Sample preparation for mass spectrometry

Abstract

This invention provides improved means and methods for preparing samples for analysis in a mass spectrometer. [Solution] The present invention provides a method for preparing a sample for an analytical procedure, wherein the sample comprises at least one protein, polypeptide, or peptide molecule, and the method comprises fragmenting the molecule using at least one mobile magnetic material.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing a sample for an analytical procedure, wherein the sample comprises at least one protein, polypeptide, or peptide molecule, and the method comprises fragmenting the molecule using at least one mobile magnetic material.

[0002] This specification references numerous documents, including patent applications and manufacturers' manuals. While the disclosures of these documents are not considered relevant to the patentability of the present invention, they are incorporated herein by reference in their entirety. More specifically, all referenced documents are incorporated by reference to the same extent as individual documents are indicated to be incorporated by reference specifically and individually. [Background technology]

[0003] In recent years, biological analytical procedures have developed at a rapid pace. In particular, mass spectrometry (MS)-based proteomics has emerged as a powerful analytical tool for a wide range of pharmaceutical and clinical applications. While traditional approaches, including immunoassays and enzyme-based assays, are limited to the analysis of single proteins at predetermined time points, untargeted mass spectrometry opens up entirely new perspectives by detecting hundreds or even thousands of proteins within a single measurement in a completely unbiased manner. Novel MS strategies are increasingly being applied, among other things, to the analysis of biomarkers in bodily fluids, such as urine, plasma, or serum, to assess and monitor the health status of an individual. A second broad area of ​​application is tracking host cell protein (HCP) impurities in therapeutic proteins throughout the production and purification process to ensure the safety and quality of formulations.

[0004] In addition to complex samples, MS-based proteomics is also used for single-protein analysis, particularly for therapeutic proteins, where extensive and comprehensive characterization of the active pharmaceutical ingredient is essential for patient safety. Because this approach focuses on the detailed sequence and composition of single proteins, including post-translational modifications (PTMs), sample preparation must be optimized with respect to the requirements for these identifications, for example, by enhancing preparation efficiency for maximum sequence coverage or by reducing the introduction of artificial modifications.

[0005] Enzymatic protein digestion presents a crucial step in most sample preparation workflows for MS-based proteomics, involving the hydrolysis of proteins on the protein backbone using specific endoproteinases (often referred to as proteases) to produce polypeptides or peptides. Most proteases used in proteomics are sequence-specific endoproteinases, which helps prevent increasing sample complexity beyond the level of MS capability. Regarding bottom-up proteomics strategies, mammalian (porcine or bovine) trypsin is the most broadly distributed protease. Trypsin specifically cleaves at the carboxyl side (C-terminus) of arginine (R) and lysine (K) residues, thus producing positively charged peptides with an average amino acid length of approximately 9. Other proteases, including LysC, GluC, AspN, ArgC, and chymotrypsin, are also used, to a lesser degree, often in MS sample preparation to complement trypsin sample information. Manufacturers aim to improve protease purity while preserving enzyme activity by using recombinant protein expression systems instead of natural proteins of mammalian or bacterial origin, but often face the challenge of expressing active proteolytic enzymes.

[0006] Complete coverage MS-based proteomics heavily relies on efficient protein hydrolysis and the resulting sequence coverage during data analysis. Therefore, almost all The sample preparation protocol relies on high protease content and very long digestion times of up to 24 hours. Therefore, protein digestion is a critical step that significantly impacts the variability of proteomics sample preparation observed across laboratories and businesses.

[0007] Several additional parameters are known to affect the quality and reproducibility of enzymatic proteolysis. In particular, the composition of the buffer solution is a crucial aspect, as proteases are highly sensitive to pH and salt conditions, as well as to the presence of washing agents and chaotropic reagents. Furthermore, while digestion efficiency can be significantly increased by higher enzyme concentrations, this simultaneously introduces further protein contamination from the enzyme source (e.g., porcine protein), thus highlighting the challenges and limitations of established digestion strategies in this field. Additionally, proteases themselves can produce undesirable signals in the mass spectrum, for example, overlapping with signals from the sample.

[0008] In contrast to classical bottom-up proteomics, the top-down proteomics approach is used to analyze intact proteins in an MS instrument. This application is very attractive because it allows for the study of complete proteins and the determination of modification patterns. However, the properties of the protein to be analyzed strongly influence the quality of the analysis, and examining complete protein samples remains difficult. Intact proteins do not behave uniformly under MS-compatible liquid chromatography (LC) conditions and are difficult to separate, which results in very complex samples and a very wide dynamic range in any case of data acquisition. Furthermore, intact proteins are only inefficiently ionized by electrospray ionization (ESI), which is the optimal method as a soft ionization technique when MS is coupled to an LC setup. Finally, intact proteins result in very complex isotopic patterns on the MS analysis side, making it very difficult to interpret the mass spectrum. As a result, top-down proteomics of complete protein samples is not applicable in a wide range of settings and is limited to specialized laboratories.

[0009] The middle-down approach represents a very attractive compromise between top-down and bottom-up proteomics, and, combined with a well-established analytical system, offers the potential for high sequencing coverage. The middle-down approach encompasses protein digestion in a more restricted manner than the bottom-up approach, targeting larger peptide fragments. Here, structure-specific enzymes, such as papain or IdS proteases for antibody analysis, are often selected to selectively cleave one or more sites on the protein. However, a major challenge is the identification of proteases generally suitable for the wide range of proteins to be analyzed. This challenge has been addressed to some extent by chemical means of protein hydrolysis.

[0010] Classical chemical methods for hydrolyzing proteins involve strong acids at high temperatures. This results in nearly random cleavage of the protein backbone. Typically, proteins are boiled in a strong acid, such as high-concentration hydrochloric acid (10M HCl), for approximately 1 to 24 hours, depending on the level of degradation to be achieved. While this process can be partially controlled by the acid concentration, temperature, and time, the hydrolysis occurs randomly, resulting in an extremely complex sample of hydrolyzed protein. This process can be carried out in a faster but less streamlined form using microwave-assisted protein hydrolysis, where microwaves are used to increase the rate of hydrolysis by exciting water. The main challenge here remains the complexity of cleavage possibilities resulting from random hydrolysis. This usually exceeds the capabilities of even the most state-of-the-art MS instruments.

[0011] Most LC-MS instruments used in bottom-up proteomics are tripsymp This method is optimized for identifying peptides, i.e., peptides obtained by the trypsin digestion of proteins. Peptide sequences of approximately 6–40 amino acids (AA) in length that produce a double-charged peptide ion (one positive charge at the K / R residue and one positive charge at the N-terminus) can be separated very efficiently using reversed-phase (C18) chromatography coupled to a high-resolution mass spectrometer via ESI. Proteases that yield shorter peptides (less than 6 AAs) typically have the disadvantage of no longer being protein-specific and instead being present in many possible proteins. Proteases that yield longer peptides (more than 100 AAs) may cause irreversible binding of the peptide to the reversed-phase chromatography column, and therefore inefficient ESI.

[0012] The goal of middle-down proteomics is to generate uniform and reproducible peptide lengths of approximately 30–100 AA across all proteins present in the sample. This then significantly reduces sample complexity, increases sequencing coverage, and produces peptide samples well-suited for reverse-phase chromatography, stable ESI, and MS analysis.

[0013] Considering the shortcomings of known approaches to sample preparation for MS, the underlying technical problem of the present invention can be understood as providing improved means and methods for preparing samples for analysis in a mass spectrometer, such methods including the fragmentation of chain molecules and polymers, including those of biological origin. [Overview of the project]

[0014] As can be seen from the examples contained herein, this technical problem is solved by the subject matter of the claims further presented below.

[0015] More specifically, in a first aspect, the present invention provides a method for preparing a sample for an analytical procedure, wherein the sample comprises at least one protein, polypeptide, or peptide molecule, and the method comprises (a) fragmenting the molecule using at least one mobile magnetic material.

[0016] The term "preparation" generally refers to the pretreatment of a sample so that its results can be analyzed. Typically, proteins and polypeptides, as well as longer peptides, are molecules whose size is not optimal for analytical purposes, such as analysis by mass spectrometry. For at least this reason, samples generally require a preparation step before being fed into the mass spectrometer. Such preparation involves reducing the size of the analyte molecule by fragmentation. Traditionally, fragmentation is carried out by adding a protease, such as trypsin. Proteases are an enzymatic means of fragmentation, and their drawbacks are outlined in the introductory section above.

[0017] Departing from the pretreatment established in such prior art fields, the preparation according to the present invention uses at least one moving magnetic body. From the perspective of implementation, this can be a single magnet in the simplest case. This option as well as alternatives thereto are described in more detail below.

[0018] Surprisingly, the inventors have found that a moving magnetic body can induce fragmentation, i.e., the breaking of one or more covalent bonds in the above molecules. This is a characteristic advantage, one of which is that the above-mentioned proteases become non-essential. In fact, in the simplest implementation of the present invention, chemical or biological agents are not used at all for the purpose of fragmenting proteins, polypeptides or longer peptides. In fact, also as outlined above herein, the prior art relies on biological agents for cleaving proteins, more specifically proteases such as trypsin. Also, the use of chemical substances such as CNBr, etc. is described for that purpose. On the other hand, the cleavage or fragmentation according to the present invention can be carried out without using an enzymatic catalyst and without using a chemical agent that induces cleavage of a protein, polypeptide or peptide.

[0019] By the terms "chemical agent" and "chemical substance", compounds such as CNBr are understood to be referred to that induce cleavage under conditions where cleavage does not occur or occurs significantly less in other situations (e.g., ambient temperature, atmospheric pressure, presence of water, buffer, salt) (further compounds capable of cleaving proteins, polypeptides or peptides are further disclosed below). In other words, such chemical substances are intentionally added for the purpose of cleaving proteins, polypeptides or peptides.

[0020] Fragmentation can directly result in stable fragments or intermediate reactive species that subsequently react with other molecules present to yield stable products. The other molecules can include water, or more generally, any molecule capable of forming them as long as stable adducts are formed with the reactive species.

[0021] The term "peptide" refers to a polycondensate of up to 30 amino acids, whereas a polypeptide contains more than 30 amino acid components. Thus, a "longer peptide" refers to a peptide consisting of 15 to 30, 20 to 30, or 25 to 30 amino acids. The term "protein" encompasses polypeptides, but extends to higher-order structures, such as non-covalent bonds or oligomers of multiple identical and / or different proteins.

[0022] Modifications may be present on any one of peptides, polypeptides, and proteins, including but not limited to post-translational modifications as they exist in biological systems. Examples include glycosylation, phosphorylation, and modifications using lipophilic moieties, such as prenylation. Upon fragmentation, the above-mentioned higher-order structures are generally lost if they can exist in proteins (oligomers), but not necessarily always. Local structures, such as secondary structures, can be retained so that they can interact with homologous binding partners of a particular domain or motif.

[0023] Considering the components, 20 proteinogenic amino acids are preferred. Additionally, amino acids that do not belong to this set of 20 amino acids, usually α-amino acids, exist naturally and can likewise be present. Examples include ornithine, citrulline, hydroxyproline, selenocysteine, methionine oxide, and deaminated asparagine.

[0024] This repertoire can be further expanded, for example, by the above-mentioned post-translational modifications. These include, for example, phosphorylated forms of serine, threonine, and tyrosine, glycosylated amino acids, methylated lysine, and methylated arginine.

[0025] Regarding linkages between amino acids, this is generally a standard back-chain peptide bond between the α-carboxylate of one amino acid and the α-amino group of the following amino acid (in the direction from N to C-terminus). Less common, but conceivable linkages include isopeptide bonds, i.e., peptide bonds containing a side-chain amino group and / or side-chain carboxylate.

[0026] Fragmentation by the method of the present invention can occur at main chain peptide bonds, but other bonds can be similarly fragmented.

[0027] In fact, the covalent bonds that are broken are not particularly limited. Single bonds and double bonds as well Triple bonds are included, and they can all exist between atoms of the same type (e.g., C=C bonds) or different atoms (e.g., CN bonds or CO bonds). Bonds found in functional groups linking components include peptide bonds, ester bonds including phosphate esters, and glycosidic bonds. Phosphate ester bonds are found, for example, in phosphorylated Ser, Thr, and Tyr residues, and glycosidic bonds are found in glycosylated amino acids. It will be understood that bonds found in such functional groups generally have lower bond energies compared to, for example, C=C bonds, and are therefore suitable for cleavage using the method of the present invention when they operate in a manner that transfers less energy to the reaction mixture.

[0028] The energies contained in covalent bonds are known, and these energies also define the energy required for cleavage, particularly in the absence of a catalyst. Most covalent bond energies fall in the range of 100–1200 kJ / mol. See, for example, Chemistry: Atoms First 2e, ISBN 978-1-947172-63-0. Some examples of covalent bond energies include the CC bond with an energy of 345 kJ / mol, the CN bond with 290 kJ / mol, and the CO bond with 350 kJ / mol.

[0029] In a preferred embodiment, the analytical procedure described above is a spectroscopic and / or spectroscopic method. A preferred spectroscopic method is mass spectrometry (MS). Preferred spectroscopic methods include NMR spectroscopy and / or UV / visible spectroscopy. One or more of these methods may be used subsequently. MS is particularly preferred.

[0030] For conventional mass spectrometry applications, the preferred size range for peptides is 5–30 amino acids. The preferred median size is approximately 12 amino acids. Therefore, it is also preferable that peptides be fragmented prior to analysis, provided their sizes exceed the aforementioned range.

[0031] However, as mentioned in the introduction above, there are cases where longer molecules, such as polypeptides with a length of 30 to 100 amino acids, can be fed into a mass spectrometer (also known as the "middle-down approach"). In these cases, the peptide does not need to be fragmented, but the polypeptide and protein do need to be fragmented to the extent that their size exceeds the size desired for these particular executions, especially when established means in the prior art of protein fragmentation cannot provide a satisfactory solution. Furthermore, obtaining fragments within this size range is highly desirable because they have greater potential to be specific (specific to a single protein) than shorter fragments. In other words, smaller fragments are sufficient for good sequence coverage. Preferably, the present invention fills this gap.

[0032] More generally, the preferred size range also depends on the choice of the analytical method (usually liquid chromatography) preceding the mass spectrometry. C18 materials are particularly suitable for peptides, as long as reversed-phase materials are used, while C8 materials can also be used for proteins. Orbit-trap instruments have the best resolution in the lower m / z range, while TOF instruments do so in the higher range. "m / z" refers to the ratio of mass (m) to charge (z), which is a characteristic that generally governs the separation of ions in a mass spectrometer.

[0033] Regarding m / z, the preferred range for fragment size is approximately 300 Thomsons to approximately 1700 Thomsons (Th). For the middle-down approach described above, and assuming that the peptide ions are more highly charged, the preferred range is approximately 600 to approximately 2000 Th.

[0034] As a result of the above, the desired number of fragments for a given starting molecule depends, on the one hand, on the size of the starting molecule, and on the other hand,

[0035] It should be noted that a given protein may not always be fragmented in the same manner when the method of the present invention is applied, as is also shown in the examples. A given molecule of a molecular species may be fragmented at a location partially or completely different from the location at which another molecule of the same molecular species is fragmented under the same conditions. Also, the number of fragments obtained may differ. This is a further advantage compared to established protein digests in the art that typically or more likely produce the same fragments for a given protein under given conditions. In particular, proteases generally exhibit sequence-dependent specificity, whereas the method of the present invention typically does not, or exhibits it to a lower and / or different degree.

[0036] As shown in Example 2, the method of the present invention yields more than 1,000 different fragments of protein having approximately 250 amino acids, compared to the low two-order-of-magnitude number obtained in conventional trypsin digestion. A wide variety of fragments are a further embodiment of the present invention and facilitate de novo sequencing by mass spectrometry, which is further disclosed below.

[0037] The non-enzymatic and non-chemical cleavage processes of the present invention can generate fragmentation patterns that were previously unattainable. In this context, the term "fragmentation pattern" refers to both the sites of fragmentation and the average size and size distribution of the fragments obtained from a given molecule.

[0038] The term "at least one protein, polypeptide, or peptide molecule" encompasses applications that use exactly or approximately one molecule, and is also referred to as a "single-molecule application." When there is no need to study or address individual molecules, but there is a need to fragment a small number of molecules, amounts of 1 fmol to 1 μmol are included in the term "at least one molecule." Also, for example, 1 μmol to 10¹⁶ per molecular species. 6 This encompasses macroscopic quantities in the range of moles.

[0039] The molecules to be cleaved may belong to a single molecular species, form a family of more closely related or less closely related molecules, or constitute a complete proteome. Indeed, the analysis of the proteome is a field of particular interest, and mass spectrometry is particularly well-suited to it. Furthermore, there is considerable room for improvement regarding the sample preparation process leading to actual mass spectrometry analysis. This is addressed by the novel fragmentation method of the present invention.

[0040] The term "mass spectrometer" has an established meaning in the art and is not particularly limited. The term "mass spectrometer" refers to a device capable of separating charged analytes based on a ratio m / z, where m is the mass of the analyte and z is the charge (in Thomsons). As is clear from the above, the analyte to be fed into the mass spectrometer is a peptide (and possibly polypeptide) obtained by fragmentation. In general, a mass spectrometer includes a device for ionizing the analyte contained in the sample, in addition to the device that performs the separation described above. Further components may be means for storing or filtering ions.

[0041] In a preferred embodiment, the sample to be processed is a liquid. Handling of the liquid is facilitated by a container. Therefore, in a preferred embodiment, at least one of the above-mentioned The molecules and the at least one magnetic material are located in a reactor. Where used herein, the term “reactor” generally refers to an enclosure or container in which the at least one molecule and the at least one magnetic material are located and capable of interacting (“reacting”) according to the present invention. Otherwise, the term is not particularly limited and includes vessels, vessels with closed bottoms, vessels with lids, vessels with closed bottoms and lids, completely closed or sealed vessels, tubular elements, and elements having at least two openings that allow a continuous liquid flow through a well-designed reactor. The reactor may also be implemented as a microfluidic device, i.e., a miniaturized device comprising one or more channels having openings with spreads or vessels and / or valves, optionally.

[0042] The inventors have found that nucleic acids are equally suitable for magnetic fragmentation. Accordingly, in a relevant aspect, the present invention provides a method for preparing a sample for an analytical procedure, wherein the sample comprises at least one nucleic acid molecule, and the method comprises (a) fragmenting the molecule using at least one mobile magnetic material.

[0043] The term "nucleic acid" has its general meaning. The term "nucleic acid" encompasses DNA and RNA, as well as chimeric molecules. In terms of length, the term "nucleic acid" includes oligonucleotides and polynucleotides. It also includes RNA molecules with specific functions and / or structural characteristics, such as mRNA, tRNA, siRNA, and miRNA.

[0044] In a preferred embodiment, the magnetic material is (a) a single magnet, or (b) an aggregate of particles, at least one of which is a magnet, wherein the aggregate of particles is mediated by the magnetic field of the at least one magnet. Briefly, in the magnetic field, the resulting aggregate behaves essentially like a single magnet. For further details, see below. In terms of size, the particles are larger than the molecules to be fragmented (and smaller than the vessel or reactor in which the method is carried out).

[0045] The magnet is a piece of ferrimagnetic, ferromagnetic, or paramagnetic material. The size of a single magnet may vary considerably and can be appropriately selected depending on the dimensions of the reactor or vessel to be used. When carrying out the method of the present invention, as will be discussed in more detail below, a reactor or vessel is preferably used that holds at least one molecule to be fragmented and at least one magnetic material.

[0046] With respect to relative size, it is preferable that the maximum dimensions of the magnetic material pass through the smallest passage or cross-section in the reactor or vessel. Being smaller than the minimum dimensions preferably means 2 / 3, 1 / 2, 1 / 3, 1 / 4, or 10% of the minimum dimensions or cross-section of the reactor, vessel, or tube. Such a mechanism generally provides free motion or substantially free motion. Preferably, the free motion or substantially free motion occurs around or along all of at least two, at least three, at least four, at least five, or preferably six axes of translational and rotational motion, and preferably, the free motion or substantially free motion includes translation along at least two axes.

[0047] To describe a point-like object, there are three degrees of freedom of motion, namely translation in three independent directions in three-dimensional space. For an extended object, there are three further degrees of freedom that can be defined with respect to three independent axes of rotation.

[0048] As far as they are present, other materials or reaction mixtures in the reactor may be taken into consideration when selecting the size of the magnetic material, and such other materials may have molecules to be cut and / or non-magnetic In addition to magnetic particles (also called non-magnetic beads; see below), the sample contains further constituent components.

[0049] For completeness, exemplary values ​​for useful magnet sizes are given herein as 0.1 mm to 10 cm, for example, 0.2 mm to 2 cm (including any of the following values ​​and ranges defined thereby: 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, and 1 cm). The same preferred sizes and size ranges apply to any magnetic material considered herein, and also to applications where multiple magnetic materials and / or magnets are used. Generally, these lengths refer to the maximum elongation of the magnetic material.

[0050] Alternatively, multiple magnets, such as those specified above, may be used. Exemplary but non-limiting numbers exist in the one- and two-digit range, for example, 2, 3, 4, 5, 6, 7, 8, 9, and 10. More than 10 magnets, such as 20, 30, 40, or 50, may also be used. These numbers refer to a single reactor or vessel, even when a vessel or reactor arrangement (e.g., a microtiter plate) is used.

[0051] It will be understood that the number of magnetic materials or magnets is limited as far as it is specified. In other words, while the method of the first embodiment may include other measures, such possibilities do not extend to the option of there being more magnetic materials or magnets than is clearly specified.

[0052] The magnets of such multiple magnets may be identical or different from each other. The same generally applies to magnetic materials, as long as multiple magnetic materials are to be used. To give a specific example, a single magnet may be combined with a single ferromagnetic or ferrimagnetic bead. This provides a more precise motion of the magnet while the parameters controlling the magnetic field remain unchanged.

[0053] There are no particular limitations on the shape of the magnets mentioned above; the preferred shape is one that does not negatively impede the free motion of the magnets. Examples of shapes include sticks, rods, rods with rounded ends, cubes, rectangular prisms, spheres, elongated and flattened ellipsoids, disks, tetrahedrons, octahedrons, dodecahedrons, and icosahedrons.

[0054] Such mechanisms must still be distinguished from the “assemblies” disclosed above. The latter refers to a mechanism in which a number of particles, usually a large number (e.g., 100 or 1000), assemble into a single magnetic material that behaves essentially like a single magnet under the influence of one or several (e.g., 10) magnets (which may be enhanced by the magnetic field according to the present invention). For completeness, similarly, multiple magnetic materials may be used, each or part of which result from such assemblies. However, this is not very likely under many circumstances. In particular, if no measures are taken to prevent this from happening, placing more than one assemblies in the same reactor or container can lead to the formation of a single, larger assembly (which will also then behave like a magnet once in a magnetic field).

[0055] In a more preferred embodiment, (a) the magnet includes or consists of a ferromagnetic material or a ferrimagnetic material, (b) the particles of the aggregate as defined above include or consist of a material selected from ferromagnetic materials, ferrimagnetic materials, superparamagnetic materials, paramagnetic materials and / or diamagnetic materials, and / or (c) the magnet and / or particles are preferably (i) chemically stable (ii) Coatings that impart qualitative properties; (ii) Coatings that impart mechanical stability or hardness; (iii) Coatings with catalysts; (iv) Coatings with nucleic acids, e.g., probes and / or primers; (v) Coatings with chelating agents, e.g., IMAC, TiO2 and ZrO2; (vi) Preferably (1) reversed-phase groups, e.g., C18, C8, benzene; (2) HILIC groups, e.g., hydroxyl groups; (3) cation exchange groups, e.g., sulfonic acid, phosphoric acid, carboxylic acid; (4) anion exchange groups, e.g., primary, secondary, tertiary and quaternary amino groups; and (5) any combination of any one of (1) to (4) (vii) Coating with selected chromatographic material; (vii) Preferably with ligand-binding proteins and / or their homologous ligands selected from globulins, particularly immunoglobulins, streptavidin, biotin, protein A, protein G, and enzymes, e.g., oxidoreductases, transferases, ligases, e.g., polymerases, hydrolases, e.g., proteases, peptidases, nucleases, saccharidases, lipases, lyases, and isomerases; and (viii) Coating with a coating selected from any one combination of (i) to (vii).

[0056] Suitable materials for the above magnets include the following elements and their alloys: neodymium-iron, neodymium-iron-boron (e.g., Nd2Fe 14B) Cobalt, gadolinium, terbium, dysprosium, iron, nickel, iron oxide, manganese-bismuth, manganese-antimony, manganese-arsenic, yttrium-iron oxide, chromium oxide, europium oxide, and samarium-cobalt. Particularly preferred materials are neodymium-iron and samarium-cobalt.

[0057] In less desirable embodiments, what is referred to as a "magnet" may also be implemented using such a paramagnetic material, particularly if the paramagnetic susceptibility of the paramagnetic material is high.

[0058] Suitable coatings according to (c)(i) include polypropylene, polyethylene, polystyrene, parylene, titanium nitride, polyimide, chloropolymer, and fluoropolymer, preferably polytetrafluoroethylene (PTFE).

[0059] In a more preferred embodiment, the at least one magnetic material performs a fluctuating or oscillating motion, preferably the motion is induced by a fluctuating or oscillating magnetic field, and preferably the magnetic field is generated by an electric current and / or an electromagnet.

[0060] In a more preferred embodiment, the magnetic material collides with the molecules. In a more preferred embodiment, at least one magnetic particle is present, and the motion of the magnetic material induces collisions between the at least one non-magnetic particle and the molecule.

[0061] The latter two embodiments may be implemented as alternative methods. With respect to the second two embodiments, it should be noted that the at least one non-magnetic particle not only collides with the molecule but also with the magnetic material. In other words, the magnetic material acts as an inducer of collisions between the at least one non-magnetic particle and the molecule, and may also cause direct collisions with the molecule. All of these effects generally contribute to fragmentation.

[0062] The term "collision" refers to a situation of temporary and sufficient spatial proximity between the magnetic material or non-magnetic particles and the molecules. Under such circumstances, energy, including kinetic energy, is transferred to the molecules. The transferred energy then subsequently induces or contributes to the fragmentation of the molecules.

[0063] The non-magnetic beads described above are different from particles that are optionally included in magnetic materials, and furthermore, they are different from paramagnetic beads that are commonly used in analysis. Non-magnetic beads are ceramic beads. These may be polymer beads, glass beads, or metal beads, and the metals are non-magnetic. In terms of size, the non-magnetic beads are preferably in the range of 1 μm to 5 μm, for example, 0.1 mm to 0.2 mm. Furthermore, it is preferable that the non-magnetic particles have the same or similar size range when compared to the size of a magnetic material or magnet.

[0064] Regarding motion, the magnetic material moves up and down and back and forth, and the motion may have regular or repeating components, but is not required, and the spatial direction is not particularly limited. In addition, the magnetic material may rotate about one or more axes, in addition to the usual translational motion. For a more precise description of the types of motion that can be assumed, see further below. As long as a reactor, i.e., a container, enclosure, or vessel is used, the magnetic material may or may repeatedly strike the walls of the reactor, but is not required. Thus, the magnetic material is always in motion (unless a particular execution of the method of the present invention otherwise specifies, for example, intermittent motion), but such motion is preferably not in a fixed direction. Also, the motion may be irregular in some cases, but generally it occurs with respect to an average position located within the enclosure or container, and the magnetic material does not leave the reactor. The motion of the magnetic material generally has one or more translational components, and the average position may be located around the center of the reactor. Therefore, unlike the motion performed by a magnetic stirrer, which is rotation, the average position of the magnet is at or near the bottom of the container containing the liquid to be mixed or stirred.

[0065] The term "vibration" refers to regular motion, while "fluctuation" is broader and includes irregular motion as well. There is no particular preference in this regard. In fact, irregular motion occurs more frequently, considering that magnets often collide not only with the molecules to be cut, but also with at least one wall of the reactor or enclosure used to contain the magnetic material and molecules. In either case, the motion ensures that, insofar as multiple molecules or a macroscopic amount thereof are processed by the method of the present invention, the magnetic material generally comes into repeated contact with the above molecules, and with all or substantially all of the molecules to be cut. As stated above, the motion of the magnetic material may also cause collisions between the above molecules and further magnetic and / or non-magnetic particles, as long as they are present.

[0066] As described above, the motion of the magnetic material is preferably induced by a fluctuating or oscillating magnetic field.

[0067] A magnetic field is a common means of controlling the position and / or motion of a magnet. Considering that the magnetic material moves according to the present invention, a fluctuating or oscillating magnetic field is used in this preferred embodiment. Any such magnetic field may be useful.

[0068] The magnetic field generated by the electric current is desirable. It is well established that electric and magnetic fields are interrelated, particularly that electric current generates a magnetic field. Consequently, controlling the electric current is a means of controlling the magnetic field it generates.

[0069] Alternatively, or more preferably, the magnetic fields may each be generated or regulated by an external magnet. The term “external” means that such a magnet is not located inside the reactor. The external magnet may be a permanent magnet. If a permanent magnet is used, the magnetic field it generates may be varied or vibrated by the corresponding movement of the external magnet with respect to the at least one magnetic material.

[0070] In a preferred embodiment, the magnetic field is generated by an electromagnet. As used herein, the term “electromagnet” encompasses, in its simplest implementation, a portion of a conductor through which an electric current flows when in use. For better control of the magnetic field, or to generate a stronger magnetic field... For this purpose, a specific execution of the electromagnet, which is the subject of the preferred embodiments disclosed below, is envisioned.

[0071] In a preferred embodiment, the current fluctuates or oscillates. This behavior can also be commonly referred to as a "wave." The quantity of the current is known as amperes.

[0072] In a preferred embodiment, the amperage of the current as a function of time is (i) a rectangular function, (ii) a sine function, (iii) a triangular function, (iv) a sawtooth function, or (v) a combination or convolution of any one of (i) to (iv).

[0073] Considering that currents oscillate or fluctuate, this also applies to patterns (i) to (v), i.e., the above rectangular and triangular functions are actually repeating rectangular and triangular functions. The term "pattern" refers to a series of events in which a given fundamental event is repeated at least once. More broadly, the repetition does not have to be an exact repetition; for example, the length of the rectangle in the time graph can change (this effectively becomes a change in frequency, and preferred frequencies and preferred time dependencies of frequencies are further specified below).

[0074] Embodiments (i) to (v) are all also referred to as "alternating current" in this specification. The above-mentioned rectangular function (also referred to as a rectangular wave or square wave), more specifically a pattern of a repeating rectangular function, is particularly preferred. Surprisingly, the inventors have found that this pattern induces particularly vigorous motion in the magnetic material, and that such vigorous motion is particularly efficient with respect to cutting. In the above-mentioned rectangular function, the time intervals between high current and low current (or the state where the current is off) may be the same or different. Means for controlling the length of the above time intervals, such as pulse width modulation (PWM), are known to those skilled in the art. It should be noted that the energy transferred to the reaction mixture is controlled not only by the frequency and amplitude of the current, but also by the relative duration of the above time intervals.

[0075] In a more preferred embodiment of the method of the first aspect, the collision transfers an amount of energy to the molecule that is sufficient to break at least one covalent bond. It is well established that collision is a means of transferring energy from one object to another. The energy received by the receiving object can be converted into the internal energy of the object (in this case, the molecule) to induce its fragmentation.

[0076] The preferred embodiments disclosed above are means for specifying the overall outcome of a particular execution selected: the energy transferred to the molecule to be cleaved may be such that at least one covalent bond in the molecule accepts the amount of energy required for dissociation.

[0077] It is estimated that the amount of energy transferred to the covalent bond by the magnetic material is equal to or less than the energy transferred to the magnetic material by the magnetic field. The energy in a magnetic field per unit volume is E mag = 1 / 2 B 2 / μ0 is defined, and for the definitions of B and μ0, see below. Next, E mag This is equal to or less than the energy of the current that produces the magnetic field. The latter energy is E curr It can be estimated that =UIt, where U is the voltage, I is the amperage of the current that generates the magnetic field, and t is the time the current flows. In other words, controlling any one of B, U, I, and t is a means of controlling the amount of energy transferred to the covalent bond by the magnetic material.

[0078] However, other means exist to specify the detailed implementation of the method of the first embodiment. This involves specifying one or more of several parameters that can be controlled or measured more directly. These parameters include the frequency and amperage of the current. This includes, and may further, include the dimensions of the reactor and coil, to the extent used. Preferably, the strength of the magnetic field in the magnetic material portion includes means of quantitatively specifying the details of the operation. In all these cases, parameter values ​​or combinations of parameter values ​​that ensure the above requirement (transfer of sufficient energy to break the bond) is satisfied are preferred. Preferred ranges for the above parameters are specified below.

[0079] In a preferred embodiment, the current fluctuates or oscillates at a given frequency, preferably 0.1 Hz to 20 MHz, more preferably 10 Hz to 2 kHz, and even more preferably 50 to 500 Hz, 90 to 300 Hz, or 100 to 200 Hz. It is understood that these scales apply not only to sinusoidal currents but to all current profiles specified herein, including, for example, repeating rectangular patterns. The term frequency may also apply to fluctuations, i.e., time-dependent behavior that is not regular (such regular time behavior is also referred to herein as “oscillation”), and is a means of characterizing the time scale of the fluctuation. In such cases, the term “frequency” is understood to refer to the average frequency of the fluctuation.

[0080] For reactors with volumes in the mL range of at least one to two orders of magnitude, and for the wells of a standard 96-well plate, lower frequencies of 80–300 Hz work particularly well, while significantly higher frequencies, such as around 1000 Hz, induce vibrations in the magnetic material while simultaneously failing to induce the full range of motion covering a substantial portion of the reactor volume. This does not mean that higher frequencies are unbeneficial; rather, higher frequencies may be used in conjunction with lower frequencies (see below).

[0081] More generally, a more preferred frequency range is one that ensures the magnetic material not only vibrates or rotates but also performs translational motion that explores the entire volume or substantially the entire volume of the material to be processed in the method of the present invention. In implementations utilizing reactors and the material to be processed in solution or suspension, the volume is the total volume of the solution or suspension as contained in the reactor. In other words, while guidance is provided above for reactors and 96-well plates having volumes in the one- to two-order-of-a-kind mL range, the frequency range may require adaptation for reactors with significantly smaller volumes, significantly larger volumes, or special geometries. For example, for smaller volumes, e.g., wells of a high-density microtiter plate (e.g., a 1536-well plate), higher frequencies, e.g., above about 1 kHz, e.g., above 200 Hz, are expected to produce magnetic material motion comparable to motion observed at lower frequencies in larger vessels. In any case, those skilled in the art, provided with the guidance provided herein, can directly explore and optimize the parameters controlling the motion of the at least one magnetic material. As further explained above, a magnetic material that performs translational motion in addition to rotation is preferred.

[0082] In a preferred embodiment, the frequency is kept constant throughout the entire duration of the method.

[0083] In an alternative, preferred embodiment, the above frequency varies as a function of time. In a more preferred embodiment, more than one frequency is applied at a given time. In such a case, each of the multiple frequencies may be selected from any of the preferred intervals given above. In the case of two frequencies, it is particularly preferred that the first frequency is 50 Hz to 500 Hz and the second frequency is 80 Hz to 20 MHz. In other words, this preferred embodiment provides a superposition of multiple frequencies.

[0084] More than one frequency includes two, three, four, five, six, seven, eight, nine, and ten different frequencies. Such multiple frequencies can be provided throughout in place of a single frequency. This means that such multiple frequencies are applied during the overall implementation of the above method. Also, multiple frequencies, or different multiple frequencies, can be applied at different time intervals within a longer period. Within the above longer period, in addition to the time intervals at which more than one frequency is applied, there may be one or more, for example, two, three, four, five, six, seven, eight, nine, or ten time intervals, at which only a single frequency is applied. Surprisingly, the inventors have found that a control system to which more than one frequency is applied functions better with respect to yield. "Yield" refers to the relative amount of fragmented molecules compared to the total number of (non-fragmented) molecules at the beginning of the above method.

[0085]

[0086] In a more preferred embodiment, the above frequency (singular) is not constant over time, and in the case of more than one frequency, the frequency (plural) is not constant over time, and preferably in a periodic manner, it is preferably switched between two or more frequencies or gradually changed (singular / plural).

[0087] An exemplary control system is (120 Hz to 1000 Hz) n , (200 Hz to 1000 Hz) n , or (100 Hz to 800 Hz) n (where n is an integer, for example, from 2 to 1000, for example, from 10 to 100, specifying the number of times the frequency pattern within the parentheses should be repeated).

[0088] The duration of the time interval when a constant frequency and / or a constant number of amperes is used is not particularly limited. Time intervals of 1 second to 1 day, for example, 1 minute to 1 hour are envisioned.

[0089] ​In a more preferred embodiment, the current has (a) an amperage I of 20mA to 100A, preferably 0.1 to 20A, and (b) the magnet has a current of 0.02 to 10 9 A / m, preferably 10-10 6 (c) Exposure to a magnetic field strength of A / m and / or (c) applied for a period of time t from 1 second to 1 week, for example, from 10 minutes to 5 hours.

[0090] As disclosed above, the amperage as a function of time varies on a time scale controlled by the frequency disclosed herein, and it should be noted that there is no constant amperage with respect to the time scale of variation. Furthermore, for practical purposes and in accordance with electrodynamic practice, alternating current can be quantified with respect to its average amperage. The above value is the average amperage in that sense. It should be noted that the average preferably exceeds the time scale of variation. In other words, as long as intermittent current is used, the average amperage is preferably within the range specified above when the current is on, and zero amperage exists when the current is off.

[0091] Magnetic field strength H determines the strength of the magnetic field and is measured in A per meter. H must be distinguished from magnetic flux density B, which is particularly relevant in situations where a core is used to enhance the magnetic field of an electric current. See below.

[0092] In a preferred embodiment, the amplitude of the fluctuation or vibration is (a) constant or (b) preferably changes over time on a time scale slower than the time scale of the fluctuation or vibration.

[0093] This embodiment refers to the amplitude of the current motion described above. The amplitude of the current oscillation or fluctuation is controlled by the amperage.

[0094] In a more preferred embodiment, the current is intermittent and / or the ampere The frequency (A) changes over time, preferably in a periodic manner. This change over time generally relates to a time scale slower than the time scale defined by the frequency of the alternating current. In other words, if the alternating current changes over time in the sense of this embodiment, the time dependence of the current is a superposition of two patterns or waves: generally faster fluctuations inherent in alternating currents, and generally slower changes.

[0095] An exemplary intermittent pattern is a repetition of on (1 minute) - off (1 minute) cycles. Other preferred time intervals are given above. The advantage of the intermittent pattern is that it is possible to keep the temperature constant or substantially constant, especially when it is observed that the contents of the reactor are heating.

[0096] In a more preferred embodiment, a power of 0 to 1000W, preferably 1 to 200W, is applied.

[0097] In a more preferred embodiment, the current is powered by a power supply. Preferably, the power supply has a potential or voltage U in the range of approximately 0 to 240V, for example, 0.1 to 75V. These values ​​refer to the average voltage applied.

[0098] In a further preferred embodiment, the electromagnet includes at least one coil, preferably the coil having (a) a plurality of, for example, 1 to 10 4 It has (b) at least 10 to 1000 windings and / or (c) at least one Helmholtz coil and / or (c) at least one core.

[0099] Exceptional numbers of coils are 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 150, 200, 300, 400, and 500.

[0100] The term "Helmholtz coil" is established in the art and refers to an arrangement of two identical coils, usually, where the rotationally symmetric axes are aligned in a line or spaced apart to occupy the same space. The magnetic field in the space between the coils is particularly homogeneous and / or particularly strong. An arrangement of at least one Helmholtz coil may also consist of two Helmholtz coils.

[0101] Further arrangements of multiple coils are known to provide an extended spatial region of particularly homogeneous magnetic fields. A further example is the Maxwell coil.

[0102] The core enhances the effect of the magnetic field. The core is preferably made of a ferromagnetic material, such as iron, especially soft iron. The magnetic flux density B is related to the magnetic field strength as follows: B = μ r μ0H is the permeability of vacuum, and therefore a fundamental physical constant. On the other hand, μ r μ is the relative permeability, which determines the degree of magnetic flux density enhancement by a given material, such as a ferromagnetic core, under the influence of a magnetic field. Typically, μ is used for ferromagnetic materials that are to be used as cores. r is 10 3 ~10 6 For example, 200,000 to 400,000, or approximately 300,000. Suitable core materials include powder metals, laminated metals, annealed metals such as annealed iron, ceramics, and solid metals.

[0103] The preferred value of B in the absence of the core is 10 -8 ~10 4 Tesla (T), for example 10 -5 It is ~1T. When a core with a specific relative permeability is used, the value of B can be multiplied by the above relative permeability. Therefore, a preferred value of B in the presence of the core is 10 -2 ~10 9 For example, 1 to 10 6 T is the value of B in general, either in the magnetic material or within the reactor.

[0104] Geometry-wise, the preferred coil is circular. Preferred diameters are 1 mm to 1 m or 1 mm to 0.5 m, e.g., 2 mm to 300 mm or 2 mm to 200 mm. Different geometries, such as square, rectangular, or triangular coils, are also conceivable (i.e., all or part of the winding is square, rectangular, or triangular). Finally, it should be noted that the coil is not an essential requirement for an electromagnet, and an arrangement of two antiparallel wires may also be used ("antiparallel" refers to the direction of the current flowing through the two wires at a given time).

[0105] In a more preferred embodiment, more than one coil, preferably 2 to 10 coils, are used. 4 Individual coils, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 coils, or 10 to 1000 coils, may be used. It is understood that each coil may have one or more windings, and preferred numbers of windings are disclosed above in this specification.

[0106] In a more preferred embodiment, the resulting fragments are suitable for mass spectrometry. This is consistent with the foregoing, namely, that fragmentation is a means of obtaining portions whose size is particularly suitable for analysis by mass spectrometry. The most appropriate size range depends on the application. The parameters disclosed above that control the motion of the magnetic material are means of controlling the size of the resulting fragments.

[0107] In a more preferred embodiment, the sample has a biological origin and preferably (i) the sample is a solution or suspension of the molecule, or comprises a solution or suspension of the molecule, for example, the sample comprises a mixture of the molecule, protein, polypeptide and / or peptide in a purified form, or a body fluid such as blood, serum, plasma, cerebrospinal fluid, sputum or urine, or comprises a body fluid such as blood, serum, plasma, cerebrospinal fluid, sputum or urine, (ii) the sample is a cell such as a prokaryotic cell or eukaryotic cell, or comprises a cell such as a prokaryotic cell or eukaryotic cell, for example, the sample is a suspension of cells, or comprises a suspension of cells, (iii) the sample is a virus, or comprises a virus, for example, the sample is a suspension of viruses, and / or (iv) the sample is a tissue, such as muscle tissue or brain tissue, or comprises a tissue, such as muscle tissue or brain tissue.

[0108] In a particularly preferred embodiment, the sample is derived from a healthy organism, a diseased organism, or an organism exposed to a stimulus, chemical, or drug. Comparing the complete proteome across multiple states (healthy vs. diseased; before vs. after exposure to a stimulus) is of particular interest in the field of diagnostics and benefits from the present invention. Stimuli may include temperature or changes thereof, gas concentration or changes thereof, and exposure to UV irradiation.

[0109] In a more preferred embodiment, the sample comprises cells and / or tissues, wherein the cells or tissues are dissolved by the motion. The inventors were previously aware that cellular materials can be dissolved under the influence of a moving magnet. See International Publication No. 2020 / 002577, which is incorporated by reference in its entirety.

[0110] In another preferred embodiment, the sample contains a virus, which is inactivated or destroyed by the aforementioned motility.

[0111] Considering viruses, and noting that the above method provides both the disintegration of viruses and the fragmentation of their proteins, the present invention also provides a convenient method for epitope mapping. As is known in the art, epitope An epitope is a region recognized by an antibody. An epitope can be, but does not have to be, a sequence of amino acids that are in close proximity in the primary sequence. In the case of a three-dimensional epitope, sequences that are separated from each other in the primary sequence may eventually become spatially close during folding, thereby defining the epitope.

[0112] The term "antibody" is not particularly limited. Preferred antibodies include all naturally occurring families of immunoglobulins, preferably of mammalian or human origin, such as IgG, IgM, and IgE, but also include camel antibodies and antigen-binding molecules with structures not found in nature (antibody fragments, single-chain antibodies, labeled antibodies, antibody conjugates, such as antibody-drug conjugates).

[0113] An exemplary method of epitope mapping according to the present invention includes a method of the first embodiment applied to a virus, followed by adding one or more antibodies of interest to the resulting mixture of fragments, enabling the fragments to bind to the one or more antibodies, and identifying the bound fragments, preferably by mass spectrometry.

[0114] Preferably, the antibodies are coupled to beads, which facilitates their handling. The antibodies may originate from individuals that have developed immunity to a particular viral pathogen.

[0115] Furthermore, after enabling the above binding, it is preferable to wash the resulting antibody-fragment complex. This removes fragments that do not bind to the antibody.

[0116] Prior to identification, it is preferable to elute the bound fragment. This breaks down the antibody-fragment complex, allowing for downstream processing of the bound fragment.

[0117] By knowing all the binding fragments, the epitope can be identified, or at least the region in which the epitope is located can be revealed.

[0118] Accordingly, the present invention provides a method for mapping an epitope, the method comprising: fragmenting a protein containing the epitope using at least one mobile magnetic material; adding at least one antibody of interest; separating the antibody-binding fragment from the unbinding fragment; and identifying the antibody-binding fragment.

[0119] Preferably, the protein is a viral protein, more preferably a capsid protein.

[0120] Notably, viral proteins do not need to be provided in the context of viral particles. This means that epitope mapping may also be performed using viral proteins or already degraded viruses as starting material.

[0121] In alternative methods, antibodies cannot be immobilized on a carrier or bound to a matrix or particles, whereas fragments obtained by the method of the present invention can be immobilized on a carrier or bound to a matrix or particles. Such carriers, matrices, or particles can be used to capture antibodies capable of binding to specific fragments. In further alternative methods, immobilization may be completely omitted.

[0122] The concept of interaction mapping is not limited to antibody / antigen interactions. In fact, the above methods can be used to characterize protein / protein interactions in general.

[0123] Therefore, the present invention can bind to a second protein or binding partner. A method for identifying a site on a first protein that is or is likely to be capable of, the method comprising: fragmenting the first protein using at least one mobile magnetic material; adding the second protein or the binding partner; separating the fragments that bind to the second protein or the binding partner from the unbound fragments; and identifying the fragments that bind to the second protein or the binding partner, thereby identifying the site.

[0124] The term "binding partner" is more general than the enumerated "second protein." In fact, this method is not limited to protein-protein interactions but can be extended to, or instead applied to, protein-nucleic acid interactions and protein-small molecule interactions.

[0125] In a more preferred embodiment, the magnetic field induces at least two motions of the magnetic material, wherein the motions have different kinetic energies, and the at least two motions occur simultaneously or at different time intervals.

[0126] This embodiment provides fine-tuning of what a magnetic material does to a sample. One example is the accompanying lysis of cells and the fragmentation of proteins, polypeptides, and peptides contained within those cells. Depending on the type of cells to be lysed and the desired size range of the fragments, different configurations of the device that controls the movement of the magnetic material at different points in time during the implementation of the method of the present invention (see further below for details of such a device, which is also the subject of the present invention) may be desirable. Generally, when lysis and protein fragmentation are carried out, nucleic acids are fragmented simultaneously.

[0127] A further example is the aforementioned disintegration of the virus, accompanied by the fragmentation of proteins contained within the viral particle. Here, changes in the settings of the above device may be beneficial, for example, once the virus has disintegrated, in order to optimize the yield of protein fragmentation and / or to ensure that fragments of the desired size are obtained.

[0128] In the epitope mapping scenario described above, a somewhat wide range of fragment sizes is of interest. In this context, this range is, for example, approximately 5 to 100 amino acids. Such a wide range, including longer and specific fragments as well as very short ones, is particularly suitable for epitope identification.

[0129] In a further preferred embodiment, the method of the first embodiment further comprises (b) a step of exposing the sample to heat, for example, boiling; a step of denaturing the sample, for example, by sonication; a step of adding a cleaning agent to the sample; and / or a step of adding a chaotropic agent to the sample, preferably, step (b) is carried out prior to or simultaneously with step (a), i.e., fragmentation according to the first embodiment. Such further measures may be selected depending on the specific application under consideration. Such further measures may improve the yield of dissolution (as long as cell material is present in the sample) and / or the yield of fragmentation.

[0130] Preferred detergents are sodium dodecyl sulfate, sodium deoxycholate, Triton X-100, Tween20, and CHAPS.

[0131] Preferred chaotropic agents are guanidine hydrochloride, urea, and thiourea. In a more preferred embodiment, the method of the first embodiment further includes the step of (c) chemically modifying the molecule and / or a fragment obtained from the molecule.

[0132] In a particularly preferred embodiment, the above chemical modification step is a step of reducing (ca) disulfide, a step of alkylating (cb) thiol groups, such as cysteine ​​residues, a step of crosslinking (cc), and (cd) any combination of (ca), (cb), and (cc). A combination of (ca) and (cb) is preferred.

[0133] These are routinely employed methods in the preparation of samples for mass spectrometry. According to the present invention, they can be performed simultaneously with fragmentation. Notably, according to the present invention, reduction and alkylation are not essential. This is particularly true for the aforementioned middle-down approach when larger fragments are of interest.

[0134] Therefore, it is particularly preferable that processes (a), (ca), and (cb) be carried out simultaneously.

[0135] In other words, in embodiments relating to the latter embodiment, the present invention provides a method for preparing a sample for analysis in mass spectrometry, wherein the sample comprises at least one protein, polypeptide, or peptide molecule, and the method comprises (1) adding a reducing agent and an alkylating agent to the sample, and (2) fragmenting the molecule using at least one mobile magnetic material. It will be understood that step (2) is carried out in the presence of the agents added in step (1). For example, steps (1) and (2) may be carried out simultaneously, or step (2) may follow directly from step (1).

[0136] In a more preferred embodiment, an inert viscous liquid; gel, such as a polyacrylamide gel or agarose gel; aerogel and / or zeolith is added to the sample (singular / plural). While we do not wish to be bound by any particular theory, embedding the molecules to be fragmented in a gel or viscous liquid is considered a means of enhancing energy transfer from magnetic materials or, as far as they are present, any non-magnetic particles to the molecules, and subsequently enhancing the yield.

[0137] When using a polyacrylamide gel, this generally involves performing polyacrylamide electrophoresis on a sample containing at least one of the above molecules. Subsequently, the region of interest may be cut from the gel, or the gel as a whole may be processed according to the first embodiment, i.e., subjected to fragmentation. However, electrophoresis is not required. Alternatively, the components necessary for forming a polyacrylamide gel may be added to the sample to be processed to form a gel, which may then be subjected to the method of the first embodiment.

[0138] If agarose is to be used, it may be added to the sample to be processed, boiled, and cooled. This results in an agarose gel with the molecules to be fragmented embedded within the gel. Alternatively, the agarose may be boiled before combining it with the sample, thereby avoiding exposure of the sample to the boiling temperature.

[0139] In a more preferred embodiment, a protease, such as trypsin, LysC, GluC, AspN, ArgC, or chymotrypsin, is added.

[0140] As described above, one of the advantages of the present invention is that chemical and enzymatic means of hydrolysis or cleavage are non-essential in that they can be replaced by "magnetic fragmentation." Furthermore, in certain cases, it may be desirable to adjust the spectrum of the fragments obtained by the magnetic fragmentation by adding proteases, which themselves are established in the art.

[0141] Furthermore, chemicals that facilitate protein cleavage may also be added. For example, CNBr is described as being cleaved at Met residues. Formic acid is preferred for Asp-Pro bonds with respect to hydrolysis, and hydroxyamine is preferred for Asn-Gly bonds. 2-Nitro-5-thiocyanobenzoic acid is cleaved at Cys residues. Furthermore, iron and Copper salts facilitate the generation of reactive oxygen species that subsequently react with proteins and decompose them.

[0142] In another preferred embodiment, one or more of the following are added to the sample: surfactant; detergent; buffer; acid; base; chaotropic agent; cosmotropic agent; salt; and solvent, preferably an organic solvent. Furthermore, additional active ingredients commonly used in sample preparation, such as phosphorylase inhibitors, may also be added.

[0143] Particularly preferred acids are acetic acid, formic acid, and trifluoro acid. Particularly preferred organic solvents are acetonitrile, ethanol, methanol, isopropanol, and trifluoroethanol.

[0144] In a more preferred embodiment, the method of the present invention further comprises (d) purifying and / or concentrating the obtained fragments, preferably by filtration, non-covalent bonding and / or covalent bonding, wherein non-covalent bonding is preferably carried out with a reversed-phase material, a normal-phase material, an ion-exchange material, an affinity-bonding material, a chelating material or a paramagnetic particle, and covalent bonding is preferably carried out using a reagent capable of forming a conjugate with any of the functional groups of the fragments, such as an amine group.

[0145] In a more preferred embodiment, the method further includes the step of reacting the sample with a probe or ligand, preferably the probe being a DNA probe and / or an RNA probe.

[0146] In a more preferred embodiment, the method further comprises (e) labeling the molecule and / or a fragment obtained from the molecule, preferably the labeling of the fragment being achieved after the purification and / or concentration of the fragment. The label used is not particularly limited. Given that mass spectrometry is a preferred analytical method according to the present invention, a label detectable by the method, and a label that can be provided in a different isotope-labeled form, is preferred. Labeling with different isotopes is a convenient method in which the label is distinguishable in the mass spectrum but behaves identically or similarly with respect to chemistry, including the chemistry that couples the label to the analyte (here including a protein, polypeptide or peptide, or a fragment delivered by the method of the first embodiment).

[0147] Therefore, in a more preferred embodiment, the labeling step is carried out by reacting a functional group of the molecule or a fragment thereof with a reagent capable of forming a conjugate with the functional group, wherein the reagent capable of forming a conjugate is preferably a tag detectable by mass spectrometry.

[0148] In particularly preferred embodiments, the functional group is selected from a primary amine group, a carboxyl group, and a thiol group.

[0149] It is particularly preferable that the functional group is a primary amine group, the tag is an active ester, such as an N-hydroxysuccinimide (NHS) ester, and / or is provided in at least two different isotopically labeled forms. Preferably, the different isotopically labeled forms have the same total nominal mass for all forms (this is also referred to as a series of "isobaric tags").

[0150] Preferably, the above tags are suitable for fragmentation in a mass spectrometer, preferably by gas-phase fragmentation, such as collision-induced dissociation (CID), electron transfer dissociation, or UV fragmentation. Notably, such fragmentation is suitable for the fragmentation of proteins, polypeptides, and peptides according to the present invention. The fragmentation should remain in a different state from the tag, which is performed by mass spectrometry, while the latter is performed prior to spectroscopic analysis and affects the aforementioned protein molecules.

[0151] This type of labeling process is generally carried out at the fragment level, where proteins and polypeptides (and possibly longer peptides) are fragmented using the method of the first embodiment, and the resulting fragments are then labeled. Stable isotopes are preferred.

[0152] Furthermore, this is not the only possibility for labeling protein molecules for analytical purposes in mass spectrometry. For example, metabolic labeling (one implementation of which is referred to as "SILAC") may be performed. This provides, for example, the incorporation of isotopically labeled components into proteins while the isotopically labeled components are synthesized on ribosomes. In such cases, the labeling step is performed prior to fragmentation, and the samples, especially differently labeled samples, may be pooled prior to fragmentation.

[0153] More than one sample may be processed using the method of the present invention, and different samples are preferably labeled differently. Different samples may be healthy samples and diseased samples, or samples taken before and after administration of an active substance or drug or exposure to an irritant, see above for details.

[0154] Differently labeled samples may be pooled. This is a means of overall enhancement in the mass spectrometer. Different labeling ensures that different samples contained in the pooled sample are distinguishable in the mass spectrum. In other words, pooling generally occurs after the labeling step. Furthermore, it is preferable to perform pooling after fragmentation, especially when the labeling procedure is applied to fragments obtained by the method of the present invention.

[0155] In a more general sense, pooling involves the addition of standard substances, which enables or makes more accurate protein quantification.

[0156] In a more preferred embodiment, the average fragment length deviates from the fragment length obtained by digestion of the above molecule using a protease, and preferably, the average fragment length obtained by the above method is greater. This is a characteristic advantage of the present invention. The average fragment length results from “magnetic fragmentation,” which is mechanistically different from enzymatic fragmentation established in the art, and furthermore, from the fact that the motion of the magnetic material can be conveniently controlled and fine-tuned by a number of parameters disclosed above. As described in exemplary form above, magnetic fragmentation can be used for average fragment sizes obtained that are larger than trypsin fragments, for example, in the range of 30 to 100 amino acids.

[0157] When using the method of the present invention compared to enzymatic or chemical fragmentation, a wider probability density of fragment sizes is even more preferable. In this embodiment, a wide range of fragment sizes can be obtained that can encompass both conventional fragment sizes, such as those obtained by trypsin digestion, and larger fragments in the sense of the middle-down approach described above.

[0158] In a second aspect, the present invention provides an analytical method comprising the step of performing mass spectrometry on the obtained fragments, one of the methods described in any of the prior claims.

[0159] The method of the second embodiment, in a preferred embodiment, includes a step of chromatographic separation of fragments by liquid chromatography (LC) prior to mass spectrometry. Preferably, this is performed as HPLC. The chromatographic material is preferably a reversed-phase material, a C18 material.

[0160] In a preferred embodiment of the method according to the second aspect, the method includes a computer implementation step of determining the sequence and / or uniqueness of the molecules. This step is achieved by analyzing the fragments, for example, by determining their sequences from mass spectra. Once the sequences of a sufficient number of fragments are known, a sequence assembly algorithm can be used to infer the original sequence of the protein or polypeptide from which the fragments originated.

[0161] In a third aspect, the present invention provides the use of magnetic materials and magnetic means for generating a fluctuating or oscillating magnetic field for fragmenting protein, polypeptide, or peptide molecules.

[0162] The preferred embodiment of the above method defines the preferred embodiment of the above use of the present invention. In a fourth aspect, the present invention provides a kit comprising (i) at least one magnetic material, and (ii) a container or array of containers configured to receive a sample comprising the magnetic material and at least one protein, polypeptide, or peptide molecule.

[0163] In a preferred embodiment, the kit further comprises or consists of (iii) a reducing agent and (iv) an alkylating agent.

[0164] The preferred reducing agents in all embodiments are dithiothreitol and tris(2-carboxyethyl)phosphine.

[0165] Preferred alkylating agents in all embodiments include haloacetic acid, haloacetamide, haloalkaneamide, and N,N-dialkylhaloalkaneamide. Preferably, the presence of the alkane is independently selected from linear or branched C1-C5 alkanes, such as methyl, ethyl, and isopropyl. The halogen (or "halo") includes chloro, bromo, and iodine. The alkyl moiety may be substituted, with hydroxyl being a preferred substituent.

[0166] These active ingredients can be used in the reduction and alkylation steps of the method of the present invention as disclosed above.

[0167] In a more preferred embodiment, the kit preferably further comprises, in addition to the alkylating agent and the reducing agent, (v) one, more or all of the surfactants, chaotropic agents, denaturants, and organic solvents; (vi) at least one buffer; (vii) nonmagnetic particles; and / or (vii) a manual containing instructions for carrying out the method of the present invention.

[0168] Preferred surfactants, preferred chaotropic agents, and preferred solvents are disclosed herein as described above. These active ingredients may have denaturing properties.

[0169] The buffer serves to establish and maintain the pH value of the sample during the various steps of the method of the present invention. An exemplary pH value is 8 to 8.5. Buffer materials suitable for handling biological molecules, such as proteins, polypeptides, peptides, and nucleic acids, are known to those skilled in the art and are available from numerous manufacturers.

[0170] The non-magnetic particles described above are further disclosed above. The particles, also referred to herein as “beads,” may be metal beads or ceramic beads.

[0171] In a preferred embodiment of the above kit, the kit is adapted for the method of epitope mapping disclosed above. Thus, the kit further comprises, or comprises, protein A and / or protein G, and optionally, one or more buffers suitable for carrying out the above method of epitope mapping. Preferably, protein A and / or protein G are immobilized on a surface or carrier, such as beads or a plate. Alternatively, the kit further comprises, or comprises, a surface or carrier having a reactive portion. Such reactive portion may be an NHS, epoxy, or carboxyl group. Such reactive portion serves to immobilize the fragment obtained in step (a) of the method of the present invention onto the surface or carrier. Furthermore, beads and plates are suitable implementations of the surface or carrier.

[0172] In this specification, the phrase "further comprising" refers to an implementation in which the kit consists of closed-number components. In the above embodiments, these are items (i) to (iii) as defined according to the fourth aspect and item (iv), which is the subject of the above embodiments.

[0173] The containers disclosed above are not particularly limited. Useful containers include those commonly used in the fields of molecular biology and in vitro diagnostics. Such containers generally contain no or substantially no contaminants, are chemically inert, and / or have surfaces with low binding capacity.

[0174] It is important in the context of the present invention that the container has at least one wall that does not shield the magnetic field. Preferably, the entire container is made of a material that does not shield the magnetic field. Suitable materials include plastics, polymers, such as polypropylene, glass, and ceramics. Metals may also be used, taking into account the requirements for magnetic permeability.

[0175] Exemplary and preferred containers are configured to hold a volume of 5 μL to 1 L, preferably 10 μL to 50 mL, and more preferably 30 μL, 40 μL, 100 μL, 150 μL, 200 μL, 250 μL, 500 μL, 1 mL, 1.5 mL, 2 mL, 5 mL, 15 mL, and 50 mL.

[0176] The containers can be arranged in arrays, for example, in common formats (96, 384, or 1546 wells).

[0177] In one embodiment, the container may be equipped with a filter layer or frit, and such a container is also referred to as a cartridge. This allows for the convenient separation of fragments (optionally, labeled fragments) from any other material. The solution containing the fragments can pass through the filter or frit by, for example, applying positive pressure to the cartridge, connecting the cartridge to a vacuum, or centrifuging. The entire process can be carried out by a liquid handling machine. The filtrate can be captured in a second container or in a dedicated compartment within the same container.

[0178] In a particularly preferred embodiment, the filter contained in the container is a molecular weight cutoff filter. Such a filter is known in the art and allows all molecules with molecular weights below the cutoff value to pass through while retaining all materials with molecular weights above the cutoff value.

[0179] A cartridge provided accordingly provides a preferred embodiment of the method of the present invention. In particular, when the above method is carried out in the above cartridge (where the magnetic material is positioned above the filter, i.e., a high molecular weight material is held), the cartridge It provides continuous removal of fragments that are below the to-off level. By passing the liquid through the cartridge while inducing the movement of a magnet, further fragmentation of the fragments that can pass through the filter does not occur.

[0180] This mechanism has distinctive advantages. It can be used to obtain fragments with a narrow size distribution. Furthermore, this mechanism can operate in a continuous, i.e., flow-through mode.

[0181] In a more preferred embodiment, the kit further comprises or consists of a labeling agent and / or a crosslinking agent.

[0182] In a further embodiment, the present invention provides a device comprising or comprising a conductor, preferably at least one coil, more preferably a Helmholtz coil, and a container or array of containers, wherein an opening in the coil is configured to accommodate the container or array of containers.

[0183] The above-mentioned container and the above-mentioned array of containers are defined in relation to the kit of the present invention. The term “to contain” means that the coil has an opening wide enough so that the container or array fits inside the opening, and as a result the contents of the container are positioned such that the magnetic field generated by the coil during use is particularly strong and / or particularly homogeneous. Preferably, and in the case of the container having a circular cross-section (for example, a cylindrical container), the inner diameter of the coil (in that case, a circular coil) is only slightly wider than the outer diameter of the container. “Slightly wider” could mean 0.01 to 10%, for example, 0.1 to 1% wider.

[0184] Similarly, in the case of an array of containers (e.g., microtiter plates), the coil may appear to be only slightly wider than the array. If the shape of the array is rectangular, a rectangular coil, such as a Helmholtz coil, may be used.

[0185] In a preferred embodiment, the device further comprises (iii) at least one magnetic material, preferably at least one magnetic material per container, or further comprises them.

[0186] Such a design provides a conductor or coil in which at least one of the magnetic materials is configured to be under the influence of a magnetic field generated by the conductor or coil during use.

[0187] It goes without saying that preferred embodiments of the magnetic material described above are disclosed herein, for example, in conjunction with the method of the first embodiment. Generally speaking, a preferred embodiment of one embodiment may be applied mutatis mutandis to specify a preferred embodiment of another embodiment.

[0188] In a more preferred embodiment, the device further includes, or comprises, a control unit configured to cause the at least one magnetic material to perform a fluctuating or oscillating motion during use. More specifically, the control unit is configured to deliver any of the preferred time profiles of current as detailed in relation to the methods of the present invention. The control unit may further include a power supply or adapter to be connected to an electrical plug.

[0189] In a further embodiment, the present invention relates to a computer implementation method for analyzing the mass spectrum obtained from a sample prepared by the method of the first or second embodiment, wherein the obtained fragments The present invention provides a method comprising the step of constructing sequences to obtain the above-mentioned protein or polypeptide sequences from which they are derived.

[0190] Preferably, such construction is carried out without the use of pre-known sequence information. To further illustrate, a common approach in mass spectral analysis is the use of a database of known protein fragments, such as trypsin fragments. The present invention enables complete and clear mapping of the entire protein sequence by providing fragments of a diverse range of sizes, thereby eliminating the need for such a database. The approach according to the present invention may also be referred to as de novo sequencing by mass spectrometry.

[0191] The drawing is shown below: [Brief explanation of the drawing]

[0192] [Figure 1] This figure shows the number of peptide or fragment identifications (IDs). Yeast and liver samples were prepared either by regular sample preparation (Reg. / standard) or by magnetic fragmentation (Mag. / standard). "Trypsin" peptides contain a K or R amino acid at their C-terminus, while "fragment" peptides can terminate with any other amino acid. [Figure 2] This figure shows the sequence coverage observed using various implementations of the present invention. [Figure 3] This is a diagram illustrating an exemplary distribution of fragment sizes obtained using the method described in Example 3. [Modes for carrying out the invention]

[0193] The examples illustrate the present invention. Examples [Examples]

[0194] Improvement of dissolution and digestion when applying the present invention method in addition to protein digestion. material Fresh Saccharomyces cerevisiae and mouse liver samples were used in quantities equivalent to 100 μg of protein. Buffers and enzymes from the iST kit (PO00001, PreOmics) were used throughout this experiment.

[0195] method Standard iST sample preparation: Sample preparation was performed according to the PreOmics standard protocol for yeast samples and the mammalian tissue protocol for liver samples. For cell lysis and protein denaturation, yeast pellets (approximately 100 μg protein content) were resuspended in 50 μl of lysis buffer, boiled at 95°C and 1000 rpm for 10 minutes, and sheared with a Diagenode Bioruptor (10 cycles, 30 seconds on, 30 seconds off). Liver samples (wet weight 1-2 mg with approximately 100 μg protein content) were resuspended in 100 μl of lysis buffer, sheared with glass beads using a Diagenode Bioruptor (10 cycles, 30 seconds on, 30 seconds off) to promote tissue lysis, and boiled at 95°C and 1000 rpm for 10 minutes. All samples were further processed according to the manufacturer's instructions. After elution, the purified peptides were dried by vacuum centrifugation and resuspended in an LC-Load. Samples were analyzed using a ThermoFisher Scientific Easy n-LC 1000 system coupled with a Thermo LTQ Orbitrap XL. Peptides were analyzed using a homemade C2C system with a 45-minute gradient applied. 18 Separate by column Tandem mass spectrometry was then performed using the DDA Top 10 method. The MS / MS data were retrieved against the yeast database using MaxQuant software with default settings, except for the selection of a nonspecific digestion mode.

[0196] iST sample preparation using cell lysis and digestion with a novel magnetic system: Yeast samples containing 100 μg of protein were resuspended in 50 μl of lysis buffer and first boiled at 95°C and 1000 rpm for 10 minutes, or directly mixed with 50 μl of trypsin / LysC solution. For cell lysis and protein digestion, the samples were incubated for 60 minutes at a magnetic flux density of approximately 1 mT and 120 Hz in a magnetic system with a 3 mm circular neodymium magnet. Next, 100 μl of stop buffer was added, and the peptides were purified and analyzed as described in standard iST sample preparation.

[0197] result See Figure 1.

[0198] essay The magnetic system significantly improves the overall peptide identification process for yeast cells and tissue samples. Compared to processes involving further boiling, this process is self-contained and does not require further boiling to achieve the best results. The system can be used directly and can replace traditional dissolution with the addition of digestive mixtures. The best results were achieved using the magnetic system in a direct combined dissolution and digestion configuration. [Examples]

[0199] Fragmentation of individual proteins by the method of the present invention (in the absence of any protease) material 50 μg of carbonic anhydrase (bovine erythrocytes; C7025-1VL) at a concentration of 10 mg / ml in ddH2O was used in the following experiments. For protein extraction and fragmentation, a prototype magnetic system with 50-winding Helmholtz coils was developed and performed using a 3 mm × 2 mm cylindrical samarium-cobalt magnet (MagnetExpert, F412SC-250). 1.2 mm steel beads or 1.4 mm ceramic beads were added as desired. For peptide purification, the iST kit was used (PreOmics, PO00001).

[0200] method The samples were incubated in a prototype magnetic system containing a cylindrical samarium-cobalt magnet and, if desired, steel or ceramic beads. The system was used for 60 minutes at a magnetic flux density of approximately 1 mT and 120 Hz. ThermoFisher Scientific Easy connected to LTQ Orbitrap XL. Fragmented peptides were directly analyzed using an n-LC 1200 system. The peptides were purified according to the iST manual and subjected to a manual C24 gradient with a 45-minute gradient applied. 18The samples were separated by column, and tandem mass spectrometry was performed using the DDA Top 10 method. The MS / MS data were searched against the carbonic anhydrase database using MaxQuant software with default settings, except for the selection of a nonspecific digestion mode.

[0201] result Peptides of various lengths were generated. Fragments with a difference of just one amino acid length are generally observed. This provides nearly complete coverage of all conceivable peptide fragment options within a given range of analysis. For example, sequences ANGERQSP, ANGERQSPV, ANGERQSPVD, ANGERQSPVDI, ANGERQSPVDID, ANGERQ Fragments such as SPVDIDT and ANGERQSPVDIDTK were measured as individual peptides.

[0202] See Figure 2. essay Using a magnetic system, proteins can be fragmented into virtually any conceivable peptide composition. With sample preparation time of just 1.5 hours from start to measurement, carbonic anhydrase was fragmented to generate over 95% peptide-based protein sequence coverage. Four combined experiments produced complete sequence coverage. The peptide fragments obtained using the method of this invention can be used to sequence proteins, as fragments of any length combination differing only by the mass of a single amino acid are generated.

[0203] Furthermore, by using these differences, the original sequence can be estimated. [Examples]

[0204] Protein fragmentation on a proteome-wide scale material A fresh Saccharomyces cerevisiae cell pellet containing approximately 100 μg was used in the following experiments. For protein extraction and fragmentation, a prototype magnetic system with 250 windings and a coil with an inner diameter of 12 mm was developed and performed using a 2 mm circular neodymium magnet. Peptide purification and preparation for LC-MS measurement were performed using iST cartridges and buffers (iST kit, PO00001) from PreOmics. Water was commercially available from Fisher Scientific (W6-212).

[0205] method Yeast pellets containing 100 μg of yeast protein were resuspended in 100 μl of ddH2O with pH 1, 4, 7, or 10, or in 90 μl of ddH2O with pH 1, 4, 7, or 10 and 10 μl of acetonitrile. For protein extraction and fragmentation, samples were incubated for 60 minutes in a magnetic system prototype with a 2 mm circular neodymium magnet, applying a magnetic flux density of approximately 1.5 mT and 120 Hz. Next, 100 μl of iST stop buffer was added, and the peptides were purified according to the manufacturer's protocol. The peptides were dried by vacuum centrifugation and resuspended in an LC-Load to a final concentration of 2.5 μg / μl. Samples were analyzed using a ThermoFisher Scientific Easy n-LC 1200 system coupled with an LTQ Orbitrap XL. A 5 μg peptide loading was subjected to a 45-minute gradient applied to a custom-made C1200 system. 18 The samples were separated by column, and tandem mass spectrometry was performed using the DDA Top 10 method. The MS / MS data were searched against a yeast database using MaxQuant software with default settings, except for the selection of a nonspecific digestion mode.

[0206] result See Figure 3.

[0207] essay The magnetic system used in this experiment produced peptides with an average length of 13.2 amino acids, longer than those produced by standard trypsin digestion (approximately 12 amino acids). This system allows for the generation of suitable peptide fragments across a wide pH range and even in the presence of organic solvents.

Claims

1. A method for preparing a sample for an analytical procedure, wherein the sample comprises at least one protein, polypeptide, or peptide molecule, and the method comprises (a) fragmenting the molecule using at least one moving magnetic body, wherein the at least one moving magnetic body performs a fluctuating motion or an oscillating motion, preferably the motion being induced by a fluctuating magnetic field or an oscillating magnetic field.

2. The method according to claim 1, wherein the magnetic field is generated by an electric current and / or an electromagnet.

3. (i) Whether the fragmentation is a non-enzymatic and non-chemical process, (ii) The method according to claim 1 or 2, wherein a chemical selected from CNBr, formic acid, hydroxylamine, and 2-nitro-5-thiocyanobenzoic acid and / or protease is added.

4. The method according to any one of claims 1 to 3, wherein the magnetic material collides with the molecules and / or at least one non-magnetic particle is present, and the motion of the magnetic material induces a collision between the at least one non-magnetic particle and the molecules.

5. The sample has a biological origin, preferably, (i) The sample is a solution or suspension of the molecule, or comprises a solution or suspension of the molecule, for example, the sample comprises a mixture of the molecule, protein, polypeptide and / or peptide in a purified form, or is a body fluid such as blood, serum, plasma, cerebrospinal fluid, sputum or urine, or comprises a body fluid such as blood, serum, plasma, cerebrospinal fluid, sputum or urine, (ii) The sample is a cell such as a prokaryotic cell or a eukaryotic cell, or contains a cell such as a prokaryotic cell or a eukaryotic cell, for example, the sample is a cell suspension, or contains a cell suspension (iii) The sample is a virus or contains a virus, for example, the sample is a suspension of a virus or contains a suspension of a virus, and / or (iv) The sample is tissue, for example, muscle tissue or brain tissue, or includes tissue, for example, muscle tissue or brain tissue. The method according to any one of the prior claims.

6. The method according to any one of the preceding claims, wherein the method further comprises (b) a step of exposing the sample to heat such as boiling, a step of denaturing the sample, a step of adding a cleaning agent to the sample, and / or a step of adding a chaotropic agent to the sample, preferably step (b) is carried out prior to or simultaneously with step (a).

7. (c) The method according to any one of the preceding claims, further comprising the step of chemically modifying the molecule and / or a fragment obtained from the molecule.

8. The method according to any one of the preceding claims, wherein the analytical procedure is mass spectrometry (MS).

9. The aforementioned chemical modification step, (Ca) Disulfide reduction process, (cb) A step of alkylating thiol groups such as cysteine ​​residues, (cc) A cross-linking process, and / or Any combination of (cd), (ca), (cb), and (cc) (combination of (ca) and (cb) (Preferably) The method according to claim 7, wherein steps (a), (ca), and (cb) are selected from and preferably carried out simultaneously.

10. The method according to any one of the preceding claims, wherein an inert viscous liquid; a gel such as a polyacrylamide gel or agarose gel; an aerogel and / or zeolis is added to the sample.

11. The method according to any one of the preceding claims, further comprising (d) purifying and / or concentrating the obtained fragments, preferably by filtration, non-covalent bonding and / or covalent bonding, wherein non-covalent bonding is preferably performed on a reversed-phase material, a normal-phase material, an ion-exchange material, an affinity-bonding material, a chelating material or a paramagnetic particle, and covalent bonding is preferably performed using a reagent capable of forming a conjugate with an amine group of any of the fragments.

12. The method according to any one of the preceding claims, further comprising the step of (e) labeling the molecule and / or a fragment obtained from the molecule, preferably the step of labeling the fragment being achieved after the step of purifying and / or concentrating the fragment.

13. The method according to claim 12, wherein the labeling step is performed by reacting the functional group of the molecule with a reagent capable of forming a conjugate with the functional group, and the reagent capable of forming a conjugate with the functional group is preferably a tag detectable by mass spectrometry.

14. An analytical method comprising the method according to any one of the prior claims, and the step of performing mass spectrometry on the obtained fragment.

15. A method for identifying a site on a first protein that is capable of or likely to be capable of binding to a second protein or binding partner, the method comprising: fragmenting the first protein using at least one mobile magnetic body; adding the second protein or binding partner; separating the fragments that bind to the second protein or binding partner from the unbound fragments; identifying the fragments that bind to the second protein or binding partner, thereby identifying the site, wherein the at least one mobile magnetic body performs a fluctuating or oscillating motion, preferably the motion is induced by a fluctuating or oscillating magnetic field, preferably the first protein is an antigen and the second protein is an antibody.

16. The use of magnetic materials and magnetic means to generate fluctuating or oscillating magnetic fields for fragmenting protein, polypeptide, or peptide molecules.

17. (i) at least one magnetic material; (ii) A container or array of containers configured to receive a sample comprising the magnetic material and at least one protein, polypeptide, or peptide molecule. A kit containing or consisting of these.

18. (iii) Reducing agent; and (iv) Alkylating agent; and optionally, (v) one, more or all of the following: surfactants, chaotropic agents, denaturants, and organic solvents; (vi) at least one buffer solution; (vii) Nonmagnetic particles; and / or (viiii) A manual including instructions for carrying out the method described in any one of claims 1 to 15. The kit according to claim 17, further comprising or comprising the following.

19. (i) a coil, preferably a Helmholtz coil; and (ii) A container or an array of containers; (iii) A control unit configured to cause the at least one magnetic material to perform a fluctuating motion or an oscillating motion when in use; and optionally (iii) at least one magnetic material, preferably at least one magnetic material per container A device that includes or consists of the following: A device in which the opening of the coil is configured to accommodate a container or an array of containers.

20. A computer implementation method for analyzing a mass spectrum obtained from a sample prepared by any one of claims 1 to 15, comprising the step of constructing a sequence of the obtained fragments to obtain a sequence of the protein or polypeptide from which they are derived.

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