only after the plasma pyrolysis and digestion processes have been completed, and these two processes can typically be performed in different locations or in different laboratory equipment, for example, at different times.

[0009] The plasma used to generate cleavage and disintegration in condensed-phase samples can be generated in a suitable gaseous environment (such as argon) to produce suitable organic molecular fragments, typically smaller than about 1000 m / z - 2000 m / z. This technique can be regioselective, favoring the formation of specific fragments, depending on processing conditions such as the total processing time and the electrical power used to generate the plasma; and, the inventors' experimental evidence does indeed show that, for a given sample of organic molecules, the sample environment, and the plasma generation conditions, the bond breaking within the organic molecules is quite consistent. Because no additional liquid, such as an enzyme solution, is required to achieve cleavage, solid samples can be treated in this manner without the migration or movement of the target molecules used for cleavage within the sample.

[0010] After plasma treatment of the condensed phase sample, for example, at least 1 second, 5 seconds, 10 seconds, 30 seconds, 1 minute, 1 hour, or 1 day after treatment, the resulting molecular fragments can be analyzed using known analytical methods, such as mass injection and mass-to-charge ratio separation, for example using direct injection, liquid chromatography-mass spectrometry, imaging mass spectrometry, or other of the aforementioned analytical techniques. Data obtained by mass spectrometry or other analytical techniques can then be used, for example, to determine the presence of specific proteins or other molecules in the plasma-treated sample, or for de novo sequencing or structural determination.

[0011] According to some aspects, the present invention provides a method for analyzing molecules in a condensed phase sample, the method comprising: generating a plasma, for example, using an electric spark to generate a plasma; exposing the condensed phase sample to the plasma to generate molecular fragments within the condensed phase sample; and detecting the fragments using a mass spectrometer or other analytical device or apparatus. Subsequently, the fragments themselves, or the molecules or the sample, can typically be characterized, identified, or analyzed using a mass spectrometer or other analytical device or apparatus associated with or including fragment detection.

[0012] Typically, plasma treatment of samples is performed externally to or away from the mass spectrometer or other analytical equipment during phase separation. The treated sample is then transferred to the mass spectrometer or other analytical equipment (or particularly to the sample introduction component of the analytical equipment, such as an ion source) and received for characterization. The plasma generated using the described technique can be referred to as cold plasma, non-thermal plasma, or cryogenic plasma. In such plasmas, the temperature distribution of electrons is typically much higher than that of heavy ions and neutral particles.

[0013] The condensed phase sample, when exposed to plasma, may be situated on a solid substrate, such as a microscope slide or other similar substrate. In some configurations, the substrate may be perforated, for example, in a mesh structure, which can facilitate gas flow to allow better contact between the plasma and the sample. In some embodiments, the condensed phase sample may be provided in or as part of an enzyme-linked immunosorbent assay (ELISA) kit, a lateral flow assay device, or other various types of assay kits and devices; said kits or devices subsequently provide a substrate on which the sample is placed, either on or inside.

[0014] The sample may specifically include organic molecules, such as one or more of the following: proteins, peptides, lipids, other organic polymer molecules, and small molecules. If the sample includes organic polymer molecules, the described technique has a significant advantage in causing the organic molecules to cleave along the polymer backbone.

[0015] Condensed phase samples can be in liquid, solid, or some mixture of liquid and solid forms, but the plasma treatment techniques described are particularly suitable for solid samples.

[0016] After exposure to plasma, at least a portion of the condensed phase sample can be removed from the substrate, for example, by mechanical removal in its existing condensed phase morphology or by dissolving part of the sample in a solvent, and then introduced into a mass spectrometer for subsequent ionization and characterization operations.

[0017] The plasma treatment described includes electric sparks, plasma, and exposed samples, and can typically be performed at or near ambient pressure, for example, at about 30 kPa to 300 kPa.

[0018] An electric spark can be generated by applying an electrical signal to one or more electrodes. Examples of AC and DC signals are described herein, but the inventors have found that AC signals are generally more effective at causing fragmentation of condensed phase samples, especially radio frequency (RF) signals. If AC or RF signals are used, their power is typically from 10 watts to 300 watts, and / or peak voltage from 1000 volts to 10000 volts, and / or frequency from 100 kHz to 5 MHz.

[0019] The one or more electrodes may include: a plasma source electrode configured to generate plasma between the plasma source electrode and the sample; and a ground plane electrode disposed on the opposite side of the sample relative to the plasma source electrode. For example, the ground plane electrode may be disposed directly below the sample, or may be separated from the sample by some or all of the thickness of a substrate. In other embodiments, all electrodes may be provided on the same side of the sample and the generated plasma.

[0020] The method may further include providing a gas flow at the electric spark, or the gas flow flowing from the electric spark to or to the sample. A variety of such gases may be used, including at least 50% or at least 90% of one or more of the following gases: noble gases, argon, helium, and nitrogen. The inventors have found that the effect is particularly significant when using argon. The gas may also contain one or more dopants (such as fluoranthene), at concentrations much lower than those of the aforementioned gases, to support the above and other functions.

[0021] One of the one or more electrodes, such as the plasma source electrode described above, may be provided by a conductor (e.g., a needle-like body) extending along a conduit to an open end of the conduit, where the conductor may slightly protrude from the conduit port, for example, 0 mm to 5 mm. In this way, an electric spark can be generated in the region near the conduit opening. Furthermore, a gas flow can also be guided along the conduit.

[0022] The condensed phase sample can be exposed to plasma for a suitable duration, which can be determined or optimized experimentally. However, based on the experimental results completed by the inventors, the duration can be any one or more of the following: at least 0.1 seconds, at least 1 second, at least 10 seconds, and at least 60 seconds.

[0023] The method may further include, prior to the step of exposing the condensed phase sample to plasma to generate fragments of organic molecules, treating the condensed phase sample with a compound selected from spin traps and spin scavengers. This implementation is particularly advantageous when the electrical signal is a DC signal. While not wishing to be bound by theory, it is believed that when generating plasma using a DC signal under the conditions described herein, at least some fragments generated from organic molecules can exist in the form of short-lived free radicals. Spin traps or spin scavengers can react with these short-lived free radicals to generate stable species, which can then be characterized and analyzed using mass spectrometry.

[0024] The present invention also provides corresponding apparatus for processing condensed phase samples for subsequent analysis (e.g., processing organic molecules in condensed phase samples). Such apparatus may include: a plasma generator configured to generate plasma using an electric spark; and a substrate carrier for receiving a substrate carrying the condensed phase sample, thereby exposing the condensed phase sample to the generated plasma, such that the plasma generates fragments of organic molecules within the condensed phase sample.

[0025] Such devices may also include a mass spectrometer or other analytical apparatus configured to receive and detect the generated debris after plasma treatment, and optionally a sample transfer mechanism configured to transfer part or all of the condensed phase sample to the mass spectrometer or other analytical apparatus after plasma treatment. The device may also include a substrate and optionally the condensed phase sample itself, which is typically situated on the substrate surface or within the substrate. Generally, the mass spectrometer or other analytical apparatus is configured to receive debris generated outside the apparatus.

[0026] The device, such as a computer system using the device, may also be configured to characterize or identify a sample using data from a mass spectrometer or other analytical device, or more specifically to identify or characterize fragments of molecules or molecules themselves, the mass spectrometer or other analytical device including data representing the detected fragments, such as specific data associated with the fragments, or mass spectrometry data or spectral data or other data representing the fragments.

[0027] The substrate may be a solid substrate, such as a microscope slide, an insulating substrate, or a substrate carrying one or more electrodes (such as the one or more ground plane electrodes described above), for providing a return path for electrical signals used in the plasma generation process. The device may further include a signal generator configured to deliver an AC or DC signal to the plasma generator to generate plasma.

[0028] If an AC signal is used, it can include one or more of the following: power from 10 watts to 300 watts; peak voltage from 1000 volts to 10000 volts; and frequency from 100 kHz to 5 MHz.

[0029] The device is further configured to provide a gas flow at or near the spark, optionally directed toward a sample. The plasma generator may include one or more electrodes, to which an AC or DC signal is applied to generate an electric spark. At least one of the electrodes (e.g., one or more plasma source electrodes described above) may be provided by a conductor extending along a conduit to an open end of the conduit, thereby generating an electric spark near the open end of the conduit while the gas flow continues along the conduit.

[0030] Although the methods and apparatus described herein may be described in detail below as using mass spectrometry to detect fragments and characterize or identify fragments, molecules and / or samples, other analytical techniques and instruments, such as nuclear magnetic resonance spectroscopy or electron paramagnetic resonance spectroscopy and corresponding instruments, may be used as alternatives to or supplements to mass spectrometry. Attached Figure Description

[0031] Embodiments of the present invention will now be described by way of example only, with reference to the accompanying drawings, which are as follows: Figure 1 The diagram schematically illustrates an apparatus for plasma-processing and digesting a sample to produce molecular fragments, which are then detected using a mass spectrometer or other analytical equipment or apparatus. Figure 2 The method of the present invention is shown, which can be implemented using the apparatus of claim 1; Figure 3The mass spectrometry intensities of various digestion products of leucine enkephalin after different plasma treatment times as described above are shown. Figure 4 The mass spectrometry intensity of various digestion products of leucine enkephalin after plasma treatment with AC signals of different powers as described above is shown. Figure 5A and Figure 5B The mass spectrometry detection intensities of various digestion products of leucine enkephalin are shown in the figure after plasma treatment with DC electrical signals of different powers as described above.

[0032] Figure 6 The mass spectrometry intensities of various digestion products of methionine enkephalin after different plasma treatment times as described above are shown. Figure 7 The mass spectrometry intensities of various digestion products of creatinine after different plasma treatment times as described above are shown. Figure 8 The mass spectrometry intensity of various digestion products of leucine enkephalin after plasma treatment as described above using a DC electrical signal is shown, wherein the leucine enkephalin was first treated with DMPO before plasma treatment. Figure 9 The mass spectrometry intensity of various digestion products of leucine enkephalin after plasma treatment as described above using a DC electrical signal is shown, wherein the leucine enkephalin was treated with 4-phosphono-TEMPO before plasma treatment. Figure 10 The mass spectrometry intensity of various digestion products of leucine enkephalin after plasma treatment as described above using DC electrical signals is shown, wherein leucine enkephalin was first treated with 3-CP before plasma treatment. Figure 11 Mass spectrometry was used to detect the intensity of various digestion products of leucine enkephalin after plasma treatment as described above, without the use of spin trapping / spin scavenging agents; Figure 12 The DESI-MSI thermograms of two m / z peaks generated by exposure to positive DC-AP plasma applied to pure leucine enkephalin, pure bradykinin, and a mixture thereof are shown. Figure 13 The MALDI-MSI thermograms of the peak at m / z 502.319 are shown in the lysates of control cells and cells modified to express the fluorescent protein mNeonGreen, where the lysates were not subjected to plasma treatment or exposed to a cryogenic argon plasma device for 1 minute; and Figure 14The MALDI-MSI thermograms of different tissues at a peak of 203.172 m / z are shown. These tissues were optionally spiked and / or exposed to a cryogenic argon plasma device for 1 minute. Detailed Implementation

[0033] For reference Figure 1 , Figure 1 An apparatus is shown for analyzing organic molecules in a condensed phase sample, more specifically, condensed phase sample 10, using a plasma processing digestion process. The condensed phase sample 10 may be a solid, a liquid, or a mixture of both, such as a gel or other mixed phase. In some cases, the sample may be a crystalline solid; in others, the sample may include: a solution containing dissolved organic molecules, tissue samples from human or animal individuals (e.g., biopsy samples), cell samples or blood samples, environmental samples (e.g., soil or plant tissue), food samples, cell cultures (e.g., cultures of mammalian cells, plant cells, insect cells, or microbial cells) or cell lysates, or engineered or artificial tissues, or many other types of condensed phase samples.

[0034] The organic molecules to be analyzed can specifically be proteins and / or peptides, or include proteins and / or peptides, but a broad range of other organic molecules (such as amino acids and other small organic molecules, lipids, carbohydrates, and other long-chain molecules) can also be processed and analyzed in a similar manner. Experiments conducted by the inventors, discussed below, show that applying the described plasma treatment process to peptides often produces C-type and Z-type ions commonly seen in mass spectrometry analysis. These ions are typical products of odd-electron cleavage, and the extent of this bond breaking and subsequent fragmentation appears to depend on factors such as the duration of plasma treatment.

[0035] The described techniques are particularly advantageous when the organic molecules are organic polymer molecules such as proteins and / or peptides, because they can be used to cleave these organic molecules along the polymer backbone, as shown in the exemplary experiments listed below.

[0036] exist Figure 1 In this configuration, the condensed phase sample is deposited in a solid form on, above, or inside the solid substrate 12 (i.e., the sample is in the form of a continuous liquid, solid, or a mixture of liquid and solid). The substrate can be a microscope slide or similar substrate, and may also be perforated, for example, in the form of a sieve, to allow continuous gas flow through the sample and better contact between the plasma and the sample. Typically, the sample rests on the substrate. To generate plasma for processing the sample, an electrical signal... SA signal generator 14 generates and applies a signal to one or more electrodes, thereby producing an electrical spark 18 (which may be described as a corona discharge in at least some embodiments), which in turn generates plasma 20 in the vicinity of the electrodes. Subsequently, the condensed phase sample 10 deposited on the substrate is exposed to the plasma 20, typically through direct contact between the plasma and the sample; this process causes the organic molecules in the condensed phase of the sample to break down, for example, without requiring desorption of the sample. This process can also be referred to as sample digestion.

[0037] In some configurations, such as Figure 1 As shown, one or more electrodes include: one or more plasma source electrodes 16 configured to generate plasma 20 between the plasma source electrodes 16 and the sample 10; and one or more ground plane electrodes 24 or grounding pads disposed on the opposite side of the sample relative to the plasma source electrodes. For example, the ground plane electrodes may be spaced from the sample by at least a portion of the substrate 12 and may form part of the substrate.

[0038] In other configurations, two or all electrodes can be provided on the same side of the sample, i.e. the same side that generates the plasma, instead of using one or more grounded plane electrodes.

[0039] The fragmentation described herein originates from the breaking of bonds between different main components of a molecule; for example, in the case of peptide molecules, this typically involves the breaking of C-C or CN bonds. If the target molecule is an organic polymer, the advantage of the described technique lies in its ability to induce fragmentation along the main chain of the organic polymer molecule, which is particularly advantageous. The term "fragmentation" as used herein does not refer to a simple ionization process achieved solely by the gain or loss of one or more electrons or protons. Although some degree of ionization may indeed occur during this process (e.g., in the liquid solution contained in the sample), this fragmentation process generally does not alter the morphology of the sample on the substrate—the sample remains in a continuous condensed phase state as before plasma treatment.

[0040] The plasma generated in the described technique can be described as cold plasma, non-thermal plasma, or cryogenic plasma. Such plasma can be conveniently generated by an electric spark under ambient or near-ambient pressure conditions.

[0041] Although the description of this invention does not rely on protein plasma cleavage affected by a specific mechanism, the inventors believe that the energy of plasma can directly break organic molecules, which can be electronic effects (such as odd electron cleavage) and space-dependent energy effects (which originate from the transfer of energy from excited gas particles to bonds and atoms).

[0042] This plasma treatment of the sample can be performed for a predetermined period of time, typically tens of seconds, but optionally at least 0.1 seconds, at least 1 second, at least 10 seconds, at least 60 seconds, or at least 120 seconds. During this treatment, the sample can remain stationary relative to the plasma and / or one or more electrodes; or, if necessary, it can be moved while remaining fixed to the substrate, for example, to improve the effectiveness or consistency of the plasma treatment. The inventors have found that longer treatment durations produce more fragmentation or achieve better disintegration, possibly including further fragmentation of fragments already generated in the early stages of treatment. Useful-grade fragmentation has been observed after only about 1 to about 10 seconds of treatment. Moreover, if suitable fragmentation can be obtained within the aforementioned time, even shorter treatment durations, such as 0.1 seconds or longer, can be used.

[0043] The desorption of the condensed phase sample into the mass spectrometer is preferably not performed simultaneously with plasma treatment. Instead, once the plasma treatment is complete and the sample is no longer exposed to the plasma treatment, part or all of the sample containing fragments generated by plasma treatment of organic molecules is introduced into the mass spectrometer 30, for example, through the ionizer 32 of the mass spectrometer. To achieve this, typically the condensed phase portion of the sample 10 is first removed from the substrate 12 before being introduced into the ionizer 32; however, optionally, depending on the design and function of the ionizer 32, the entire substrate is placed in the ion source of the mass spectrometer to achieve this purpose.

[0044] Then, a mass spectrometer is used to detect and / or characterize the sample fragments generated by the plasma treatment described above. For example, the mass spectrometer 30 can output data such as the peak intensity of a specific mass-to-charge ratio of the fragments to a computer 34. The computer 34 uses this data to identify, quantify, or obtain structural information of one or more original organic molecules present in the condensed phase sample. Alternatively, the computer 34 can use this data to characterize the sample in other ways by measuring the generated fragments using mass spectrometry, such as through fingerprinting, statistical analysis, or AI techniques. It should be noted that such detection and / or characterization of the fragments by a mass spectrometer can include various mass spectrometry techniques, which can separate and further fragment the fragments generated by plasma treatment to obtain more structural information. Other analytical techniques, such as NMR, can also be used to characterize the fragments.

[0045] In some implementations, imaging mass spectrometry can be used to detect or characterize fragments at multiple locations on the sample (e.g., along a line, in a grid, or at other distributed locations). This method of using imaging mass spectrometry is particularly useful when the sample is solid after plasma treatment, and the treated solid sample can then be used directly as the sample for imaging mass spectrometry analysis, thus maintaining the integrity of the sample structure and preventing displacement during plasma treatment until the material is extracted for mass spectrometry analysis. If imaging mass spectrometry is used, it typically involves the use of a moving stage that moves the substrate through the mass spectrometer's inlet or ionization site (such as the location indicated by the ion beam), typically in grating mode, but other scanning modes may also be used. With such a moving stage, the substrate and sample can be mounted together on the moving stage for the described plasma treatment before subsequent imaging mass spectrometry analysis.

[0046] It should be noted that, although Figure 1 The illustration shows the use of mass spectrometry to detect and / or characterize sample fragments generated after plasma treatment. However, other applicable analytical methods and corresponding equipment can also be used to analyze the sample after plasma digestion. Such methods and equipment include: nuclear magnetic resonance (NMR) methods and NMR analyzers, various spectroscopic methods and spectroscopic analyzers (e.g., spectrometers operating in the infrared, ultraviolet, and / or visible light regions, spectrometers operating using Raman spectroscopy, and spectrometers operating using electron paramagnetic resonance (EPR)); and these other methods and equipment can also perform imaging pattern analysis of the sample, similar to the imaging mass spectrometer described above.

[0047] like Figure 1 As shown, a gas flow 40 can be provided to flow through the electric spark 18 toward or to the sample 10. The gas flow 40 can help generate and maintain plasma via the electric spark, and the gas flow 40 can also facilitate contact between the plasma and the sample. The gas can be of various types and gas combinations, which will be further described below.

[0048] Figure 1In this design, the conduit 42 (e.g., a plastic or glass tube) has an open end or nozzle 44 facing or reaching the sample 10, and an electric spark is generated near or within the open end of the conduit. For example, the electric spark is generated by a plasma source electrode 16 arranged as a needle, which extends along the conduit and terminates at a tip discharge point 22 near the open end of the conduit. The plasma source electrode 16 may terminate within the conduit 42 or at the end of the conduit 42, or it may extend slightly beyond the end of the conduit. In some experiments conducted by the inventors, the conduit 42 may be a plastic tube with an inner diameter of about 10 mm, and the plasma source electrode may be a stainless steel corona needle that runs along the inside of the conduit and extends outward from the open end by about 2 mm. In these experiments, the distance between the end of the plasma source electrode and the sample is about 1 mm. Of course, the geometric parameters such as the aforementioned extension distance and the distance from the discharge point to the sample can be adjusted as needed.

[0049] In other configurations, a ground plane or second electrode is provided around the tip of the conduit 42 or at other locations on the sample that are on the same side as the plasma source electrode, instead of using the ground plane electrode 24 located on the side of the sample 10 or substrate 12 opposite to the plasma source electrode 16.

[0050] although Figure 1 The configuration shown depicts a single conduit 42 for delivering the gas flow 40, with a single plasma source electrode extending along the conduit to near its end for generating plasma. However, various other geometries are also possible. For example, various other shapes and forms of electrodes can be used, which are not necessarily required to extend along the conduit delivering the gas flow and can be positioned independently outside any such conduit. Furthermore, multiple such electrodes can be used, or a single electrode with multiple discharge points 22 (e.g., comb-shaped or brush-shaped electrodes) can be used. Similarly, multiple conduits delivering the gas flow can be used to aid in the generation and maintenance of plasma and to guide the plasma toward the solidified phase sample.

[0051] More broadly, one or more plasma source electrodes 16, including discharge point 22 and optionally including other components (e.g., conduit 42), can be described at least as a plasma generator 50, which is provided with an electrical signal by signal generator 14. S To generate plasma. In some embodiments, specific means may also be provided to carry or partially carry the sample to the mass spectrometer 30 and / or ionizer 32 after plasma treatment, and in Figure 1In this context, it is typically referred to simply as a sample transfer mechanism 60. This can take the form of a robot or other mechanical device, and can involve liquid transfer, such as using the aforementioned pipette, or other liquid transfer methods, or the transfer of solid materials from a sample or the entire substrate. In particular, it can utilize an automated mechanism to automatically transfer the plasma-treated sample from the plasma treatment device to the mass spectrometer without human intervention. Figure 1 As shown, the apparatus may also include a substrate carrier 70 for receiving a substrate and / or sample in a suitable or correct position for plasma processing by the plasma generator 50. For example, such a substrate carrier 70 may include a frame of suitable size and shape for accommodating the substrate 12.

[0052] To generate plasma, various electrical signals can be used. S And its related circuit configuration, including the configuration of the substrate supporting the sample. For example, in some configurations, during processing, signals... S This can be a DC signal, or optionally a DC signal with a substantially constant voltage or a DC signal with varying voltage. For example, the voltage of a DC signal is approximately 1000 volts to 10000 volts. Such a DC signal can also have a duty cycle during which the voltage is low or zero for a portion of the time (e.g., 50% of the processing period) and at other times (e.g., the other 50% of the non-processing period) the voltage is at a higher level, thereby generating plasma. If a signal in DC form is used... S In this case, a conductive return path is usually required for the sample. For example, a microscope slide or other insulating component with a conductive plane or conductive grid is selected for the substrate 12, thereby providing a locally grounded plane electrode in contact with the sample and for the electrical signal. S Construct a return path from the ground plane electrode ( Figure 1 (Not shown); or a conductive substrate employing a similar connection method may be selected. In some configurations, this can be achieved by conveniently using a whole or fragment of a commercially available microscope slide as the substrate 12, on which electrodes in the form of a thin conductive layer (e.g., indium tin oxide) are provided, with the sample supported on the upper surface of the substrate.

[0053] The inventors discovered that, although DC signals S It can be used to generate a plasma effective for the organic molecules in lysed samples for subsequent mass spectrometry analysis and fragment detection, but generating a suitable plasma is challenging, except at relatively low effective electrical power (e.g., about 1 watt or less). Therefore, in other configurations, the signal... SThis can be an AC signal, or more specifically, a radio frequency (RF) signal. For example, the peak-to-peak voltage of an AC signal can be approximately 1000 volts to 10000 volts, more specifically approximately 2000 volts to 5000 volts. The AC signal is typically sinusoidal, or at least has a dominant or predominant frequency pattern. Thus, the frequency of the AC signal (or the frequency of the dominant or predominant pattern) can be approximately 10 kHz to 10 MHz, or more specifically approximately 100 kHz to 1 MHz, or 100 kHz to 5 MHz, or more broadly, a radio frequency AC signal. The electrical power of the AC signal can be in the tens of watts range, for example, 1 watt to 1000 watts, or more specifically 10 watts to 300 watts. The RF signal can also be in the form of a digital RF signal.

[0054] If the signal is in AC form S When used with a suitable AC frequency and electrical arrangement, it is not necessary to provide any conductive return current path directly from the sample. For example, instead of placing the sample on the conductive surface or other electrodes of the substrate 12, the ground plane electrode 24 can be used as a grounding pad, for example, placed on the lower surface of the insulating substrate 12 or other location that is not in direct contact with the sample. This grounding pad 24 typically shares an electrical ground terminal with the signal generator 14, or a more direct or specialized electrical connection method can be used to achieve the connection between the two.

[0055] In some specific embodiments proposed by the inventors, a ForceTriad (RTM) electrosurgical signal generator manufactured by Medtronic Inc. is used to provide radio frequency AC signals. S This signal is used to generate an RF AC signal containing a damped sine pulse at a frequency of approximately 472 kHz. The damped sine pulse has a random repetition center at approximately 21.7 kHz and a total duty cycle of 4.6%. Throughout the processing, the average power is approximately 20 to 120 watts, and the peak-to-peak voltage of signal S is approximately 2000 to 3700 volts.

[0056] As mentioned above, Figure 1The configuration provides the presence of a specific gas 40 located at or flowing through the spark 18 and / or discharge point 22. Using a suitable gas species in this gas enables or assists in the generation and maintenance of plasma, and the gas flow can also assist in guiding the plasma onto or into the sample for contact with the sample. The inventors have found argon to be a suitable gas stream, but other rare gases (e.g., helium) and other relatively reactive gases (e.g., nitrogen) can also be used, as well as mixtures of the above gases. For example, the gas may comprise at least 50% or at least 90% of one or more of the following gases: rare gases, argon, helium, neon, and nitrogen. The inventors have found that argon is particularly effective in supporting the fragmentation of organic molecules in a sample for mass spectrometry, possibly due in part to its lower breakdown voltage compared to air. Gas 40 may also contain one or more dopants (e.g., fluoranthene) at much lower concentrations to support the above and other functions.

[0057] The gas volumetric flow rate or gas velocity, for example at the outlet or nozzle 44, can be determined as needed and according to other aspects of the equipment used (such as the size of nozzle 44, signal strength, etc.). S The electrical power, as well as the distance and geometry between the nozzle 44, the discharge point 22, and the sample 10, can be adjusted. However, a flow gas source pressure of about 10 kPa (0.1 bar) higher than the plasma ambient pressure, or a flow rate of about 0.1 L / min–1.0 L / min, can typically be used.

[0058] In similar Figure 1 In this configuration, gas can be directed along conduit 42 to spark 18 and / or discharge point 22, but such gas flow or positioning at these points can be achieved in other ways, such as placing the sample and electrodes within a closed container filled with gas 40, delivering gas from other directions (e.g., laterally across the sample) using one or more conduits or nozzles, and so on. However, it can be advantageous to ensure that the direction of gas flow is directly from spark 18 toward the sample, thereby allowing the plasma to contact the sample under the impetus of the gas flow.

[0059] The advantage of the described apparatus for generating organic molecular fragments for mass spectrometry analysis is that it can be performed under ambient pressure, thus avoiding the use of any vacuum or pressure vessels. However, if desired, the configuration described above can also be implemented under partial vacuum or high pressure, for example, in the range of about 30 kPa to about 300 kPa.

[0060] The condensed phase sample 10 used for the plasma treatment described above can be prepared in various ways. For example, in the experiment described below, an acetate solid sample of organic molecules (such as peptides) is dissolved in a 30:70 mixture of water and methanol, then pipetted onto a suitable microscope slide substrate 12, allowed to dry, and then subjected to plasma treatment. However, various other sample preparation methods can also be used, including methods with little or no treatment. For example, biopsy or sample cell cultures, blood or other tissue samples, environmental samples, food samples, etc., can all be applied to suitable substrates for plasma treatment.

[0061] However, in some instances, condensed phase samples can undergo the aforementioned plasma treatment without being applied to a specific substrate, such as in vitro or in vivo, for example, during surgical procedures in humans or animals. For instance, during surgery, an electrosurgical instrument can be used to perform plasma treatment, generating plasma during the procedure and simultaneously performing the aforementioned fragmentation, after which fragments can be automatically or manually removed from the surgical site for mass spectrometry analysis.

[0062] The inventors also discovered that adding certain reagents or substances to a sample can generally increase yield, or increase the yield of specific fragments, and / or improve the identification of such fragments in the mass spectrometer output data. For example, samples can be treated with compounds selected from spin traps and spin scavengers. These compounds are particularly useful when organic molecules generate short-lived free radicals (species with at least one unpaired valence electron) after plasma treatment, as these free radicals may decompose before being detected in the mass spectrometer. In particular, spin traps or spin scavengers can react with these short-lived free radicals to generate stable species that can be detected. In this document, a "spin trap" refers to a diamagnetic compound that can react with unstable (short-lived) free radicals to form a stable addition product existing as a further radical. A "spin scavenger" refers to a free radical that can react with unstable (short-lived) free radicals to form a stable addition product existing as a diamagnetic (i.e., non-radical) compound. These terms are known to those skilled in the field of free radical detection. "Spin scavenger" is also known as "radical scavenger".

[0063] While the concept of using radical addition reactions to aid in the detection of short-lived radicals is known in analytical methods such as electron paramagnetic resonance (EPR) spectroscopy, mass spectrometry detects changes in mass rather than unpaired spins. Specifically, the inventors propose treating the condensed phase sample with spin traps and spin scavengers before it enters the mass spectrometer. One advantage of this approach is that the spin traps or scavengers are already present when fragments are generated, so they can be captured or removed almost instantaneously. In contrast, in most existing analytical methods (such as EPR spectroscopy), the use of spin traps and scavengers involves adding the compound after the radicals have been generated, meaning there is a risk that particularly short-lived radicals may decompose before having a chance to react with the compound.

[0064] The inventors have discovered that certain plasma treatment methods are more likely to generate free radicals, and therefore more likely to stabilize fragments using spin traps or spin scavengers. For example, when plasma is generated using a DC signal under the conditions described herein, at least some fragments generated in organic molecules can exist in the form of free radicals. In contrast, under the conditions used by the inventors to date, no products have been observed formed after the reaction of peptides with spin traps or spin scavengers followed by treatment with plasma generated using an AC electrical signal. Therefore, when treating condensed phase samples using a DC signal, treatment with compounds selected from spin traps and spin scavengers can be particularly advantageous.

[0065] "Spin trapping agents" and "spin scavengers" are known in the prior art. The specific type of spin trapping agent or spin scavenger that can be used in this invention is not particularly limited, as long as it can react with a free radical generated from a given sample to form a detectable, stable adduct. If the compound is a spin trapping agent, it can be, for example, a nitrone or a nitroso compound. Hereinafter, "nitrone" refers to the N-oxide of an imine. Any cyclic or acyclic configuration of a nitrone spin trapping agent can be used in this invention. Suitable cyclic nitrones include structures having 5 or 6 ring atoms, wherein the nitrogen atom of the N-oxide forms part of the ring. In some embodiments, the nitrone is a pyrroline N-oxide, wherein the pyrroline ring may optionally be substituted with one or more alkyl groups, more preferably 5,5-dialkyl-1-pyrroline-N-oxide. For example, each alkyl group may be independently selected from C1-C4 alkyl groups. More preferably, the nitrone is 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In other embodiments, the N-oxide group does not form a ring structure. For example, the nitrone may be α-aryl-NC1-4 Alkyl nitrones, preferably α-phenyl-N-butyl nitrone, more preferably α-phenyl-N-tert-butyl nitrone (PBN). Other suitable nitrone spin traps include α-pyridine-N'-oxide-N-tert-butyl nitrone (POBN), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO). Similarly, any suitable nitroso compound spin trap can be used in this invention. In this document, "nitroso compound" refers to a nitrous acid (-N=O) group attached to an organic fragment. Suitable nitroso compounds include halo-nitrosobenzenesulfonic acids, wherein the halogen is chlorine, bromine, or iodine, preferably bromine. For example, the nitroso compound may be 3,5-dihalo-4-nitrobenzenesulfonic acid, more preferably 3,5-dibromo-4-nitrobenzenesulfonic acid (DBNBS). Alternatively, the nitroso compound may be 2-C 1-4 Alkyl-2-nitrosopropane, more preferably 2-methyl-2-nitrosopropane (MNP).

[0066] If the compound is a spin scavenger, then it can be an aminooxy radical. Any cyclic or acyclic configuration of aminooxy spin scavenger can be used in this invention. Suitable cyclic aminooxy radicals include structures having 5 or 6 ring atoms, wherein the nitrogen atom in the aminooxy group forms part of the ring. In some embodiments, the cyclic aminooxy radical is a piperidinyloxy radical, wherein the piperidinyl ring may optionally be substituted with one or more alkyl groups and may optionally be substituted with one or more phosphonoxy groups. For example, the cyclic aminooxy radical can be 2,2,6,6-tetraalkyl-1-piperidinyloxy or 4-phosphonoxy-2,2,6,6-tetraalkyl-1-piperidinyloxy radical, wherein each alkyl group is independently selected, for example, from C1-C4 alkyl groups. In these embodiments, the cyclic aminooxy radical is preferably (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) or 4-phosphono-2,2,6,6-tetramethyl-1-piperidinoxy-4-phosphono-TEMPO (4-phosphono-TEMPO). In other embodiments, the cyclic aminooxy radical is a pyrrolidinoxy radical, wherein the pyrrolidinium ring is optionally substituted with one or more alkyl groups and optionally substituted with one or more cyano groups. For example, the cyclic aminooxy radical may be a 3-cyano-2,2,6,6-tetraalkyl-1-pyrrolidinoxy radical, wherein each alkyl group is independently selected from, for example, C164-C2 ... 1- C4 alkyl. In these embodiments, the cycloaminooxy radical is preferably 3-cyano-2,2,5,5-tetramethyl-1-pyrrolidinoxy (3-cyano-proxyl, or 3-CP).

[0067] The method of processing the condensed phase sample and the compound is not particularly limited, as long as the compound remains in contact with the sample while it is exposed to plasma. For example, the compound can be provided as a solution in water and / or a non-aqueous solvent, wherein the solution is applied to the condensed phase sample (e.g., by dropping the solution onto the sample). For example, in the experiment described below, a spin trapping agent or spin scavenger is dissolved in a 30:70 mixture of water and methanol, then dropped onto a solid peptide sample on a microscope slide substrate 12 and allowed to dry. Alternatively, the compound can be applied to the condensed phase sample in solid form. In these embodiments, the compound and the condensed phase sample are preferably combined to form a homogeneous mixture. In other embodiments, a solution of the compound is mixed with a solution of the condensed phase sample to form a mixture, which is then dried. Other methods suitable for processing the sample with this compound will be apparent to those skilled in the art. In the processing steps, the weight ratio of the compound to the organic molecule can be about 1:1, for example, in the range of 0.1:1 to 10:1.

[0068] After performing any necessary preparation steps discussed above, the condensed phase sample 10 is treated in the same manner as previously described to generate fragments of organic molecules within the condensed phase sample. This treatment may be performed on the sample for an experimentally determined period of time, the specific duration of which is intended to generate molecular fragments that have a specific purpose or importance for characterizing the molecules or sample, and to achieve an appropriate concentration level. Other parameters that can be optimized empirically or experimentally to generate molecular fragments of suitable concentration and specific purpose may include voltage, power, current, and / or electrical signals. S The AC frequency, as discussed above, as well as the composition and flow rate of gas 40, the structuring or surface treatment of the sample on the substrate, etc.

[0069] Once the condensed phase sample is processed by plasma 20, a portion of the sample is transferred to a mass spectrometer for analysis, for example... Figure 1 As shown. For example, if the sample is a crystalline solid formed from the aforementioned evaporated solution, then after plasma treatment, a small amount of solvent can be added dropwise to the sample again to dissolve it, and the resulting solution can then be transferred to the ionizer 32 or other sample introduction element of the mass spectrometer. In other instances, the substances in the sample can be in liquid form and can be transferred directly to the mass spectrometer, or solid substances from the sample can be transferred to the mass spectrometer while still solid. In some instances, some or all of the substrate carrying the treated sample can be transferred as a whole to the ionizer 32 and placed therein, or used to inject sample material into the mass spectrometer.

[0070] Advantageously, the mass spectrometer can be an imaging mass spectrometer to fully utilize the plasma processing techniques described above. During processing, it should be ensured that the sample remains structurally intact, especially when the sample is in solid form.

[0071] The method described in this invention is summarized in Figure 2 The figure illustrates a flowchart of the steps for analyzing organic molecules in a condensed-phase sample. In step 110, the condensed-phase sample is prepared. As mentioned above, this preparation process can be simplified to a minimum; for example, the sample can be applied to a suitable substrate, or a sample already present in situ can be used, such as an in-situ sample from a human or animal body. Sample preparation may involve the addition of other reagents or substances, such as spin trapping or spin scavenging materials discussed above.

[0072] In step 120, the plasma treatment is performed, typically for a predetermined duration, using plasma generation parameters such as electrical signal power and frequency, and plasma generation parameters (such as electrical signal power and frequency) determined to be suitable for producing the desired level and characteristics of organic molecule cleavage within the sample, as well as other parameters (such as gas type and flow rate).

[0073] In step 130, a portion or all of the plasma-treated sample is transferred to a mass spectrometer to detect organic molecular fragments generated by the plasma treatment. This step may include measuring specific known and expected fragments, or more broadly acquiring mass spectra or other data for further analysis.

[0074] Finally, in step 140, the original sample is subjected to some characteristic analysis using mass spectrometry data, such as by determining the presence, absence, or concentration of specific molecules in the original sample, through fingerprinting, statistical or AI techniques, or by other means, at least in part, by using data related to fragments generated by plasma processing in the mass spectrometer. In some cases, mass spectrometry data can be used to help identify or characterize unknown or uncharacterized molecules in the sample.

[0075] Several different experiments conducted by the inventors to demonstrate the efficacy and embodiments of the present invention are now described. At least some of these embodiments involve the cleavage of organic polymer molecules along the polymer backbone, and the subsequent detection of the cleaved components of the backbone.

[0076] Figure 3 This demonstrates the use of radio frequency AC signals. S The results of plasma digestion and subsequent mass spectrometry analysis of the leucine enkephalin sample using plasma generated in an argon gas flow are described above. The AC signal was generated by the aforementioned ForceTriad (RTM) signal generator, with an output power of 35 watts, an AC frequency of 4723 kHz, and a duty cycle as described previously. The experimental setup is also detailed above, and as... Figure 1 As shown, electrode 16 is a stainless steel corona discharge needle that extends approximately 2 mm from the open end of a 10 mm inner diameter plastic tube serving as conduit 42; an argon gas flow is provided at a pressure 10 kPa higher than ambient pressure, flowing along the tube over the needle tip and toward the sample. The experiment was conducted under ambient conditions without any specific measures to isolate it from the laboratory ambient air.

[0077] The experiment was repeated six times, each time using a fresh, untreated sample and different plasma treatment times: 0 seconds, 5 seconds, 30 seconds, 60 seconds, 240 seconds, and 480 seconds. After each treatment, a solvent was dropped onto the sample to dissolve a portion of the treated leucine enkephalin, and then a fraction of the solute was injected into a Bruker TIMS-ToF mass spectrometer with a mass-to-charge ratio accuracy of approximately + / - 0.005 Da.

[0078] Figure 3 The table shows the intensities of digestion products generated by mass spectrometry decomposition of C1-C4 fragments of leucine enkephalin after a specified processing time, along with the corresponding intensities of sodium adduct ion digestion products. This is standard practice in mass spectrometry; C1 fragments are generated by the breakage of the first NC bond in the peptide chain from the N-terminus (excluding the NC bond at the primary amine's N-terminus itself), while C2-C4 fragments are generated by the breakage of the three CN bonds following the peptide chain. Figure 3 In the analysis, the intensities of these digestion products were normalized by the M+H ion intensities measured simultaneously by a mass spectrometer (i.e., the parent ion corresponding to the entire undigested molecule). It can be seen that significant digestion of all the fragment species shown occurred after only 5 seconds of processing. However, in this case, the normalized product intensities peaked after 240 seconds of processing and then decreased again, which could be due to further fragmentation of the measured products into smaller possible fragment products.

[0079] Figure 4 The results of experiments on leucine enkephalin samples are shown under the same experimental setup and conditions, but this time the processing time for all samples was 60 seconds, and each sample was processed using plasma generated by an operating ForceTriad (RTM) signal generator at different power levels. Similarly, the digestion product intensities measured by Bruker mass spectrometry were normalized relative to the measured M+H ion intensities. It is evident that the digestion product intensity increases with increasing signal power, with significant peaks observed at 70 watts and 100 watts, which could be due to specific characteristics of the signal generator operation.

[0080] Figure 5A and 5B The corresponding experiments for leucine enkephalin samples are shown respectively, where DC signals were used respectively. Figure 5A ) and AC signal ( Figure 5B This drives electrode 16 to generate plasma. Figure 5A Through experimental tuning of a constant DC signal, visually optimal plasma 20 was achieved in terms of intensity and range, at a voltage of 5 kV and a current of approximately 0.1 mA. Figure 5AThe aforementioned ForceTriad (RTM) signal generator was used in conjunction with a 120-watt output AC signal to achieve the best visual effect in terms of intensity and range of plasma 20, which was significantly stronger than that achievable using a DC signal. Samples were treated with plasma for 5 seconds, 60 seconds, and 240 seconds, respectively.

[0081] In both graphs, the intensities of the digestion products are plotted on the same (left) scale, but without normalization for the precursor ion M+H; the precursor ion is plotted on a different (right) scale. Clearly, using the AC signal yields higher digestion product intensities and lower precursor ion intensities, resulting in a significantly better signal-to-noise ratio for the normalized data. It should also be noted that although the intensities of different fragment types are roughly on the same order of magnitude or intensity level in both DC and AC modes, some significant differences exist. Therefore, either signal type can be selected depending on the specific fragment to be measured. In particular, the fragment-to-precursor ion intensity ratio is much larger in the AC signal case, indicating that the AC method is significantly more efficient.

[0082] Figure 6 and Figure 3 Similarly, the difference lies in the plasma digestion and subsequent mass spectrometry analysis of the slightly heavier peptide (methionine endorphin) samples, with processing times of 5 seconds, 60 seconds, 240 seconds, and 480 seconds, respectively. Figure 3 and Figure 4 Similarly, all data were normalized based on the intensity of the M+H parent ion. In this case, only sodium adducts of c2, c3 and c4 ions were shown. The intensities of c2 and c3 ions increased significantly over time, but the intensity of c4 ions did not change much after the first 5 seconds.

[0083] Figure 7 This diagram illustrates the application of the described plasma digestion and mass spectrometry techniques in a creatinine sample prepared using the same method as the peptide sample described above. Creatinine is not a peptide, and its molecular weight is much smaller than the peptides used above; however, plasma digestion still produced useful fragments over all processing times of 5, 60, 240, and 480 seconds. The digestion product intensities (left axis) in the figure are not normalized, therefore the intensity of the parent ion M+H is also shown (right axis). Based on the mass-to-charge ratio, the fragments plotted in the figure are preliminarily identified as sodium adduct ions of the following substance: 109.11 = C4H 12 N2, 97.07 = C3H 10 N2, 81.04 = C2H6N2 and 69.04 = CH6N2.

[0084] Figures 8 to 10The illustrations show the application of the described plasma digestion and mass spectrometry techniques in leucine enkephalin samples treated with different spin trapping and spin scavenging agents. In each figure, the vertical axis represents the intensity of the digestion products for each fragment type output by the mass spectrometer (normalized for the intensity of the parent ion M+H in each case), and the identified fragments are labeled with their mass / charge ratio.

[0085] In particular, for Figure 8 The experiment used DMPO (a spin trapping agent), while Figure 9 and Figure 10 In the experiments, 4-phosphono-TEMPO and 3-cyano-proxyl (both spin scavengers) were used. In each experiment, 2 µL of a spin trapping / spin scavenging solution with a concentration of 1 mg / mL, dissolved in a 30:70 volume ratio mixture of water and methanol, was pipetted onto a solid leucine enkephalin sample on a glass slide. After drying, the treated samples were then subjected to... Figure 5A Under the same plasma generation and processing conditions, plasma was generated using a DC signal, and the generated debris was analyzed by mass spectrometry using a Bruker TIMS-ToF mass spectrometer (mass-to-charge ratio accuracy of approximately ±0.005).

[0086] Figure 11 Results from the same experiment are shown, but without applying any spin trapping / spin scavenging agent to the leucine enkephalin sample. The data were also normalized to the intensity of the parent ion M+H. From Figures 8 to 11 It can be seen that after 60 seconds of plasma exposure, both the spin trapping agent and the spin scavenger enabled the detection of a large number of different fragments, while in the absence of a spin trapping agent / spin scavenger, no significant level of fragments was detected during this exposure time. Apart from the addition peaks of the leucine enkephalin-spin trapping agent / spin scavenger adducts observed in each case (leucine enkephalin + DMPO: 667.3456, leucine enkephalin + 4-phosphono-TEMPO: 809.384, leucine enkephalin + 3-cyano-proxyl: 697.655), the remaining observed fragments have not yet been analyzed.

[0087] To further illustrate the invention, a direct current-argon plasma (DC-AP) power supply consisting of a 5 kV HVDC power supply (Stanford Research Systems PS350) is connected to 1 The resistor and brass T-connector (Swagelok) containing the corona needle (encased in an insulating plastic sheath) and gas tubing were used. Well-characterized peptide standards (such as leucine enkephalin and bradykinin, Merck, 1 mg / mL) were used as standards to study the effects of plasma; a constant volume (2.5 µL) of the standard was spotted onto an indium-tin oxide (Merck, 70-100) glass slide. After drying, the needle tip is exposed to positive or negative corona-induced plasma, with a distance of 12 mm between the tip and the ITO slide, for an exposure time of 5 seconds to 4 minutes. Activating the DC-AP source under a 0.5 bar argon flow will produce a purple glow discharge at the tip of the corona needle.

[0088] Subsequently, the DC plasma-treated substrates were directly analyzed using DESI imaging, or the DC plasma-treated substrates were dissolved in 60 µL of a 30:70 water:methanol mixture and analyzed by direct injection ESI-MS or LC-MS. Standards exposed to DC argon plasma produced consistent, unique, and spatially resolved mass peaks in subsequent DESI-MS analyses. ITO slides were divided into four regions, with standards coated onto each region—these regions were exposed to plasma for 0, 5, 60, or 240 seconds prior to DESI analysis, respectively. In this specific example, although the resulting peaks were not easily resolved by MS / MS, they clearly demonstrated the practicality of plasma-mediated peptide reactions—and also showed that increasing plasma exposure time increased peak intensity. See Results. Figure 12 The peak at a mass-to-charge ratio of 293.21 appears only when leucine enkephalin is exposed to DC-AP, while the peak at a mass-to-charge ratio of 516.28 appears only when bradykinin is exposed to DC-AP. The intensity of both peaks increases with increasing plasma exposure time.

[0089] To understand the fragmentation mechanism and resolve the structures of identified peaks, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was added to the standard before exposure to plasma. DMPO is a commonly used spin-trapping compound, typically employed in electron paramagnetic resonance (EPR) spectroscopy, capable of selectively reacting with molecules possessing unpaired electrons—most typically those radical compounds generated under DC argon plasma exposure. Adding an equiweight / weight-volume ratio of DMPO to the standard before exposure generates spin-trapping radicals, which are readily detectable by LC-MS.

[0090] Data analysis revealed numerous chromatographic peaks absent in the control samples but present in the exposed samples. The main peak (667.35 m / z) generated by applying a plasma source to the leucine endorphin-DMPO standard was presumed to be an addition product of these two compounds, linked to the phenolic hydroxyl group of the peptide via a Forrester-Hepburn mechanism. Another major peak (263.14 m / z) appearing at 8.50–8.60 min in the chromatogram was also resolved; this peak was labeled as a coupling product of the leucine-endorphin α1 ion and DMPO, followed by oxidation.

[0091] The described method using a DC-AP source on a solid-phase sample can produce spatially resolved, consistent fragments that are present only in the exposed peptide. If a spin-trapping compound such as DMPO is added before exposure, DMPO binds to the peptide to produce predictable fragments; this reaction also produces several byproducts, which may originate from the breakdown of the DMPO-peptide conjugate or from reactions between DMPO and peptide breakdown products.

[0092] Using radio frequency (RF) current to generate plasma allows for sample digestion without the need for expensive conductive ITO slides. In a further demonstration of the invention, a modified electrosurgical handheld device (Erbe) was used to generate cryogenic RF argon plasma. This handheld device was modified by replacing the blade with a corona needle. A short section of polypropylene tubing with an inner diameter of approximately 10 mm was used to guide the argon flow from the needle tip, which itself protruded slightly. The handheld device was connected to a unipolar socket of an RF AC current generator. A glass slide containing dried standards was placed on a distributed grounding pad connected to the generator. Activating the device under 0.1 bar argon flow conditions produced a purple plasma that covered an area of ​​approximately 30 mm in diameter directly below the corona needle tip. After exposure, the standards were dissolved in 60 μL of 50% acetonitrile solution and analyzed by direct injection.

[0093] The resulting digestion products include “classical” abc-xyz peptide fragments (particularly C- and Z-type fragments), as well as neutral loss products such as [M-COOH+H]+. The presence of C- and Z-type fragments suggests that this mechanism relies more on free electron or radical-mediated reactions than on DC devices. For example, specific cleavage methods such as electron transfer dissociation (ETD) and electron capture dissociation (ECD) typically generate these fragments via charge-long-range cleavage characterized by odd-numbered electron reactions. Increasing the exposure time of RF-AP tends to decrease the intensity of high-mass-number (>500) m / z peaks while increasing the intensity of low-mass-number (<150) m / z peaks (see Table 1) – for example, prolonged exposure time leads to a decrease in the intensity of the peak at 464.19 (labeled [c4+Na]+), but an increase in the intensity of the peak at 120.08 (labeled [Phe-COOH-H2+H]+).

[0094] Table 1 below lists several significant peaks and their corresponding annotations. The peaks produced include C-type fragments, as well as derivatives of A-type, C-type, Z-type, and Y-type fragments, and products formed after the neutral loss of the parent ion.

[0095] Table 1: Significant fragments and annotations obtained from direct injection analysis of leucine enkephalin exposed to RF-AP source. And its absolute strength.

[0096] Subsequent experiments on a series of standard substances (including bradykinin (Table 2), mNeonGreen (Table 3), ubiquitin, L-phenylalanine, DL-β-phenylalanine, and creatine) also showed that these compounds produced characteristic peaks after exposure to the RF-AP source.

[0097] Table 2: Significant fragments, annotations, and other characteristics obtained from direct injection analysis of bradykinin exposed to RF-AP sources. Absolute strength.

[0098] Table 3: Significant fragments and their absolute intensities obtained from direct injection analysis of mNeonGreen exposed to RF-AP sources. Spend The RF-AP instrument was also applied to Thermo Fisher Scientific Expi293F cells expressing the mNeonGreen monomeric fluorescent protein. In this case, both transfected and control cells were washed three times with ammonium acetate solution and then lysed by repeated freeze-thaw cycles in a -80°C freezer. The lysate was spread onto a microscope slide, exposed to an RF argon plasma for 1 minute, then dissolved and filtered before direct injection-ESI analysis. This analytical procedure revealed specific peaks in the lysate of transfected cells exposed to the argon plasma, which were absent in the lysate of unexposed mNeonGreen cells and the lysate of exposed control cells. These results were also replicated by MALDI imaging analysis of the exposed lysate, showing the generation of localized peaks.

[0099] Finally, the argon plasma device was applied to tissue samples. Four mouse kidney samples were first washed twice in 25 mL of a 70:30 ethanol:water solution for 30 seconds each time, then once in 25 mL of 100% ethanol for 15 seconds each time, once in 25 mL of a 90:9:1 ethanol:glacial acetic acid:water solution for 10 seconds each time, and finally twice in 25 mL of water for 2 seconds each time. 1 μL of 1 μg / mL mNeonGreen standard solution was added to two of the tissue samples, while the control tissue and the tissue sample containing mNeonGreen fluorescent protein were subjected to RF argon plasma treatment for 1 minute each. Subsequently, 7 mg / mL CHCA was used as a matrix and spread on a glass slide, and analyzed by MALDI. In this way, it is possible to distinguish between tissues exposed to plasma and those not exposed. See [link to relevant documentation] Figure 13 The figure shows the MALDI-MSI thermograms of the 502.319 m / z peak in control cells and cell lysates modified to express mNeonGreen fluorescent protein, where the lysates were either unexposed to plasma or exposed to a cold argon plasma apparatus for 1 minute. This ion was present at a level above noise only in cell lysates containing mNeonGreen.

[0100] We were also able to distinguish between the mNeonGreen standard added to tissue samples and two types of control tissues exposed but without the standard, as well as unexposed tissue samples with the standard added. Four strong and correlated peaks were observed at m / z of 158.11, 189.16, 203.17, and 204.17, which distinguished the plasma-exposed standard from the control tissues. The 204 m / z peak is likely the 13C isomer of the 203 m / z peak and is in a single-charge form. See also Figure 14The figure shows the MALDI-MSI thermogram formed by the peak at 203.172 m / z in tissue (optionally supplemented with the fluorescent protein mNeonGreen or exposed to a cryogenic argon plasma device for 1 minute). This ion is present above noise levels only in plasma-exposed tissue supplemented with the fluorescent protein mNeonGreen.

[0101] In summary, this invention provides a method for digesting proteins using spark plasma or cold plasma (e.g., low-temperature argon plasma). A significant advantage of this method compared to existing techniques is the substantial reduction in sample preparation time and workload, eliminating the need for reagents, including enzymes. Using spark plasma and / or cold plasma in this manner also preserves the spatial integrity of macromolecules, avoiding the spatial imbalance that can occur with liquid-phase digestion, and enables high-throughput imaging of proteins in condensed-phase samples.

[0102] Although specific embodiments have been described, many alternatives and variations will be apparent to those skilled in the art without departing from the scope of the invention as defined in the claims. For example, while mass spectrometry may be particularly used for detecting debris in order to characterize or identify debris, molecules, and / or samples, as described above, other analytical techniques and equipment, such as nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy, and various optical spectroscopic techniques, may also be used alone or in combination for this purpose.

Claims

1. A method of analysing an organic molecule in a condensed phase sample, the method comprising: generating a plasma with an electrical spark; exposing a condensed phase sample to the plasma to generate fragments of the organic molecule; and detecting the fragments with a mass spectrometer.

2. The method of claim 1, wherein, The condensed phase sample is on a solid substrate when exposed to the plasma.

3. The method of claim 1 or 2, wherein, The condensed phase sample is a solid sample during exposure to the plasma to generate fragments of the organic molecule.

4. The method according to any of the preceding claims, wherein, The fragments of the organic molecule are generated within the condensed phase sample and are released from the sample for detection by the mass spectrometer only after the condensed phase sample has been exposed to the plasma.

5. The method according to any of the preceding claims, wherein, At least a portion of the condensed phase sample is removed from the substrate after the condensed phase sample has been exposed to the plasma and introduced into the mass spectrometer for subsequent ionisation and detection of the generated fragments.

6. The method of claim 1, wherein, The electrical spark, plasma and exposed sample are at one or more of the following pressures: ambient atmospheric pressure and 30 kPa to 300 kPa.

7. The method according to any of the preceding claims, wherein, The electrical spark is generated by applying an electrical signal to one or more electrodes.

8. The method of claim 7, wherein, The one or more electrodes comprise a plasma source electrode configured to generate a plasma between the plasma source electrode and the sample and a ground plane electrode disposed on an opposite side of the sample relative to the plasma source electrode; and optionally wherein the ground plane electrode is further separated from the sample by at least a portion of the substrate.

9. The method of claim 7 or 8, wherein, The electrical signal is a DC signal.

10. The method of any one of claims 1 to 8, wherein, The electrical signal is an AC signal.

11. The method of claim 10, wherein, The AC signal has a power of 10 Watts to 300 Watts and / or a peak voltage of 1000 Volts to 10000 Volts.

12. The method of claim 10 or 11, wherein, The AC signal has one or more of the following characteristics: is a radio frequency signal; has a frequency of 100 kHz to 5 MHz.

13. The method of any one of the preceding claims, further comprising flowing a gas stream past the electrical spark and to the sample.

14. The method of claim 13, wherein, The gas comprises at least 50% or at least 90% of one or more of the following gases: a noble gas, argon, helium and nitrogen.

15. The method according to claim 13 or 14, when dependent on any one of claims 7 to 12, wherein, At least one of the one or more electrodes is provided by a conductor extending along a conduit to an open end of the conduit such that the electrical spark is generated proximate the end of the conduit and the gas stream flows along the conduit to the open end.

16. The method according to any of the preceding claims, wherein, The condensed phase sample is exposed to the plasma for one or more of the following: at least 1 second, at least 10 seconds and at least 60 seconds.

17. The method according to any of the preceding claims, wherein, Prior to the step of exposing the condensed phase sample to the plasma to generate fragments of the organic molecule, the method further comprises treating the condensed phase sample with a compound selected from a spin trap and a spin scavenger.

18. The method of claim 17, wherein, The compound is a spin trap, preferably wherein the spin trap is a nitrone or a nitroso compound, more preferably 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) or N-tert- butyl nitrone (PBN).

19. The method of claim 17, wherein the compound is a spin scavenger, preferably the spin scavenger is an aminooxy group, more preferably (2,2,6,6-tetramethylpiperidin-l-yl)oxy (TEMPO), 4-phosphonooxy-2,2,6,6-tetramethyl-l-piperidinyloxy-4-phosphonooxy-TEMPO (4-phosphonooxy-TEMPO), or 3-cyano-2,2,5,5-tetramethyl-l-pyrrolidinyloxy (3-CP).

20. The method of any one of claims 17-19, wherein, In the step of treating the condensed phase sample with a compound selected from a spin trap and a spin scavenger, the weight ratio of the compound to the organic molecule is from 0.1:1 to 10:

1.

21. The method according to any of the preceding claims, wherein, The organic molecule comprises one or more of: a protein, a peptide, a lipid, and an organic polymeric molecule.

22. The method of claim 21, wherein, The organic molecule is an organic polymeric molecule, and the fragments of the organic molecule generated are derived from cleavage along the polymer backbone.

23. A method of performing imaging mass spectrometry, the method comprising the steps of any one of the preceding claims, wherein detecting fragments with a mass spectrometer comprises detecting fragments at multiple locations of the sample using an imaging mass spectrometer.

24. The method of any one of the preceding claims, further comprising characterizing or identifying the sample, the organic molecule, or the fragments with data from the mass spectrometer, the data from the mass spectrometer representing or comprising information of the detected fragments.

25. An apparatus for analyzing an organic molecule in a condensed phase sample, comprising: a plasma generator configured to generate a plasma with an electrical spark; a substrate support for receiving a substrate with the condensed phase sample, exposing the condensed phase sample to the generated plasma, thereby causing the plasma to generate fragments of the organic molecule within the condensed phase sample; and a mass spectrometer configured to subsequently receive and detect the generated fragments.

26. The apparatus of claim 25, wherein the substrate is a solid substrate.

27. The apparatus of claim 25 or 26, configured such that the mass spectrometer receives the generated fragments only after the end of the exposure of the condensed phase sample to the generated plasma.

28. The apparatus of any one of claims 25 to 27, further comprising a signal generator configured to deliver an AC or DC electrical signal to the plasma generator to generate the plasma. The electrical signal is an AC signal, and the AC signal has one or more of the following characteristics: a power of 10 to 300 Watts; a peak-to-peak voltage of 1000 to 10000 Volts; a frequency of 100 kHz to 5 MHz or is a radio frequency signal.

29. The apparatus of claim 28, wherein, 30. The apparatus of any one of claims 25 to 29, further configured to provide a gas flow, the gas flow flowing through the electrical spark and optionally to the sample. The plasma generator comprises one or more electrodes to which an AC or DC signal is applied.

31. The apparatus of any one of claims 25 to 30, wherein, ​ 32. The apparatus of claim 31, wherein, At least one of the one or more electrodes is provided by a conductor extending along the conduit to the open end of the conduit such that the electrical spark is generated proximate the end of the conduit and the flow of gas is along the conduit.

33. The apparatus of claim 31 or 32, wherein, The one or more electrodes include a plasma source electrode configured to generate the plasma between the plasma source electrode and the sample and a ground plane electrode disposed on an opposite side of the sample relative to the plasma source electrode.

34. The apparatus of claim 33, wherein, The ground plane electrode is spaced apart from the sample by at least a portion of the substrate.

35. The apparatus of any one of claims 25 to 34, further comprising a sample transfer mechanism configured to deliver some or all of the condensed phase sample to a mass spectrometer after the sample is subjected to plasma treatment.

36. The apparatus of any one of claims 25 to 35, wherein the mass spectrometer is an imaging mass spectrometer.

37. The apparatus of any one of claims 25 to 36, further comprising the condensed phase sample on the substrate.

38. The apparatus of any one of claims 25 to 37, wherein the organic molecules comprise one or more of: proteins, peptides, lipids, and organic polymeric molecules.

39. The apparatus of claim 38, wherein the organic molecules are organic polymeric molecules and the detected fragments of the organic molecules originate from cleavage along a polymer backbone.

40. The apparatus of any one of claims 25 to 39, configured to characterize or identify the sample, the organic molecules, or the fragments using data from the mass spectrometer representative of the detected fragments.

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