Systems and methods for improved sequence coverage in large polypeptide analysis

Abstract

In some embodiments, methods for increasing polypeptide sequence coverage include removing inter-chain disulfide bonds from a first sample and retaining substantially all intra-chain disulfide bonds, and performing a first mass spectrometry (MS) analysis on the first sample to determine a first amino acid sequence, the interchain and intrachain disulfide bonds of the second sample are removed and a second MS analysis is performed to determine a second amino acid sequence, and the first and second sequences are combined to determine a combined amino acid sequence of the polypeptide.

Description

[0001] Related US applications

[0002] This application claims the benefit of priority to U.S. Provisional Application Nos. 63 / 442,645 and 63 / 466,880, filed February 1, 2023 and May 16, 2023, respectively, the entire contents of which are hereby incorporated by reference. Technical Field

[0003] The present disclosure relates to polypeptide sequence measurement in mass spectrometry, and in particular to improved sequence coverage in large polypeptide analysis. Background Art

[0004] Mass spectrometry (MS) is an analytical technique used to determine the elemental composition of a substance. Specifically, MS measures the mass-to-charge ratio (m / z) of ions produced by a test substance. MS can be used to identify unknown compounds, determine the isotopic composition of elements in a molecule, determine the structure of a specific compound by observing its fragmentation, and quantify the amount of a specific compound in a sample. Mass spectrometers detect ions, so during mass analysis, the test sample must be converted into ionic form.

[0005] When a polypeptide passes through an MS system and undergoes fragmentation, the different m / z values ​​of the polypeptide's fragments can be measured as distinct peaks in the spectrum. These peaks can be interpreted and attributed to specific fragment ions originating from the polypeptide. In this process, the sequence of the polypeptide can be determined.

[0006] Conventionally, large polypeptides are enzymatically digested into smaller peptides before sequence analysis by mass spectrometry. This analytical approach is known as a bottom-up approach. This approach generally requires fewer fragments for sequence reconstruction, making it easier to analyze by mass spectrometry. However, this approach includes an additional preliminary step of enzymatic digestion of the polypeptide, which can be time-consuming and can also be prone to introducing artifacts.

[0007] Alternatives to the bottom-up approach include direct sequencing of large polypeptides (top-down approach) or performing a limited proteolytic digestion step (middle-down approach) without the risk of introducing artifacts. However, all of these approaches have drawbacks in the analysis of large polypeptides, such as proteins, which can have a complex structure with a primary structure and optionally secondary structure, tertiary structure, and even higher levels of structure often connected via mechanisms such as inter- and intra-chain disulfide bonds. Therefore, it is important to devise methods for optimally sequencing such molecules in innovative ways to increase sequence coverage.

[0008] Summary of the Invention

[0009] Some embodiments relate to a method for sequencing a polypeptide, the method comprising: removing one or more interchain disulfide bonds and retaining substantially all intrachain disulfide bonds of a first sample of the polypeptide to produce a first set of subunits; performing a first mass spectrometry (MS) analysis on the first set of subunits to determine a first amino acid sequence of the polypeptide, removing one or more interchain disulfide bonds and one or more intrachain disulfide bonds of a second sample of the polypeptide to produce a second set of subunits; performing a second MS analysis on the second set of subunits to determine a second amino acid sequence of the polypeptide; and combining the first amino acid sequence and the second amino acid sequence to determine a combined amino acid sequence of the polypeptide.

[0010] Some embodiments relate to methods in which a fragmentation process is utilized to determine the amino acid sequence.

[0011] Some embodiments relate to methods wherein the fragmentation scheme is selected from one of electron activated dissociation and electron capture dissociation, ultraviolet light dissociation, and electron transfer dissociation.

[0012] Some embodiments concern methods wherein removing one or more interchain disulfide bonds comprises removing substantially all interchain disulfide bonds.

[0013] Some embodiments concern methods wherein the second sample is the same as the first sample.

[0014] Some embodiments relate to methods wherein the first sample and the second sample are subsamples of the same sample.

[0015] Some embodiments concern methods wherein the first sequence comprises at least one segment from the middle portion of the polypeptide.

[0016] Some embodiments relate to methods wherein the second sequence comprises at least one moiety blocked by an intrachain disulfide bond.

[0017] Some embodiments relate to methods wherein the polypeptide is a protein.

[0018] Some embodiments relate to methods wherein the polypeptide is an antibody.

[0019] Some embodiments relate to the method wherein the antibody is one of a monoclonal antibody (mAb), a bispecific antibody (bsAb), and an antibody drug conjugate (ADC).

[0020] Some embodiments relate to methods wherein tandem mass spectrometry is used for MS analysis.

[0021] Some embodiments relate to methods wherein disulfide bonds are removed by using a chemical agent.

[0022] Some embodiments relate to methods wherein the chemical agent is selected from DTT (dithiothreitol), TCEP (tris(2-carboxyethyl)phosphine), or mercaptoethanol.

[0023] Some embodiments relate to methods wherein the removal of disulfide bonds is performed electrochemically.

[0024] Some embodiments relate to methods wherein enzymatic digestion of the sample is performed prior to removal of disulfide bonds.

[0025] Some embodiments relate to methods wherein the enzyme performing the enzymatic digestion is one of IdeS, IgdE, IdeZ, Kgp, and SpeB.

[0026] Some embodiments concern methods wherein removing one or more interchain disulfide bonds of the first sample comprises removing substantially all interchain disulfide bonds.

[0027] Some embodiments relate to methods wherein removing one or more interchain disulfide bonds and one or more intrachain disulfide bonds of the second sample comprises removing substantially all interchain and intrachain disulfide bonds.

[0028] Some embodiments relate to a method for polypeptide sequencing, the method comprising: connecting a portion of a polypeptide sample using a chemical reaction; and performing MS analysis on the sample to determine the amino acid sequence of the polypeptide.

[0029] Some embodiments relate to methods that further perform MS analysis on a second sample of the polypeptide without utilizing a chemical reaction to determine a second amino acid sequence of the polypeptide, and combine the amino acid sequence with the second amino acid sequence to determine a combined amino acid sequence of the polypeptide.

[0030] Some embodiments relate to methods wherein the chemical reaction is a reaction with a cross-linking agent.

[0031] Some embodiments relate to methods wherein the chemical reaction is a reaction with a metal cation.

[0032] Some embodiments relate to a method for biopolymer sequencing, the method comprising: utilizing physicochemical interactions to link a portion of a biopolymer sample; and performing MS analysis on the sample to determine the amino acid sequence of the biopolymer.

[0033] Some embodiments relate to methods that further perform MS analysis on a second sample of the biopolymer without utilizing physicochemical interactions to determine a second amino acid sequence of the polypeptide, and combine the amino acid sequence with the second amino acid sequence to determine a combined amino acid sequence of the polypeptide.

[0034] Some embodiments relate to methods wherein the physicochemical interaction is an ion-ion interaction.

[0035] Some embodiments relate to methods wherein the ion-ion interaction is the result of an ion-ion reaction.

[0036] Some embodiments relate to methods wherein the ion-ion reaction is performed in the gas phase.

[0037] Some embodiments relate to methods wherein the reactant polypeptide is a polypeptide having an opposite charge to the analyte biopolymer.

[0038] Some embodiments relate to the method wherein the biopolymer is one of a polypeptide and a polynucleotide.

[0039] Some embodiments concern methods wherein the subunit polypeptide has a mass greater than 20 kDa.

[0040] Some embodiments relate to methods and 24 wherein the analyte polypeptide has a mass greater than 20 kDa.

[0041] Some embodiments relate to methods and 32 wherein the sequence comprises at least a portion of the middle of a protein.

[0042] Some embodiments relate to a method for sequencing a middle portion of a polypeptide, the method comprising: removing substantially all interchain disulfide bonds of a sample of the polypeptide and retaining substantially all intrachain disulfide bonds to produce subunits, and performing mass spectrometry (MS) analysis on the subunits to determine the amino acid sequence of the middle portion of the polypeptide.

[0043] Some embodiments relate to methods wherein the middle portion of the polypeptide spans a portion greater than 50 amino acids from the terminus of the polypeptide.

[0044] Some embodiments relate to methods wherein the middle portion of the polypeptide spans a portion greater than 70 amino acids from the terminus of the polypeptide.

[0045] Some embodiments relate to methods wherein the middle portion of the polypeptide spans a portion greater than 100 amino acids from the terminus of the polypeptide.

[0046] Some embodiments relate to a method for sequencing polypeptides from a sample, the method comprising: removing one or more interchain disulfide bonds and retaining substantially all intrachain disulfide bonds from a first portion of the sample of the polypeptides to produce a first group of subunits; removing one or more interchain disulfide bonds and one or more intrachain disulfide bonds from a second portion of the sample of the polypeptides to produce a second group of subunits; separating the first group of subunits from the second group of subunits; performing mass spectrometry (MS) analysis on the first group of subunits to determine a first amino acid sequence of the polypeptides; performing a second MS analysis on the second group of subunits to determine a second amino acid sequence of the polypeptides; and combining the first amino acid sequence and the second amino acid sequence to determine a combined amino acid sequence of the polypeptides.

[0047] Various aspects of the embodiments may be further understood by reference to the following detailed description taken in conjunction with the associated drawings, which are briefly described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The drawings are not necessarily drawn to scale or to be exhaustive. Instead, emphasis is generally placed on illustrating the principles of the embodiments described herein. The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate several embodiments consistent with the present disclosure. Together with the description, the drawings serve to explain the principles of the present disclosure.

[0049] In the attached figure:

[0050] Figure 1A is a schematic representation of a polypeptide 100 according to some embodiments.

[0051] Figure 1B A method 150 of preparing two samples for mass spectrometry analysis of a polypeptide 100 is schematically illustrated according to some embodiments.

[0052] Figure 1C A preparation method 160 is shown utilizing a reduction protocol without IdeS digestion, according to some embodiments.

[0053] Figure 1D Schematic diagrams illustrating polypeptides of other examples of middle portions according to some embodiments are shown.

[0054] Figure 2 is a flow chart of a method 200 for sequencing a polypeptide based on a product of a preparation method according to some embodiments.

[0055] Figure 3 is a graphical representation of the results of sequencing fully reduced polypeptide subunits according to some embodiments.

[0056] Figure 4 is a graphical representation of the results of sequencing partially reduced polypeptide subunits according to some embodiments.

[0057] Figure 5 and Figure 6 The effect that intrachain disulfide bonds can have on the dissociation of polypeptides according to some embodiments is shown.

[0058] Figure 7 is a schematic diagram of the dissociation of three polypeptides 710, 720, and 730, according to some embodiments.

[0059] Figure 8A is an alternative schematic diagram of an intrachain disulfide bond, according to an embodiment.

[0060] Figure 8BA structure is shown in which the peptide backbones are linked using cross-linker 810.

[0061] Figure 8C Shown is a reaction between a cross-linker 820 and a polypeptide 830 according to some embodiments.

[0062] Figure 8D An exemplary reaction according to such embodiments is shown, wherein the reaction between polypeptide 830 and metal 850 forms metal-polypeptide complex 860 .

[0063] Figure 8E The formation of additional connections according to some embodiments is shown.

[0064] Figure 9 A flow chart is shown of a method 900 for improving top-down sequence coverage using chemical reactions with reagents, according to some embodiments.

[0065] Figure 10 Shown is a flow chart of a method 1000 for performing physicochemical interactions to achieve top-down sequence coverage improvement, according to some embodiments.

[0066] Figure 11 A liquid chromatogram is shown, which shows the separation of the two components and the mass analysis of each component.

[0067] Detailed description of the invention

[0068] It should be understood that, for the sake of clarity, the following discussion will set forth various aspects of the embodiments of the applicant's teachings, while omitting certain specific details where it is convenient or appropriate to do so. For example, the discussion of similar or similar features in alternative embodiments may be slightly omitted. For the sake of brevity, well-known ideas or concepts may also not be discussed in detail. Those skilled in the art will recognize that some embodiments of the applicant's teachings may not require the details of certain specific descriptions in each embodiment, and the details are set forth herein only to provide a thorough understanding of the embodiments. Similarly, it will be apparent that, without departing from the scope of this disclosure, the described embodiments may be susceptible to change or variation according to common common sense. The following detailed description of the embodiments should not be considered to limit the scope of the applicant's teachings in any way.

[0069] The following detailed description refers to the accompanying drawings. The same or similar reference numerals may be used in the accompanying drawings or the specification to represent the same or similar parts. In addition, unless otherwise specified, similarly named elements can perform similar functions and can be similarly designed. Details are set forth to provide an understanding of the exemplary embodiments. Embodiments can be implemented without some of these details, such as alternative embodiments. In other cases, well-known technologies, processes, and components are not described in detail to avoid obscuring the described embodiments.

[0070] In this disclosure, the following terms may be used along with the following corresponding definitions.

[0071] Tandem mass spectrometry (MS / MS) is a method in which mass spectrometry analysis can be performed in multiple stages, for example, starting with the isolation of a precursor ion, which can then undergo an ion reaction or fragmentation, and then the resulting products are detected in a second mass spectrometry experiment. The same mass analyzer can be used to perform both experiments, or multiple mass analyzers can be placed in series to perform different experiments.

[0072] Fragmentation is a type of ionic reaction in which the reaction products include fragments of the ion.

[0073] A polypeptide is a polymer of amino acids linked together by peptide bonds. In some cases, multiple polypeptides can be linked to each other covalently (e.g., by disulfide bonds) or non-covalently to form a larger polypeptide. In some cases, each of these polypeptides can be considered a subunit of a larger polypeptide.

[0074] A polypeptide sequence or sequence is a sequence of amino acids that extends from one end to the other in a polypeptide in a specific order. In the case of a complex polypeptide containing multiple polypeptide chains, the sequence may include the sequence of each polypeptide chain.

[0075] The term protein is used interchangeably with polypeptide and refers to large polypeptides with an atomic mass greater than 10 kDa.

[0076] Bottom-up mass spectrometry is an MS sequencing method in which intact proteins are digested into peptides before introduction into a mass spectrometer for sequencing.

[0077] Top-down mass spectrometry is an MS method in which proteins are introduced directly into a mass spectrometer for sequencing without first being digested.

[0078] Mid-down mass spectrometry is an MS method based on limited proteolytic digestion of proteins to produce larger polypeptides with an atomic mass of approximately 20 kDa, which are then introduced into a mass spectrometer for sequencing.

[0079] Collision-induced dissociation (CID) is a mass spectrometry technique that deposits energy into ions by passing them into a gas bath, thereby inducing fragmentation of the target ions in the gas phase.

[0080] Electron capture dissociation (ECD) is a fragmentation technique in which peptide ions react with free, low-energy electrons, resulting in electron capture, energy release, and in some cases, dissociation of the ion.

[0081] Electron transfer dissociation (ETD) is a fragmentation technique in which a peptide cation reacts with a reagent anion, followed by electron transfer, resulting in a fragmentation process similar to ECD.

[0082] UV photodissociation (UVPD) is a method in MS where ions are excited by high-energy photons, causing them to fragment.

[0083] The terminal portion of a polypeptide can include a portion adjacent to the N- or C-terminus of the polypeptide.

[0084] The middle portion of a polypeptide can include the portion beginning about 50 amino acids away, or, when the polypeptide includes an intrachain disulfide bond or other linkage, the portion extending from the inner end (away from the end) of the intrachain disulfide bond or linkage.

[0085] Complete reduction refers to the removal of substantially all interchain and intrachain disulfide bonds of a polypeptide.

[0086] Partial reduction refers to the removal of substantially all interchain disulfide bonds while retaining substantially all intrachain disulfide bonds of the polypeptide.

[0087] Next, various embodiments are described in detail. In recent years, top-down and middle-down MS have continued to gain more attention. An exemplary area of ​​interest is the sequence analysis of monoclonal antibodies (mAbs). For certain mAb assays, close to 100% sequence coverage is often required. Conventional bottom-up methods using multiple proteases can meet this demand, but can be time-consuming and error-prone. On the other hand, existing top-down and middle-down methods can only result in partial sequence coverage, which is mainly due to poor sequence coverage in the middle part of the protein. Some embodiments aim to improve this situation.

[0088] Typically, CID fragmentation can be used for both top-down and middle-down analysis. However, sequencing methods based on CID fragmentation are known to suffer from incomplete sequence coverage due to rather preferential fragmentation of certain sites.

[0089] Other fragmentation techniques, such as ECD, ETD, and UVPD, can be used to perform top-down and middle-down analyses and can produce better sequence coverage.

[0090] Some improved techniques according to various embodiments are described in conjunction with the following figures.

[0091] Figure 1A is a diagrammatic representation of a polypeptide 100 according to some embodiments. The polypeptide 100 can be, for example, a mAb. Figure 1A As shown, polypeptide 100 may include multiple polypeptide chains, such as heavy chain 114 and light chain 115. In addition, polypeptide 100 may include multiple intrachain disulfide bonds 112 and multiple interchain disulfide bonds 113. As shown, intrachain disulfide bonds 112 can form linkages between amino acids within the same chain, while interchain disulfide bonds 113 can form linkages between different polypeptide chains. In addition, polypeptide 100 may also include a hinge region 168, which connects its antigen-binding fragment (Fab) to its crystallizable fragment (Fc).

[0092] Figure 1B Schematically illustrated is a method 150 of preparing two samples for mass spectrometry analysis of a polypeptide 100, according to some embodiments. Method 150 illustrates such preparation for mid-down analysis of antibodies using techniques such as ECD, ETD, and UVPD of mAbs and involving a step of limited proteolytic digestion as further described below.

[0093] like Figure 1B As shown, method 150 begins at step 151, where a stock solution of an antibody sample is obtained. The stock solution can then be subjected to an optional limited proteolytic digestion step (e.g., IdeS digestion; (step 152)), which can convert the intact antibody sample into a digested sample 153, including some subunit types. IdeS (immunoglobulin degrading enzyme from Streptococcus pyogenes) is an engineered recombinant protease overexpressed in Escherichia coli. IdeS digestion can specifically cleave IgG molecules below the hinge region 168 to produce a digested sample 153, which can include F(ab')2 (antigen binding fragment) and Fc (crystallizable fragment), such as Figure 1B shown.

[0094] Next, at step 156, a first subsample can be removed from the digested sample 153 and exposed to a complete reduction process to produce a first set of subunits 140. In some embodiments, during the complete reduction process, substantially all interchain and intrachain disulfide bonds are removed. In some other embodiments, a high percentage of these bonds can be removed, and less than 10% of the subsample can contain one or more disulfide bonds. Detailed techniques for performing the complete reduction are described below.

[0095] like Figure 1BAs shown, a first set of subunits 140, resulting from complete reduction of the first subsample, may include subunits comprising completely reduced polypeptides, such as LC (light chain), Fd' (heavy chain portion of Fab), and Fc / 2.

[0096] Alternatively, at step 158, a second subsample can be removed from the digested sample 153 and exposed to a partial reduction process to produce a second set of subunits 159. In some embodiments, during the partial reduction process, substantially all or a high percentage of the interchain disulfide bonds 113 can be removed, leaving substantially all or a high percentage of the intrachain disulfide bonds 112 intact. Some more detailed techniques for performing partial reduction are described below. Figure 1B As shown, the second set of subunits 159 may include subunits such as LC, Fd', and Fc / 2 having intact intrachain disulfide bonds.

[0097] Some embodiments utilize a preparation method that includes a reduction protocol without IdeS digestion, thereby generating subunit sets that include light and heavy chains. Figure 1C A preparation method 160 utilizing this reduction scheme without IdeS digestion is shown according to some embodiments. In method 160, unlike in method 150, the starting sample may not be subjected to IdeS digestion. Instead, as described above, a first subsample and a second subsample may be taken from the starting sample and exposed to a complete reduction and a partial reduction process, respectively. As a result, the resulting subunit group may include not only a significant percentage of light chains 161, but also a significant percentage of heavy chains 162, such as Figure 1C shown.

[0098] Some embodiments enable sequencing of different portions of a polypeptide, such as the middle portion. The middle portion 170 of the polypeptide, Figure 1B and 1C In the case of a light chain such as shown, it can be defined as the portion of the polypeptide between the two intrachain disulfide bonds. In the case of a heavy chain such as heavy chain 162' (which is produced by partial reduction), Figure 1C As shown, the middle portion 170 can be defined as the portion of the polypeptide between the two outer intrachain disulfide bonds 112a and 112d, but excluding the portion blocked by intrachain disulfide bonds 112b and 112c.

[0099] Figure 1D Schematic diagrams illustrating other examples of polypeptides according to some embodiments of the middle portion are shown. In FIG1D , the polypeptide is shown as a straight line connecting the N-terminus and the C-terminus. Figure 1D As shown, in various embodiments, the middle portion of a polypeptide can be defined as the portion of the polypeptide that is 50, 70, or 100 amino acids (AA) away.

[0100] In some embodiments, the products of the above preparation methods can be subsequently subjected to MS analysis to sequence the polypeptide. Figure 2 is a flow chart of a method 200 for sequencing a polypeptide based on a product of a preparation method according to some embodiments.

[0101] In step 210, a sample (such as a protein sample) is subjected to limited proteolytic digestion, which can produce a sample of large polypeptides. This process corresponds to Figure 1B 152 of method 150. The method then bifurcates and separates into two different pathways for performing two different sets of operations on the two subsamples of the sample.

[0102] Considering the first approach, in step 220, in the first subsample, both interchain and intrachain disulfide bonds are removed and a first set of subunits is obtained. This step corresponds to Figure 1B Step 156 of method 150.

[0103] In step 222, ECD*MS / MS is performed to sequence the first group of subunits. As a result, a first sequence corresponding to the first group of subunits is obtained in step 224. The asterisk in ECD* indicates that other techniques such as ETD or UVPDMS / MS can be used instead of ECD MS / MS.

[0104] Next, considering the second approach, in step 230, in a second subsample of polypeptides, interchain disulfide bonds are removed while intrachain disulfide bonds are retained, thereby obtaining a second set of subunits. This process corresponds to Figure 1B Step 158 of method 150.

[0105] In step 232, ECD*MS / MS is performed to sequence the second set of subunits. As a result, in step 234, a second sequence corresponding to the second set of subunits is obtained.

[0106] At step 240, the first sequence and the second sequence are combined to obtain a combined sequence corresponding to the protein in the sample.

[0107] It should be noted that method 200 can start with a protein or a portion of a protein or a large polypeptide and the method can still follow the same steps of preparing reduced subunits, obtaining the sequences of these subunits, and combining the sequences to obtain a combined sequence.

[0108] For the example of the preparation of IDES subunits of NIST mAbs having (a) fully reduced disulfide bonds during full reduction and (b) partially reduced disulfide bonds during partial reduction, the method of removing interchain and intrachain disulfide bonds can be performed according to the following steps.

[0109] (a) Preparation of the fully reduced NIST mAb IdeS subunit:

[0110] a. Mix 40 μL of NIST mAb stock solution (10 μg / μL) with 360 μL of water to produce NIST mAb (1 μg / μL).

[0111] b. Add 2 μL of LideS enzyme (25 units / μL) to 50 μL of NIST mAb (1 μg / μL) and incubate the mixture at 37°C for 2 hours.

[0112] c. Add 157 μL of denaturing reagent (at 50 mM

[0113] 7.2 guanidine in Tris, pH 7-8) and vortex.

[0114] d. Add 23 μL of 1 M DTT to the mixture and incubate at 60°C for 30 minutes.

[0115] (b) Preparation of partially reduced NIST mAb subunits:

[0116] e. Mix 10 μL of NIST mAb stock solution (10 μg / μL, 67 pmol / μL) with

[0117] 119.3 μL of 0.01M PBS buffer was mixed to produce NIST mAb

[0118] diluent (5 pmol / μL).

[0119] f. Add 4 μL of deS enzyme (25 units / μL) and incubate the mixture at 37°C

[0120] 60 minutes.

[0121] g. After incubation, 10 μL of reducing agent (TCEP, 50 mM) was added to the solution.

[0122] Incubate at 37 °C for 30 min.

[0123] h. Add 4.1 μL of 243 mM iodoacetamide and incubate at 37°C in the dark for 30 minutes.

[0124] The above techniques can result in high yields of partially or fully reduced subunits. For example, in some embodiments, the yield is greater than 90%.

[0125] Figure 3 In accordance with method 200 (reference Figure 2) of the embodiment described in steps 220, 222 and 224, the fully reduced polypeptide subunit Fd' (reference Figure 1B ) is a graphic representation of the sequencing results. Figure 3 The top portion of the figure shows the amino acid sequence of an exemplary subunit NIST mAb having a breakage site identified in a complete reduction experiment according to some embodiments. The light-colored boxed region 311 at the top represents amino acids belonging to the portion of the sequence blocked by intrachain disulfide bonds before complete reduction. The figure shows good sequence coverage of region 311, but poor sequence coverage of the middle portion 310 (black boxed region) of the polypeptide subunit.

[0126] Figure 3 The bottom of the figure is a graph of the number of fragment ions identified in the middle portion 310 (ie, the region between the two intrachain disulfide bonds at the different cleavage sites of the fully reduced Fd' subunit, respectively). Figure 3 The top black box region shows poor fragmentation identification in the middle portion 310 of the polypeptide subunit.

[0127] Figure 4 In accordance with method 200 ( Figure 2 ) of the embodiment described in steps 230, 232 and 234, the partially reduced polypeptide subunit Fd' (reference Figure 1B ) is a graphic representation of the sequencing results. Figure 4 The upper portion of FIG. 5 shows the amino acid sequence of an exemplary subunit NIST mAb having a cleavage site identified in a partial reduction experiment according to some embodiments. Figure 4 The dark-boxed region at the top represents amino acids belonging to the middle portion 410, which is not blocked by intrachain disulfide bonds. The light-boxed region 411 at the top of the figure represents amino acids belonging to the portion of the sequence blocked by intrachain disulfide bonds. The figure also shows the poor sequence coverage in region 411 covered by intrachain disulfide bonds.

[0128] Figure 4 is a graphical representation of the number of fragment ions identified in the middle portion 410 (i.e., the region between the two intrachain disulfide bonds at the different cleavage sites of the partially reduced Fd' subunit, respectively). As shown, the graph demonstrates good fragment identification of the middle portion of the polypeptide subunit.

[0129] Therefore, if Figure 3 and Figure 4 As demonstrated in , according to various embodiments, combining sequences of fully reduced and partially reduced subunits can provide improved polypeptide sequence coverage. Specifically, various embodiments provide methods for improving sequence coverage of the middle portion of a polypeptide, resulting in a more complete sequence of the polypeptide as a whole.

[0130] In some embodiments, the amino acid sequence can be determined using fragmentation techniques, which can include electron activated dissociation, ECD as described above, ETD, or UVPD.

[0131] In some embodiments, the fully reduced subunit and the partially reduced subunit are produced from portions of the same original sample of protein.Thus, the fully reduced sample and the partially reduced sample are subsamples of the same sample.

[0132] In some embodiments, the protein being sequenced is an antibody, which can be a monoclonal antibody (mAb), a bispecific antibody (bsAb), or an antibody drug conjugate (ADC). The disclosed embodiments can be applied to these situations and provide improved sequence coverage.

[0133] In some embodiments, disulfide bonds are removed by using a chemical agent. Examples of suitable chemical agents may include DTT (dithiothreitol), TCEP (tris(2-carboxyethyl)phosphine), or mercaptoethanol.

[0134] In some embodiments, removing disulfide bonds is performed electrochemically.

[0135] In some embodiments, enzymatic digestion of the sample is performed prior to removing disulfide bonds. In some embodiments, the enzyme performing the enzymatic digestion can be IdeS, IgdE, IdeZ, Kgp, or SpeB.

[0136] It should be noted that the mAb examples given in some embodiments are for illustrative purposes and that the methods of these embodiments can be applied to different types of proteins, protein portions, or polypeptides.

[0137] Figure 5 and Figure 6 The effect that intrachain disulfide bonds can have on the dissociation of polypeptides according to some embodiments is shown.

[0138] More specifically, Figure 5 Shown is polypeptide 520 dissociating at fragment 526. As a result, two product ions 522 and 524 are generated.

[0139] On the other hand, Figure 6 A polypeptide 620 undergoing fragmentation is shown according to some other embodiments. Polypeptide 620 includes a backbone ion 622 in which a break 626 is generated. Polypeptide 620 includes an intrachain disulfide bond 625 connecting two portions of backbone ion 622 separated by break 626. Due to the presence of this linkage of intrachain disulfide bond 625, after dissociation, a single product ion 622 is generated. According to some embodiments, the linked polypeptide is indicated as Figure 6Improved polypeptide sequence coverage is provided in the polypeptide portion of portion 623.

[0140] Thus, because the additional linkage provided by intrachain disulfide-bonded polypeptides is beneficial, in some additional embodiments, different techniques are used to achieve similar advantages.

[0141] Figure 7 7 is a schematic diagram of the dissociation of three polypeptides 710, 720, and 730, as further described below and according to some embodiments. More specifically, polypeptide 710 is a linear polypeptide that undergoes a single dissociation via break 716. On the other hand, polypeptide 720 is a linear polypeptide that has undergone multiple dissociations via breaks 726 and 728. Finally, polypeptide 730 is a nonlinear polypeptide due to the presence of an additional link 735 and similarly undergoes multiple dissociations via breaks 726 and 728.

[0142] Peptides 710 and 720 illustrate how multiple fragmentation events can redistribute the total ionic intensity from the middle portion of the polypeptide to the outer portion of the polypeptide. In particular, in polypeptide 710, break 716 produces two fragments 712 and 714. Fragments 712 and 714 are both long fragments.

[0143] On the other hand, in polypeptide 720, two breaks 726 and 728 produce a long fragment 724, a short terminal fragment 721, and a short internal fragment 722 due to secondary fragmentation.

[0144] According to some embodiments, polypeptide 730 having a connection (such as additional connection 735) can substantially protect longer fragments from secondary fragmentation. This will correspondingly improve sequence coverage during MS / MS sequencing because the additional connection provides a linkage between the short end fragment 721 and the internal fragment 722.

[0145] According to these embodiments, providing additional linkages improves the sequence coverage of the middle portion 733 .

[0146] Figure 8A is an alternative schematic diagram of an intrachain disulfide bond according to an embodiment. In this embodiment, the distance between the cysteine ​​residues forming the disulfide bond is shown closer, which is consistent with Figure 6 In this more realistic illustration, the peptide backbones are shown forming a knot. This illustration helps illustrate an embodiment in which artificial connections between the peptide backbones are introduced as described below.

[0147] Figure 8BA structure is shown in which a cross-linker 810 is used to connect peptide backbones. In this embodiment, cross-linker 810 is designed to react with a specific set of amino acid residues. In some embodiments, lysine is used as the target amino acid given its relatively high prevalence of approximately 6%. In some embodiments, N-hydroxysuccinimide esters or sulfosuccinimide esters have been reported as cross-linkers in which lysine is targeted.

[0148] Figure 8C Shown is a reaction between a cross-linking reagent 820 and a polypeptide 830, which provides cross-links 822. After this process, the resulting analyte resembles a polypeptide with intrachain disulfide bonds. The cross-linking reagent correspondingly increases the sequence coverage of the polypeptide.

[0149] In this case, a first mass spectrometry analysis can be performed on a subsample that did not react, and another mass spectrometry analysis can be performed on a subsample that did react. Alternatively, only the subsample that reacted can be analyzed, which, according to some embodiments, improves sequence coverage in a specific region of interest. These embodiments may not require the sample to have disulfide bonds and can be applied to other proteins and peptides.

[0150] In some embodiments, the reactant may be a metal cation, and the polypeptide may have a site that accepts the reactant. Figure 8D An exemplary reaction according to such embodiments is shown, wherein a reaction between a polypeptide 830 and a metal 850 forms a metal-polypeptide complex 860. Metals and polypeptides are known to form donor-acceptor bonds. Other types of bonds, such as ionic bonds, are also possible. The resulting complex can then be stabilized and thus protected from secondary fragmentation in specific regions of the polypeptide sequence where the complex is formed. Thus, according to these embodiments, the process can be used to achieve the goal of increasing sequence coverage.

[0151] In some embodiments, as Figure 8E As shown, additional linkage is provided by a gas phase reaction, for example, in a reaction between an analyte such as a positively charged polypeptide 830A and a reactant such as a negatively charged polypeptide 830B. In these embodiments, the two ions can be attracted to each other and form a non-covalent complex 870. Such a reaction can be performed in an ion trap having the ability to capture positive and negative ions.

[0152] In some embodiments, a precursor ion corresponding to the analyte ion is first selected and delivered to an ion-ion reaction device. A reactant ion is also selected and also delivered to an ion-ion reaction device. The reactant and analyte ions are then allowed to mix and interact in the reaction chamber to form ion-ion reaction products. Optionally, the method may include selecting a specific product corresponding to a complex formed by an analyte and a single or multiple reactant ions. The complex is then further fragmented and the resulting products analyzed. Similar to a cross-linking agent, this reaction does not require the analyte polypeptide to contain a disulfide bond. However, unlike the case of disulfide bonds or cross-linking, in this case, the location of the reaction can be random. However, if the size of the analyte polypeptide is substantially larger than the reactant polypeptide, a sufficient portion of the analyte polypeptide can undergo the fragmentation reaction unimpeded, thereby preventing secondary fragmentation events in the area where the complex is formed.

[0153] In some embodiments, it can be advantageous to use an electron capture mechanism for fragmentation because such reactions require a positive charge. In view of the fact that the reactant ions are negatively charged, the likelihood of reactant ion fragmentation is greatly reduced in cases where the fragmentation method may require electrons for the reaction. In the region where the reactant ions interact with the analyte ions, the total charge can be zero, and therefore, this portion of the complex will be neutral. This can also greatly reduce the efficiency of the reaction, further preventing unwanted fragmentation of the reactant polypeptide. Due to this phenomenon, two series of products can typically be detected: one series of products corresponding to the fragments observed after fragmentation of the unreacted analyte ions and another series of products in which the fragments also contain modifications of the reactant polypeptide. In this case, other products, such as fragments of the reactant polypeptide, may be unlikely. Since only two series of fragments are formed under this method, signal dilution can be minimal, and improved sequencing of the middle portion of the polypeptide can be achieved in this scheme.

[0154] Some embodiments can be used to sequence other biopolymers such as polynucleotides. For example, single-stranded DNA and RNA polymer molecules can be composed of many nucleotide bases and, due to secondary fragmentation, can encounter similar problems when sequencing the middle portion of a polynucleotide. These molecules can be preferentially analyzed in negative mode, so reactions with positively charged reactants can be appropriate. Such reactants can be positively charged polypeptides.

[0155] Figure 9 A flow chart is shown of a method 900 for improving top-down sequence coverage using a chemical reaction with a reagent, according to some embodiments. The chemical reaction can stitch together different parts of a polypeptide.

[0156] In some embodiments, the chemical reaction is carried out in the liquid phase. In some embodiments, the reactant can be a metal cation. In some embodiments, the metal cation forms other types of bonds, such as donor-acceptor bonds or ionic bonds. In some embodiments, a cross-linking agent can be used, which can form a covalent bridge between two amino acids in a polypeptide.

[0157] Method 900 begins with the optional step of performing complete removal of disulfide bonds from a polypeptide at step 910. The method then bifurcates and separates into two distinct pathways.

[0158] In one approach, in step 920, method 900 utilizes a chemical reaction to join portions of a polypeptide. In step 922, the resulting subunits are subjected to ECD*MS / MS analysis. In step 924, a first sequence of the subunits is obtained.

[0159] In another approach, in step 930, the subunits are subjected to ECD*MS / MS analysis without using a chemical reaction to link the polypeptides. In step 932, a second sequence of the subunits is obtained.

[0160] In step 940, the first sequence and the second sequence are combined to obtain combined sequence coverage of the polypeptide. According to these embodiments, the combined sequence can provide improved polypeptide sequence coverage.

[0161] In some embodiments, Figure 9 A similar objective of the embodiment is achieved by linking portions of a polypeptide through physicochemical interactions, which can provide secondary linkages that link multiple fragments of the polypeptide together. Such physicochemical interactions linking portions of the polypeptide can be achieved through ion-ion reactions between the analyte and the ionic reactant.

[0162] in particular, Figure 10 A flow chart of a method 1000 for performing physicochemical interactions to achieve top-down sequence coverage improvement according to some embodiments is shown. The physicochemical interactions can link different parts of a polypeptide.

[0163] Method 1000 begins with the optional step of performing complete removal of disulfide bonds from a polypeptide at step 1010. The method then bifurcates and separates into two distinct pathways.

[0164] In one approach, in step 1020, physicochemical interactions are performed to link the polypeptide portions. In step 1022, the resulting subunits are subjected to ECD*MS / MS analysis. In step 1024, a first sequence of the subunits is obtained.

[0165] In another approach, in step 1030, the subunits are subjected to ECD*MS / MS analysis without performing physicochemical interactions to link the polypeptide portions. In step 1032, a second sequence of the subunits is obtained.

[0166] In step 1040, the first sequence and the second sequence are combined to obtain a combined sequence of the polypeptide. According to these embodiments, the combined sequence can provide improved polypeptide sequence coverage.

[0167] In certain embodiments, denaturation and reduction of a monoclonal antibody (mAb) can be performed to produce fully reduced and disulfide-linked light chains (LC) in the same sample. The concentration of guanidine hydrochloride (Gnd), the temperature of incubation, and the duration of incubation are key factors in determining the ratio of fully reduced subunits to disulfide-linked subunits. A final concentration of 2M Gnd and an incubation temperature and time of 45°C and 30 minutes, respectively, resulted in the co-formation of fully reduced LC subunits and disulfide-linked LC subunits of NISTmAb. According to the teachings of the present invention, in a single high-resolution MRM experiment, these two species can be separated (e.g., by chromatographic separation, such as liquid chromatography) and targeted for fragmentation. Analysis of the combined results of these two species resulted in ultra-high sequence coverage (>85%) in a single injection.

[0168] Aliquots of 10 μg / μL NISTmAb were mixed with guanidine hydrochloride (Gnd) and dithiothreitol (DTT) at final concentrations of 2 M and 50 mM, respectively. The mixture was incubated at 45°C for 30 minutes. After incubation, 4 μL of the final solution (approximately 4 μg) was immediately injected for liquid chromatography-mass spectrometry (LCMS) analysis.

[0169] An ACQUITY UPLC Protein BEH C4 column (2.1 x 50 mm, 1.7 μm, The subunits of NISTmAb were separated using a HPLC-MS / MS / MS system (Waters, Inc.). The HPLC gradient used for the subunit separation is shown in Table 1. A flow rate of 0.3 mL / min was used for all HPLC runs. The column was maintained at 60°C in an ExionLC system (SCIEX) oven. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The initial concentration of the gradient consisted of 80% A and 20% B, which gradually decreased to 10% A and 90% B over 15 minutes, and then returned to 80% A and 20% B at 20 minutes.

[0170] Time (min) A(％) 8(％) initial 80 20 2 80 20 14 60 40 15 10 90 17 10 90 17.5 80 20 20 80 20

[0171] Table 1: LC gradients used for subunit separation

[0172] MS analysis was performed using a ZenoTOF 7600 system (SCIEX). According to the present teachings in this work (e.g., electron-based dissociation, such as ECD*), two charge states of the fully reduced (24+ and 19+) and disulfide-linked (18+ and 15+) LC subunits were targeted for fragmentation.

[0173] A final concentration of 2 M Gnd and an incubation temperature and time of 45°C and 30 minutes, respectively, resulted in the co-formation of the fully reduced subunit and the disulfide-linked LC subunit of NISTmAb in the same sample (e.g. Figure 11 These two forms of LC subunits can be Figure 11 The two species identified were chromatographed as shown in Figure A. The mass spectra of the two species identified are described in Figure 11 Figures B and C of the LC subunits show a clear difference in the charge state distribution. The disulfide-linked LC subunits carry less charge on average than their fully reduced counterparts, which is due to the lower degree of sequence unfolding caused by the presence of two intrachain disulfide bonds. Automated protein deconvolution using Biologics Explorer software confirmed the masses of the fully reduced (23127.3 Da) LC subunit and the disulfide-linked (23123.6 Da) LC subunit. In a single mass spectrometry experiment, two charge states from each form of the LC subunit were targeted for fragmentation. Processing of this data, either separately or together, resulted in reliable disulfide bond mapping and ultra-high sequence coverage of the starting peptide.

[0174] In some embodiments, the physicochemical interaction is carried out by a gas phase reaction. The physicochemical interaction can be the result of an ion-ion reaction. In some embodiments, the reactants can be oppositely charged polypeptides.

[0175] In some embodiments, the analyte polypeptide has a mass greater than 20 kDa.The sequence may then comprise at least the middle portion of the protein.

[0176] It will be appreciated by those skilled in the art that various changes may be made to the above-described embodiments without departing from the scope of the present invention.

[0177] Although some aspects have been described in the context of systems or apparatuses, it is apparent that these aspects may also represent a description of a corresponding method, where a block or apparatus corresponds to a method step or a feature of a method step. Similarly, aspects described in the context of a method step also represent a description of a corresponding block or item or feature of a corresponding apparatus.

[0178] The foregoing description of the embodiments is for illustrative purposes only. It is not exhaustive and does not limit the embodiments to the precise forms disclosed. Although several exemplary embodiments and features have been described, modifications, adaptations and other implementations may be possible without departing from the spirit and scope of the embodiments. Therefore, unless otherwise expressly stated, the description relates to one or more embodiments and should not be interpreted as limiting the embodiments as a whole. This is the case regardless of whether the present disclosure states that a feature is associated with "a", "the", "one", "one or more", "some" or "various" embodiments. As used herein, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" may include plural forms. In addition, stating that a feature may be present indicates that the feature may be present in one or more embodiments.

[0179] In the present disclosure, the terms "comprises," "includes," "contains," and "having," when used after a group or system, represent open inclusion and do not preclude the addition of other unrecited members to the group or system. Furthermore, unless otherwise specified or inferred from the context, the conjunction "or," if used, is not exclusive but inclusive, meaning and / or.

[0180] Also, as these terms are used, a group may include one or more members and a subgroup of a group may include one or more than one, including all members of the group.

[0181] The disclosed compositions, systems, methods, and apparatus are not limited to any specific aspect or feature or combination thereof, nor do the disclosed compositions, systems, methods, and apparatus require the presence of any one or more specific advantages or problems to be solved. Any theory of operation is provided for ease of explanation, but the disclosed compositions, systems, methods, and apparatus are not limited to such theory of operation.

[0182] In light of the above teachings, modifications and variations may be made, or modifications and variations may be obtained by implementing the embodiments. For example, the steps described need not be performed in the same order as discussed or with the same degree of separation. Likewise, various steps may be omitted, repeated, combined, or performed in parallel as needed to achieve the same or similar goals. Similarly, the described system does not necessarily include all the parts described in the embodiments, and may also include other parts not described in the embodiments. Therefore, the embodiments are not limited to the above details, but are defined by the appended claims according to the full scope of their equivalents. In addition, the present disclosure is intended to all novel and non-obvious features and aspects of the various disclosed embodiments, both individually and in various combinations and sub-combinations with each other.

[0183] While the present disclosure has been described in detail in conjunction with specific embodiments, many alternatives, modifications, and variations will be apparent from the foregoing description. It is therefore intended that the appended claims will encompass any such alternatives, modifications, and variations that fall within the true spirit and scope of the present disclosure.

Claims

1. A method for sequencing a polypeptide, the method comprising: removing one or more interchain disulfide bonds and retaining substantially all intrachain disulfide bonds of a first sample of the polypeptide to produce a first set of subunits; performing a first mass spectrometry (MS) analysis on the first group of subunits to determine a first amino acid sequence of the polypeptide, removing one or more interchain disulfide bonds and one or more intrachain disulfide bonds of a second sample of the polypeptide to produce a second set of subunits; performing a second MS analysis on the second group of subunits to determine a second amino acid sequence of the polypeptide; as well as The first amino acid sequence and the second amino acid sequence are combined to determine a combined amino acid sequence of the polypeptide.

2. The method of claim 1, wherein the amino acid sequence is determined using a fragmentation method.

3. The method of claim 2, wherein the fragmentation method is selected from one of electron-activated dissociation and electron-capture dissociation, ultraviolet light dissociation, and electron transfer dissociation.

4. The method of any one of claims 1-3, wherein removing one or more interchain disulfide bonds comprises removing substantially all interchain disulfide bonds.

5. The method of any one of claims 1-4, wherein the second sample is the same as the first sample.

6. The method of any one of claims 1-4, wherein the first sample and the second sample are subsamples of the same sample.

7. The method of any one of claims 1 to 6, wherein the first sequence comprises at least one segment from the middle portion of the polypeptide.

8. The method of any one of claims 1-7, wherein the second sequence comprises at least one portion blocked by an intrachain disulfide bond.

9. The method of any one of claims 1-8, wherein the polypeptide is a protein.

10. The method of claim 9, wherein the polypeptide is an antibody.

11. The method of claim 10, wherein the antibody is one of a monoclonal antibody (mAb), a bispecific antibody (bsAb), and an antibody drug conjugate (ADC).

12. The method of any one of claims 1-11, wherein tandem mass spectrometry is used for MS analysis.

13. The method of any one of claims 1-12, wherein the disulfide bonds are removed by using a chemical agent.

14. The method of claim 13, wherein the chemical agent is selected from DTT (dithiothreitol), TCEP (tris(2-carboxyethyl)phosphine) or mercaptoethanol.

15. The method of any one of claims 1-12, wherein removing disulfide bonds is performed electrochemically. The method of claim 15 , wherein enzymatic digestion of the sample is performed prior to removing disulfide bonds.

17. The method of claim 16, wherein the enzyme performing the enzymatic digestion is one of IdeS, IgdE, IdeZ, Kgp, and SpeB.

18. The method of any one of claims 1-17, wherein removing one or more interchain disulfide bonds of the first sample comprises removing substantially all interchain disulfide bonds.

19. The method of any one of claims 1-18, wherein removing one or more interchain disulfide bonds and one or more intrachain disulfide bonds of the second sample comprises removing substantially all interchain and intrachain disulfide bonds.

20. A method for sequencing a polypeptide, the method comprising: connecting a portion of the polypeptide sample using a chemical reaction; as well as The samples were subjected to MS analysis to determine the amino acid sequence of the polypeptide.

21. The method of claim 20, further comprising performing MS analysis on a second sample of the polypeptide without utilizing the chemical reaction to determine a second amino acid sequence of the polypeptide, and combining the amino acid sequence with the second amino acid sequence to determine a combined amino acid sequence of the polypeptide.

22. The method of any one of claims 20-21, wherein the chemical reaction is a reaction with a cross-linking agent.

23. The method of any one of claims 20-21, wherein the chemical reaction is a reaction with a metal cation.

24. A method for sequencing a biopolymer, the method comprising: connecting a portion of the sample of the biopolymer using a physicochemical interaction; as well as The samples were subjected to MS analysis to determine the amino acid sequence of the biopolymer.

25. The method of claim 24, further comprising performing MS analysis on a second sample of the biopolymer without utilizing physicochemical interactions to determine a second amino acid sequence of the biopolymer, and combining the amino acid sequence with the second amino acid sequence to determine a combined amino acid sequence of the polypeptide.

26. The method of any one of claims 24-25, wherein the physicochemical interaction is an ion-ion interaction.

27. The method of claim 26, wherein the ion-ion interaction is the result of an ion-ion reaction.

28. The method of claim 27, wherein the ion-ion reaction is performed in the gas phase.

29. The method of any one of claims 24-25, wherein the reactant polypeptide is a polypeptide of opposite charge to the analyte biopolymer.

30. The method of any one of claims 24-29, wherein the biopolymer is one of a polypeptide and a polynucleotide.

31. The method of any one of claims 1-19, wherein the subunit polypeptide has a mass greater than 20 kDa.

32. The method of any one of claims 20-30, wherein the analyte polypeptide has a mass greater than 20 kDa.

33. The method of any one of claims 1-32, wherein the sequence comprises at least a portion of the middle of the protein.

34. A method for sequencing a middle portion of a polypeptide, the method comprising: A sample of the polypeptide is removed of substantially all interchain disulfide bonds and retains substantially all intrachain disulfide bonds to produce subunits, which are subjected to mass spectrometry (MS) analysis to determine the amino acid sequence of the midportion of the polypeptide.

35. The method of claim 34, wherein the middle portion of the polypeptide spans more than 50 amino acids from the end of the polypeptide.

36. The method of claim 34, wherein the middle portion of the polypeptide spans more than 70 amino acids from the end of the polypeptide.

37. The method of claim 34, wherein the middle portion of the polypeptide spans more than 100 amino acids from the end of the polypeptide.

38. A method for sequencing polypeptides of a sample, the method comprising: removing one or more interchain disulfide bonds and retaining substantially all intrachain disulfide bonds from a first portion of the sample of polypeptides to produce a first set of subunits; removing one or more interchain disulfide bonds and one or more intrachain disulfide bonds of a second portion of said sample of said polypeptides to produce a second set of subunits; separating the first group of subunits from the second group of subunits; performing mass spectrometry (MS) analysis on the first group of subunits to determine a first amino acid sequence of the polypeptide; performing a second MS analysis on the second group of subunits to determine a second amino acid sequence of the polypeptide; as well as The first amino acid sequence and the second amino acid sequence are combined to determine a combined amino acid sequence of the polypeptide.

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