Method for heat-assisted enzymatic digestion

Abstract

The present disclosure relates to a method of digesting a sample comprising a protein, the method comprising: adding the sample to a device, the device containing a buffer and a solid support surface comprising a surface coating, wherein the surface coating immobilizes an enzyme while reducing undesired interactions between the sample and the solid support surface; immobilizing the enzyme on the surface coating for digesting a protein of the sample; and heating the sample to complete a heat-assisted digestion of the protein.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 022,056, filed May 8, 2020, entitled “Methods for Heat-Assisted Enzyme Digestion,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to methods for heat-assisted biomolecular sample processing (e.g., digestion and affinity ligand purification). More specifically, this disclosure relates to the use of coatings (such as heterofunctional coatings) in conjunction with heat from sample processing for bioanalysis (e.g., cleaning, sample release) to conjugate biomolecules onto the surface of a solid carrier. Background Technology

[0004] Biomolecules are complex molecules that require sophisticated workflows for analysis. These workflows involve multiple steps, including various sample preparation steps such as sample cleaning and protein digestion. Achieving substantially complete digestion without compromising sample quality (e.g., without introducing byproducts requiring additional cleaning) is challenging. For example, some enzymes used for digestion can become unstable when used in solution, leading to byproducts. Performing digestion at elevated temperatures can improve workflow throughput. However, enzymes are generally most efficient within specific temperature ranges due to heat-induced denaturation or other conformational changes. Within these specific temperature ranges, protein autolysis can increase significantly. Furthermore, the hardware of the heat source can be difficult to benchmark and evaluate for reproducibility. Summary of the Invention

[0005] Biomolecules are complex molecules that require sophisticated workflows for analysis. These sophisticated workflows involve multiple steps, including various sample preparation steps such as sample cleaning and protein digestion. Achieving substantially complete digestion without compromising sample quality (e.g., without introducing byproducts that require additional cleaning) is challenging. For example, some enzymes used for digestion may become unstable when used in solution, leading to byproducts.

[0006] However, enzymes are typically most efficient within a specific temperature range due to heat-induced denaturation or other conformational changes. But within this specific temperature range, protein autolysis can increase significantly. Furthermore, the hardware of the heat source may make it difficult to benchmark and evaluate reproducibility.

[0007] Immobilization of these enzymes helps stabilize them. However, conventional techniques for enzyme immobilization result in secondary interactions with the surface of the carrier, which can affect digestion efficiency and sample recovery.

[0008] Immobilized enzymes can increase digestion temperature and reduce the drawbacks associated with elevated temperatures. The result is increased workflow without sacrificing digestion quality.

[0009] Generally, this disclosure relates to a rapid digestion method that is heat-assisted to efficiently complete digestion within minutes while providing high-fidelity peptide profiling. This rapid digestion method is reliable and suitable for biological research or protein therapeutic characterization in a controlled environment. High fidelity is defined as a digestion workflow result that provides >95% sequence coverage, <10% missed cleavage, and comparable modification (thermally induced modification of approximately <5%, and in some examples less than 1%, after digestion at 25°C to 100°C) for therapeutic proteins compared to a conventional solution-based digestion workflow.

[0010] High-fidelity digestion workflows are used for peptide mapping, disulfide bond mapping, center-up, center-down, and bottom-up proteomics, protein identification, protein quantification, bioanalysis, and other digestion-related applications or applications (e.g., applications using affinity ligands). The method includes steps such as using a thermostable enzyme, a digestion buffer (or other type of buffer), a pyrogen, and obtaining a sample by cleaning or reaction quenching in preparation for downstream analysis (fluorescence, UV, LC-MS detection).

[0011] This disclosure provides a method for rapid enzymatic digestion under heat-assisted conditions. The rapid digestion method can be used for protein / peptide analysis. Generally, the method includes one or more of the following: a thermostable enzyme, a digestion buffer, a pyrogen, and steps of preparing the sample for downstream analysis (fluorescence, UV, LC-MS detection) by cleaning or reactive quenching. For example, some methods include the following components: an immobilized enzyme on a solid support with excellent thermostability and hydrophilicity (minimizing nonspecific binding); a digestion buffer that supports the kinetics of enzyme (such as trypsin) activity at elevated temperatures while minimizing heat-induced modification of the peptide; and a device (e.g., vial, plate, or column) that ensures maximum heat transfer efficiency and minimizes nonspecific binding.

[0012] This disclosure provides a method for processing a sample containing proteins, the method comprising: adding the sample to a device containing a buffer and a solid carrier surface including a surface coating, wherein the surface coating immobilizes an enzyme and an affinity ligand while reducing undesirable interactions between the sample and the solid carrier surface; immobilizing an enzyme on the surface coating for digesting proteins in the sample; immobilizing an affinity ligand on the surface coating for targeted capture of proteins in the sample; digesting the proteins in the sample with the immobilized enzyme on the surface coating; targeted capture of a portion of the sample with the immobilized affinity ligand on the surface coating; and heating the sample to activate protein digestion. In some embodiments, the step of targeted capture of a portion of the sample occurs before protein digestion. In some embodiments, the step of targeted capture of a portion of the sample occurs during digestion. In some embodiments, the step of targeted capture of a portion of the sample occurs after digestion. And in some embodiments, the step of targeted capture of a portion of the sample occurs before, during, and / or after protein digestion. In some embodiments, the buffer is selected from the group consisting of Tris, BIS-Tris, 2-ethanesulfonic acid (MES), HEPES, triethanolamine, and trimethylamine. The buffer may contain divalent ions, such as CaCl2. The buffer solution may contain a polyol selected from, but not limited to, the group consisting of glycerol, xylitol, propylene glycol, butylene glycol, or erythritol. In some embodiments, the buffer solution contains xylitol and CaCl2. In some embodiments, methionine is added to the buffer solution. In some embodiments, the buffer solution further contains one or more additives, such as, for example, xylitol, methionine, and / or CaCl2. Attached Figure Description

[0013] The present technology will be more fully understood through the following detailed description taken in conjunction with the accompanying drawings, in which:

[0014] Figure 1 It is based on the thermal spectrum curve of this disclosure.

[0015] Figure 2 It is based on the thermal spectrum curve of this disclosure.

[0016] Figure 3A This is a graph showing the effect of adding arginine to the digestion buffer on the recovery rate of hydrophobic peptides. Figure 3B This is a graph showing the effect of adding arginine to the digestion buffer on digestion efficiency. Figure 3C This is a graph showing the effect of adding dimethyl-arginine to the digestion buffer on the recovery rate of hydrophobic peptides. Figure 3D This is a graph showing the effect of adding dimethyl-arginine to the digestion buffer on digestion efficiency.

[0017] Figure 4 This is a graph showing the recovery rate of a mixture of hydrophobic peptides after culture with immobilized enzymes, according to this disclosure.

[0018] Figure 5 This is a diagram of trypsin released after being cultured in digestion buffer at 70°C.

[0019] Figure 6A , Figure 6B and Figure 6C This is a graph showing the LC-MS chromatogram generated after NIST mAb was digested under varying conditions.

[0020] Figure 7 It is a graph showing the percentage of missed cuts and the percentage of sequence coverage according to a comparison example of this disclosure.

[0021] Figure 8A , Figure 8B and Figure 8C This is a graph comparing digestion examples at different temperatures.

[0022] Figure 9A and Figure 9B It is a comparison of Ca 2+ A graph showing the effect of concentration on digestion efficiency and nonspecific binding.

[0023] Figure 10A and Figure 10B This is a graph comparing the effects of pH on digestion efficiency and deamidation percentage.

[0024] Figure 11A and Figure 11B This is a graph comparing the effects of Tris concentration on digestion efficiency and nonspecific binding.

[0025] Figure 12A and Figure 12B This is a graph comparing the effects of polyols on digestion efficiency and nonspecific binding. Figure 12C This is a graph showing a comparison of digestion results using glycerol and xylitol.

[0026] Figure 13 This is a graph showing the effect of methionine on preventing artificial oxidation.

[0027] Figure 14 This is a graph showing the effect of acetonitrile on reducing artificial deamidation.

[0028] Figure 15 This is a graph showing a comparison of digestion efficiency between examples.

[0029] Figure 16 A typical reaction setup in a 200 μL PCR tube is shown.

[0030] Figure 17 Showing from Figure 15 The size of the PCR tubes used.

[0031] Figure 18 This is a flowchart of an example of the current workflow for trypsin digestion. Detailed Implementation

[0032] Compared to conventional solution digestion typically performed at 37°C, protein digestion at elevated temperatures presents problems due to incomplete digestion, nonspecific binding to resins, and thermally induced modification of peptides. This disclosure facilitates the generation of a workflow highly similar to solution digestion, but requiring only minutes, by engineering immobilized enzymes with specific surface chemicals. The digestion buffer has been modulated to improve peptide recovery and enzyme activity.

[0033] It is worth noting that the digestibility of this disclosure is 90% to 95%, compared to only 65% ​​to 70% for the main competing product, SMART Digest. TM (Available from Thermo Fisher Scientific, Waltham, MA) This is significantly higher than Smart Digest. TM In comparison, the non-specific binding efficiency of peptides using the techniques disclosed herein is reduced by 10% to 30%. This disclosure is also applicable to the reduction / alkylation of reagents, while Smart Digest... TM Incompatible.

[0034] The enzymatic digestion of proteins is widely used in the biochemical research community, where the resulting peptides can reveal amino acid modifications or abundances of proteins in their relevant physiological contexts. With the emergence of antibody therapeutics in the biopharmaceutical industry, this technology has proven valuable in bioprocess and quality control assessments to support the identification and monitoring of key quality attributes reflecting the purity and safety characteristics of specific therapeutics. However, the digestion of large proteins (e.g., antibodies with a molecular weight of 150 kDa) can take hours to complete and often requires optimization of numerous experimental parameters, including incubation time, temperature, and the protein-to-enzyme ratio. This lengthy process limits throughput, as timely decisions are typically required during the discovery phase or during manufacturing. Another concern with this process is that the lengthy digestion process can induce artificial modifications to the protein or peptide, adding extra work to constrain these modifications. In the case of trypsin digestion, autolysis over time can adversely interfere with downstream analyses.

[0035] Attempts to increase workflow throughput have spurred some recent advancements in automating workflows. Liquid processors can now handle 96 or even 384 samples simultaneously; however, the digestion itself still takes several hours. On the other hand, the most common downstream peptide analyses require 60- to 120-minute liquid chromatography gradients for adequate separation, limiting processing to a maximum of 24 samples per day. A large number of samples queuing can lead to potential peptide loss due to stability or adsorption issues. This, in turn, affects the consistency and accuracy of platform analyses.

[0036] Attempts to perform digestion at elevated temperatures were also investigated. The underlying principle is an enzymatic reaction, which, like any other chemical reaction, follows the rules of the Arrhenius equation (Equation 1):

[0037]

[0038] Where k is the kinetic rate constant of the reaction, A is the Arrhenius constant, and G is the standard activation free energy (kJ / M). -1 The standard activation free energy (15-70 kJ / m²) depends on the entropy factor and enthalpy factor, where R is the gas law constant and T is the absolute temperature. For every 10°C increase in temperature, the typical standard activation free energy (15-70 kJ / m²) increases. -1 This can lead to an increase in rate between 1.2 and 2.5 times. However, due to heat-induced denaturation or other conformational changes, enzymes are generally most efficient within their preferred temperature range. Using sequencing-grade trypsin as an example, the preferred temperature for a 1-hour digestion assay of casein is 50°C to 55°C. However, at such temperatures, trypsin autolysis increases dramatically over time, resulting in a 60% loss of activity within 2 hours.

[0039] The heat source can be one that provides uniformity (uniform heat transfer) and uniform heat distribution. In the example of immobilized enzymes, it is necessary to ensure that the dispersion device operates in a stirring or flow mode that interacts with the protein to achieve homogeneous or uniform contact. The ramp time available from the heat source is also a factor in selecting the heat source. Short ramp times are advantageous. In the form of dispersed immobilized enzymes, the established digestion temperature is reached in 5 minutes or less for 10 minutes of digestion. In one example of immobilized trypsin, it is preferable to reach 75°C in 5 minutes or less and maintain the temperature at 75°C.

[0040] In some examples, the heat source can be any source that supports continuous heating at 50°C to 100°C for 5 minutes or longer. Some examples include ovens, incubators, shakers, or hot mixers. In some examples, for consistent and optimal results, the Eppendorf ThermoMixerC (based on Peltier technology) can be used, preferably with the option of a heated lid. The heated lid can facilitate digestion that takes a long time (e.g., more than 30 minutes).

[0041] Efficient heat transfer between consumables and the heat source is also a factor in selecting the heat source. In one example of dispersed immobilized trypsin, the thickness of the consumables used is related to their digestive performance. The preferred temperature depends on the selected enzyme or combination of enzymes. In the example of immobilized trypsin, T m and initial temperature (T) onset This can help determine the optimal temperature for heat digestion. The preferred temperature for trypsin is T. onset To T m between.

[0042] Microwave-assisted and infrared-assisted enzyme digestion appear to improve digestion kinetics by reducing the time to just 5 minutes; however, these studies are most helpful for small to medium-sized proteins (less than 100 kDa), and the quality of the digested products has not been rigorously evaluated. Furthermore, hardware like microwaves, used as heat sources, makes it difficult to benchmark and evaluate reproducibility.

[0043] Immobilized enzymes in dispersed or column / tube form are also commercially available. The advantage of immobilization is improved thermal stability by inhibiting thermal denaturation, allowing digestion to be completed at higher temperatures within a shorter timeframe. However, to date, reported workflows have achieved only limited digestion efficiency (percentage of all detectable trypsin peptides to total peptides) in proteins, ranging from 30% to 80%, compared to digestion in solution. Most protocols associated with immobilized enzymes advocate eliminating pretreatment (denaturation, reduction, and alkylation), claiming sufficient denaturation through heat. However, this is only applicable to smaller or heat-sensitive proteins, as incomplete digestion of larger proteins or proteins with multiple disulfide bonds will not be effectively denatured by heat. Furthermore, the extent of heat-induced modification and the reproducibility of these new methods have rarely been rigorously and extensively evaluated.

[0044] Figure 18This is a flowchart illustrating a peptide mapping workflow 700. In some examples, the peptide mapping workflow 700 includes four sections. The first section 702 includes unfolding a sample containing an analyte of interest (such as a protein). The second section 704 includes desalting the sample, which contains the unfolded analyte of interest. The third section 706 includes digesting the analyte of interest in the sample. Here, the apparatus for digesting the analyte of interest includes the heterofunctional coating of this disclosure. After digesting the analyte of interest, the fourth section 708 includes collecting the sample containing the digested analyte of interest.

[0045] In some examples, the first part 702 and the second part 704 may depend on the analyte of interest. For example, the first part 702 and the second part 704 may be considered as pretreatment steps, and based on the analyte of interest (such as a protein), the first and second parts may not be necessary.

[0046] A desirable heat-assisted rapid digestion method would efficiently complete digestion within minutes while providing high-fidelity peptide profiling. This rapid digestion method would be reliable and suitable for biological research or protein therapeutic characterization in a controlled environment. In this context, high fidelity is interpreted as providing a digestion workflow with >95% sequence coverage, <10% missed cleavage, and comparable modification percentage (typically <5%) for therapeutic proteins compared to conventional solution-based digestion workflows. This disclosure provides a method for rapid enzymatic digestion under heat-assisted conditions. The method essentially comprises the following components: an immobilized enzyme on a solid support with excellent thermostability and hydrophilic properties (minimizing nonspecific binding); a digestion buffer that supports the kinetics of trypsin activity at elevated temperatures while also minimizing heat-induced modification of the peptide; and a device (vial, plate, or column) that ensures maximum heat transfer efficiency and minimizes nonspecific binding as much as possible.

[0047] This disclosure provides an enhanced method for rapid enzymatic digestion under heat-assisted conditions. The method essentially comprises the following components: an immobilized enzyme with excellent thermostability and hydrophilicity (minimizing nonspecific binding) on ​​a solid support; a digestion buffer that supports the kinetics of trypsin activity at elevated temperatures while minimizing heat-induced modification of the peptide; and a device (vial, plate, or column) that ensures maximum heat transfer efficiency and minimizes nonspecific binding. The complete digestion workflow begins with desaturation of the protein in 8M guanidine or 6M urea. After reduction and alkylation, the protein is desalted and subjected to digestion. The digestion mixture consists of the immobilized enzyme and the protein in a digestion buffer at a favorable concentration. Relevant factors for the digestion buffer may include temperature, pH, metal concentration, and additives.

[0048] The buffer solution disclosed herein may contain a buffering agent for pH control, a protein sample dispersant, and metal ions. The metal ions may be divalent metal ions, preferably Ca2+. 2+ Buffers may include Tris, BIS-Tris, MES, HEPES, triethanolamine, and trimethylamine. Protein sample dispersants may be selected from polyols, such as glycerol, xylitol, propylene glycol, butylene glycol, or erythritol. Metal ions and protein sample dispersants may be additives in the buffer. For example, CaCl2, methionine, xylitol, and / or glycerol may be additives in the buffer.

[0049] After digestion, the immobilized enzyme is removed by centrifugation or filtration. The peptide is recovered as a supernatant and submitted for downstream analysis: LC-UV or LC-MS.

[0050] Immobilized enzymes on coatings (or affinity ligands in some examples), as described in more detail in Appendix A, offer thermal stability. For digestion, chemical immobilization methods via covalent interactions stand out because this ensures minimal enzyme leakage and, more importantly, better thermal stability. Many of these immobilized enzymes are provided in column form, which typically requires a multidimensional LC system for operation. Operating temperatures range from 37°C to 60°C, and information regarding their lifetime and reproducibility is lacking. Smart Digest TM It is a well-known, commercially available product in a distributed form, with an operating temperature of 70°C.

[0051] Figure 1 It is based on the thermal spectrum curves of various aspects of this disclosure. Specifically, Figure 1 It showed free trypsin, Smart Digest TM NanoDSC thermograms of trypsin and a preferred immobilized enzyme (e.g., trypsin) prototype (e.g., a coated silica-based solid carrier according to the invention), wherein T m - The temperature at which half of the protein is unfolded, and T onset - The temperature at which protein digestion begins. For example, with Smart Digest. TM ( Figure 1 Compared to the prototype with immobilized trypsin (i.e., a silica-based solid carrier with a coating), it even showed better thermal stability.

[0052] Figure 2 It is based on the thermal spectrum curves of various aspects of this disclosure. Figure 2 NanoDSC thermograms of immobilized enzyme prototypes with different modifications are shown. Unmodified trypsin exhibits a T1 of approximately 65 °C. mSpot-modified trypsin (butyl acrylate, s-methylisothiourea, and phenylglyoxal) has slightly improved T. m At 1°C to 2°C, cross-linked trypsin (glycerol 1,3-diglyceryl glycol diacrylate and triethylene glycol diacrylate) has significantly improved T m 5℃ to 7℃. To further improve the thermal stability of trypsin, cross-linking agents that react nonspecifically with amines can inhibit structural changes that occur under heating conditions. Figure 2 Similarly, modifiers covalently bound to specific amino acids not located at the active site can also create steric hindrance during thermal denaturation. Figure 2 ).

[0053] Immobilized enzymes on the support impart enhanced surface properties, such as nonspecific binding, conjugation chemistry, and digestion efficiency. The solid support can be constructed from a hydrophilic surface with a porous structure that minimizes nonspecific binding, thereby ensuring digestion efficiency and peptide recovery. Important physical parameters include the particle size and pore size of the solid support, which affect protein diffusion into the pores and their contact with the enzyme. For the chosen material, hydrophilic modification is optimized to allow nonspecific binding to be tuned to an optimal level. Figure 4 This is a graph showing the recovery rate of a mixture of hydrophobic peptides after culture with immobilized enzymes, according to this disclosure. Figure 4 The recovery rate of the hydrophobic peptide was demonstrated after mixing with the selected immobilization carrier for only 5 minutes.

[0054] In some examples, the solid carrier is porous, and the pore size is approximately... to approximately Within a certain range. Pore size can affect the diffusion of proteins into the pores and the degree of contact between proteins and enzymes.

[0055] The surface of a solid support can consist of multiple particles, each with a particle size ranging from about 1 micrometer to about 200 micrometers. Particle size can affect protein diffusion and the degree of protein-enzyme contact. Solid supports are materials used to immobilize enzymes. In some examples, consumables containing reaction components include multiwell plates, vials, or columns. Consumables are coated to reduce nonspecific binding.

[0056] Another consideration for immobilization is the stability of the enzyme conjugated on the support. Released enzymes typically exhibit significantly reduced thermal stability at elevated temperatures, and digestion efficiency decreases over time as the amount of enzyme immobilized on the support diminishes. Worse still, if downstream analysis is performed using UV analysis, it can introduce significant noise into the analysis of autolysate, particularly for trypsin. Figure 5This is a graph showing the release of trypsin after incubation in digestion buffer at 70°C. The preferred product exhibits minimal enzyme leakage during digestion. Figure 5 ).

[0057] In some examples, immobilized trypsin showed no autolysis at low temperatures (25°C to 45°C), very low autolysis (less than 1%) at temperatures above 65°C at pH values ​​greater than 7, and no autolysis at lower pH values.

[0058] Immobilized enzymes may comprise one or a combination of enzymes (e.g., trypsin, chymotrypsin, Lys-C, Glu-C, Arg-C, Asp N, papain, pepsin, elastase, IdeS, streptokinase, and PNGase) immobilized on particles, chips, or surfaces capable of maintaining a specific elevated temperature between 25°C and 110°C without negatively impacting enzyme activity. In some examples, the elevated temperature range can be further narrowed to allow heating above 45°C, preferably between 65°C and 75°C and not exceeding 85°C. The enzyme acquires thermal stability through the immobilization process. In the example of trypsin, the melt temperature (Tm) can be increased by 30°C to 50°C after immobilization. Tm can be further increased by site modification and by cross-linking and conjugating small polymers or ligands to trypsin. In some examples, the solid carrier with the immobilized enzyme exhibits less than 30% nonspecific binding to all proteins / peptides present during digestion, and the immobilized enzyme remains stable during the digestion process with less than 20% leaching.

[0059] In some examples, the modified enzyme is in solution. The enzyme may include a single enzyme or a combination of enzymes (e.g., trypsin, chymotrypsin, Lys-C, Glu-C, Arg-C, Asp N, papain, pepsin, elastase, IdeS, streptomycin, and PNGase).

[0060] The digestion efficiency of immobilized enzymes is a combination of surface properties. Using NIST mAb as a model protein, the percentage of trypsin peptides (without missed cleavage) in the total peptides detected after digestion is used to reflect digestion efficiency. The percentage of missed cleavage reflects the overall digestion efficiency. Smart Digest is used in the recommended workflow. TM The reported sequence coverage is only 50% to 76% of IgG1. Figure 6A , Figure 6B and Figure 6C This is an LC-MS chromatogram showing the NIST mAb digested under varying conditions. Specifically, Figure 6A , Figure 6B and Figure 6C It shows NIST mAb in ( Figure 6A Digestion in solution for two hours; Figure 6B 10-minute Smart Digest TM Digestion;( Figure 6C The LC-MS chromatogram of the product after 10 minutes of prototype digestion. Figure 7 This is a graph showing the percentage of missed cuts and the percentage of sequence coverage according to a comparative example of this disclosure. Specifically, Figure 7 The prototype was shown to have increased hydrophilicity modification levels compared to Smart Digest. TM The percentage of missed cuts and the percentage of sequence coverage were compared between the two. The expected prototype with improved thermal stability should be able to provide robust digestion under optimized conditions (missed cuts < 10%, sequence coverage > 90%) (Figure 6 and...). Figure 7 By adjusting the hydrophilicity of the surface, the digestion efficiency can be optimized to find the "optimal point" that balances digestion efficiency and peptide recovery rate. Figure 7 ).

[0061] This disclosure may include pretreatment prior to digestion. A pretreatment step may be present before protein digestion. In some examples, proteins that can be readily denatured by heat and introduced during digestion do not require pretreatment. For proteins requiring pretreatment, reduction and alkylation following denaturation are common steps for fully unfolding the protein.

[0062] Digestion conditions play a crucial role in the digestion of immobilized enzymes at elevated temperatures. Based on the choice of immobilization carrier, digestion parameters (such as pH, temperature, and digestion time) that optimally function with free enzymes in solution will exhibit unique characteristics in heated environments. Additives can help address improved kinetics, thermally induced modification of peptides, peptide adsorption onto the immobilization carrier, and aggregation of slurries or proteins. Using heat-assisted denaturation, complete digestion can be achieved without pretreatment (including denaturation, reduction, and alkylation with guanidine or urea) without needing to determine the extent of missed cleavage. In this disclosure, as a result of the percentage of missed cleavage, complete digestion is no more than 10%, meaning that the protein has been completely digested and more than 90% of the resulting peptides have no missed cleavage sites. This is a stringent standard and also distinguishes the quality of this disclosure and its utilization from other disclosures.

[0063] In this disclosure, pretreatment is optional, depending on the analyte of interest (e.g., protein). Desalting to remove pretreatment is optional, as the resin is compatible. Samples can be pretreated (including denaturation, reduction, and alkylation) prior to digestion to achieve high-fidelity peptide profiling. Although the entire process, including pretreatment, does not exceed 2 hours, it includes digestion.

[0064] After immobilization, the enzyme exhibits improved thermal stability, and an optimal temperature needs to be determined for the selected enzyme because, to achieve maximum activity, a temperature of T... m They may exhibit unique correlations or no correlation with temperature. Empirically, for trypsin, a temperature close to its T value is preferred. m Digestion is carried out at 70°C to 75°C for 10 minutes. Figure 8A , Figure 8B and Figure 8C This is a graph comparing digestion examples at different temperatures. Specifically, Figure 8A , Figure 8B and Figure 8C Showing comparisons of Smart Digest TM and immobilized trypsin in ( Figure 8A 60℃; Figure 8B 70℃; Figure 8C LC-MS chromatogram after digestion of NIST mAb at 80℃ for 10 minutes.

[0065] Below this temperature, significant incomplete digestion will occur. Figures 8A to 8C Without being bound by theoretical limitations, digestion efficiency will reach high values ​​at temperatures above 70°C, and it is expected that shortening the digestion time will achieve similar digestion completeness. However, this may also require a slightly longer reaction mixture equilibration time, which could lead to thermally induced degradation or adsorption, or other undesirable modifications.

[0066] In some examples, the buffer composition includes calcium ions (Ca). 2+ It can pass through 1mM to 10mM Ca in the solution. 2+ To improve the activity of free trypsin. While not theoretically constrained, the effect of ionic strength on trypsin may exist at an "optimal" concentration, regardless of the inorganic salt. Once immobilized, the enzymatic reaction can proceed at much higher temperatures. For example, using trypsin, the enzymatic reaction can be carried out at 70°C. In some examples where kinetics are significantly improved, it may be necessary to re-evaluate the catalyst Ca2+. + The concentration. Figure 9A and Figure 9B It displays Ca 2+ A graph showing the effect of Ca2+ concentration (1 mM to 50 mM) on digestion efficiency and nonspecific binding. In one example, digestion efficiency was significantly improved when the Ca2+ concentration increased from 1 mM to 50 mM. Figure 9A However, Ca 2+ The most severe loss was also observed on hydrophobic peptides (m / z 934) at 50 mM. Figure 9BFor most proteins, especially antibody therapeutics with very complex peptide profiles, 10 mM to 20 mM Ca2+ is recommended. 2+ It can achieve a balance between enzyme activity and non-specific binding.

[0067] Most enzymes exhibit pH preferences. For example, trypsin in solution exhibits maximized activity at pH 8.0. Buffers used to create such environments are mostly zwitterionic compounds, whose pKa values ​​change, affecting buffering capacity. At increasing temperatures, pKa values ​​typically decrease, which can induce pH shifts in digestion buffers. For example, from 25°C to 75°C, the pH of a 50 mM Tris solution decreased by 0.6 (Table 2, shown below).

[0068] Figure 10A and Figure 10B This is a graph comparing the effects of pH on digestion efficiency and deamidation percentage. Figure 10A In the example, the pH of the immobilized trypsin was changed from 7.6 at 25°C to 7.0 at 75°C. Figure 10A There are cases where a lower pH is preferred. For example, it is known that heated digestion accelerates deamidation, but this can be mitigated by adjusting the pH to a slightly lower value. Figure 10B Alternatively, you can simply shorten the digestion time to avoid it.

[0069] In some examples, the preferred pH of the enzyme can be the same regardless of the temperature at which digestion occurs. However, across a wide temperature range, the buffer has varying abilities to maintain the same pH. In the example of immobilized trypsin, the buffer pH is 7.6 at room temperature. At 75°C, the buffer pH drops to 7.0. However, under the influence of heat, digestion can occur more rapidly, thus achieving high-quality digestion results within 10 minutes even if the trypsin is not in its preferred state.

[0070] Ionic strength is another buffer composition condition. Each specific concentration of buffer salt provides the ionic strength necessary for enzymatic reactions to occur. Tris buffers are commonly used for digestion, but the concentration of Tris needs to be adjusted for the enzyme selected for digestion. Figure 11A and Figure 11B This is a graph comparing the effects of Tris concentration on digestion efficiency and nonspecific binding. In the example of immobilized trypsin, 100 mM Tris provided the best results for a balanced digestion efficiency and enhanced peptide recovery (Figure 11).

[0071] Once an immobilized enzyme is selected for digestion, a heated mixer is required to prevent physical sedimentation of the resin, thus ensuring uniform and efficient heat distribution during the process. However, accelerated aggregation can occur between particles and proteins; for example, denatured proteins may aggregate faster than in solution, with hydrophobic portions more readily exposed to heat. Polar additives can play a crucial role in preventing adverse interactions between particles or proteins and in cultivating a water-like environment. Indeed, small polyols (including but not limited to glycerol, xylitol, erythritol, propylene glycol, and butylene glycol) can stabilize aggregation at high temperatures and high salt concentrations and provide high peptide recovery rates due to their hydrophilic properties. Figure 12A and Figure 12B This is a graph comparing the effects of polyols on digestion efficiency and nonspecific binding. Figure 12C This is a graph showing a comparison of digestion results using glycerol and xylitol. In some examples, higher hydrophilicity corresponds to higher digestion efficiency (Figure 12).

[0072] In this disclosure, enzyme activity controls include cofactors / stabilizers and ionic strength. In the example of immobilized trypsin, the calcium concentration can be modulated to ensure enzyme activity. In the example of immobilized trypsin, the Tris concentration can also be modulated according to activity.

[0073] In this disclosure, nonspecific binding controls are suitable for immobilized enzymes. In examples of immobilized trypsin, 5% glycerol and other substances have the effect of mitigating the adsorption of hydrophobic peptides onto the resin surface. In examples of immobilized trypsin, the concentrations of calcium and Tris can be modulated to achieve minimal nonspecific binding.

[0074] Since process-induced modification associated with elevated temperatures is always a problem, there are additives that can avoid the effects of this problem. Figure 13 This is a graph showing the effect of methionine on preventing artificial oxidation. For example, adding a sufficient amount of an oxidizing agent (such as methionine) can adequately reduce artificial oxidation. Figure 13 Regarding deamidation, the addition of an organic solvent (e.g., 10% acetonitrile) can modulate the dielectric strength and mitigate method-induced deamidation. Figure 14 ).

[0075] The kit may include a digestion buffer to support enzyme activity at elevated temperatures and minimize heat-induced modification of the protein. In some examples, methionine-containing peptides are converted to oxidized variants at a relative conversion rate of less than 1%. In some examples, asparagine residues are deamidinated to isoaspartic or aspartic acid at a relative conversion rate of less than 5% (preferably less than 2%) during the kit procedure. In some examples, artificial oxidation of methionine does not exceed 1% and deamidation does not exceed 5% within 10 minutes of heat digestion at pH 7.5.

[0076] Oxidizing scavengers (such as methionine) can be added to the digestion buffer. In one example of immobilized trypsin, 50 mM methionine was able to eliminate heat-induced modification compared to digestion in solution.

[0077] Deamidation is a pH-driven process. Lowering the pH or adjusting the composition of the buffer can prevent heat-induced deamidation. In the example of immobilized trypsin, 10% acetonitrile significantly reduced artificial deamidation.

[0078] To achieve complete digestion, time-dependent studies of each enzyme involved in protein digestion are required. In the example of immobilized trypsin, digestion can be completed within 1 to 10 minutes.

[0079] According to this disclosure, heat-assisted digestion can be completed within minutes and is therefore compatible in both dispersion and linear column formats. For reactions in 200 μL vials, rise time may be problematic, as the mixture may take several minutes to reach the set temperature. Rise time may vary depending on the tubes and heat mixer used. Robust heat transfer efficiency is important for achieving high reproducibility of this process. Commercially available PCR tubes, primarily made of polypropylene, exhibit varying wall thicknesses. Figure 15 This is a graph comparing the digestion efficiency between examples. Specifically, Figure 15 It displays Smart Digest TM A graph comparing digestion efficiency with five different vendors' immobilized prototypes in PCR tubes (i.e., silica-based solid carriers with coatings of the present invention). Figure 16 A typical reaction setup in a 200 μL PCR tube is shown. Cross sections A, B, and C correspond to... Figure 17 The cross-sectional dimensions. Figure 17 Showing from Figure 15 The size of the PCR tubes used. The thickness at the bottom of the tube (size C) appears to have the least impact on digestion. For example, AXYGEN has the thickest A and B sizes, which may lead to poor heat transfer and thus the worst digestibility, such as... Figure 15 As shown.

[0080] Changes in wall thickness can significantly affect digestibility. Figure 15 In addition, the tube material needs to be considered. For example, when using QuanRecovery vials and QuanRecovery plates (available from WaterTechnologies Corporation, Milford, MA), polypropylene may cause nonspecific binding problems. These problems can be resolved by introducing plasma treatment. While blocking reagents can sometimes be useful in the presence of bovine serum albumin, surfactants, organic solvents, and detergents, they can introduce significant noise or peptide loss into downstream analyses. If processing larger quantities of samples at once is required, 200 μL PCR tubes can be easily converted to PCR plates, or even 384-well plates.

[0081] This disclosure also includes steps following digestion. For modified enzymes, quenching is required to terminate the reaction. Trypsin digestion can be stopped by freezing or by lowering the pH of the reaction to below pH 4 by adding formic acid, acetic acid, or trifluoroacetic acid.

[0082] If the immobilized enzyme is in dispersion form, the solid carrier needs to be removed by centrifugation, filtration, or magnetic bead removal. The choice of filtration membrane or device should minimize non-specific binding of the peptide or other analyte of interest.

[0083] Automated digestion systems employing plate-based digestion with liquid handling capabilities may include denaturation, reduction, alkylation, desalting equipment, and digestion performed on a heater. If immobilized enzymes are used, they can be removed via positive pressure manifold or vacuum-assisted filtration. If enzymes in modified solution are used, the reaction can be quenched for downstream analysis.

[0084] Automated systems may include flow-through digestion, in which proteins are digested as they pass through a column, chip, or any surface, with or without pretreatment.

[0085] Resin can be removed from the reaction mixture using various methods, including centrifugation and filtration. Filtration may introduce sample loss because membranes made of cellulose acetate or polyvinylidene fluoride (PVDF) are known to absorb peptides. Inert materials or modified membranes should be considered to achieve maximum recovery.

[0086] In some examples, adding low concentrations (approximately 5 mM to 100 mM) of arginine, dimethylarginine, guanidine, or other derivatives that exhibit ionization properties to the digestion buffer can significantly reduce sample loss. These relatively low concentrations of ionization reagents reduce nonspecific binding during filtration without significantly affecting the enzyme reaction. Alternatively, these reagents can be added during the filtration step to pretreat the filter or to filter simultaneously with the digest to minimize nonspecific binding. These measures ensure good recovery of hydrophobic peptides. For example, Figure 3A and Figure 3B The effects of introducing low concentrations (20 mM and 50 mM) of arginine additives on the recovery of hydrophobic peptides were shown. Figure 3A ) and digestion efficiency ( Figure 3B The impact of ). Figure 3C The effects of introducing 5 mM and 20 mM dimethylarginine on the recovery rate of hydrophobic peptides were presented. Figure 3D The effects of introducing 5 mM and 20 mM dimethylarginine on digestion efficiency are presented.

[0087] In some examples, this technology provides a method for processing samples containing proteins. The method may include the following steps: adding a sample to a device containing a buffer and a solid carrier surface including a surface coating, wherein the surface coating immobilizes an enzyme and an affinity ligand while reducing undesirable interactions between the sample and the solid carrier surface; immobilizing an enzyme on the surface coating for digesting proteins in the sample; immobilizing an affinity ligand on the surface coating for targeting and capturing a portion of the sample; digesting the proteins in the sample with the immobilized enzyme on the surface coating; targeting and capturing that portion of the sample with the immobilized affinity ligand on the surface coating; and heating the sample to activate protein digestion.

[0088] The step of targeting and capturing that portion of the sample can occur before, during, or after the step of digesting the proteins in the sample. In some embodiments, the step of targeting and capturing a portion of the sample occurs before, during, and / or after the step of digesting the proteins (e.g., two or all three of the indicated digestion time periods). The method may also include adjusting reaction conditions to determine whether the step of targeting and capturing that portion of the sample occurs before, during, or after the step of digesting the proteins in the sample.

[0089] Reaction conditions may include buffer composition, reaction temperature, or reaction pH. The solid support surface may be particles. In some examples, the enzyme and affinity ligand are immobilized on the same particles. Heating the sample to activate the purification or digestion of proteins in the sample may include digesting the proteins in the sample with the immobilized enzyme at elevated temperatures. When processing a sample, more than one buffer may be present. For example, a targeted capture elution buffer and a digestion buffer may be present.

[0090] The methods described above can utilize any type of affinity ligand. Examples of usable affinity ligands include, but are not limited to, immunoglobulin-binding proteins, such as protein A, protein G, protein L, or mixtures thereof. Affinity ligands can also be antigen conjugates, such as antibodies, nanobodies, or mixtures thereof. Affinity ligands can also be aptamers.

[0091] The affinity ligand can be immobilized on a solid support, wherein a coating covers the surface of the solid support. The coating can provide at least 5 μmol / m 2 The surface coverage is improved, and undesirable interactions between the protein sample and the solid support surface are reduced, while also enabling covalent binding of affinity ligands. The solid support on which the affinity ligands are immobilized can be non-porous, porous, or magnetic, and can take the form of membranes, particles, bulk materials, device surfaces, or microchip surfaces. The immobilized affinity ligands are then filled into devices. Devices can be plate wells, pipette tips, channels on microchips, or tubes with glass frit at one or both ends.

[0092] Those skilled in the art will understand that various changes in form and detail may be made without departing from the scope of the technology disclosed herein. For example, alternative modifications include:

[0093] • Form factors of the reaction mixture (50 μL, 100 μL, 200 μL, 500 μL, 1000 μL)

[0094] Immobilized resin filled into columns, tips, and filter plates

[0095] • Enzymes other than trypsin (chymotrypsin, pepsin, proteinase K, lysC, IdeS, Glu-C, Arg-C, Asp N, papain, streptomycin, and PNGase F, and combinations thereof)

[0096] • Enzymes modified with different hydrophilic cross-linking agents and modifiers;

[0097] • For some proteins, it is not necessary to perform every step of the digestion workflow. Proteins without disulfide bonds do not require reduction and alkylation, and are often denatured by heating themselves.

[0098] Alternative uses (e.g., for biomedical applications) include:

[0099] • Preparation of bioanalytical samples

[0100] • Preparation of released glycan samples

[0101] Subunit analysis sample preparation

[0102] • Proteomics Sample Preparation

[0103] result

[0104] Example 1. Thermal stability of immobilized enzymes obtained by NanoDSC

[0105] NanoDSC is routinely used to study the thermodynamics of proteins or polymers. In a NanoDSC experiment, a protein sample in a buffer solution is scanned at a specific heating rate (°C / min), during which peptide bonds and interactions are broken. The development process continues until the protein is completely denatured. The temperature at the midpoint of the development process determines when half of the molecules have developed, indicating the protein's thermal stability. In this example, we used trypsin to investigate the beneficial effects of immobilization. For all samples, the NanoDSC run was set to scan at 1°C / min, from 10°C to 100°C. Each sample contained approximately 1 mg / mL of trypsin, and approximately 330 μL was used for two scans, with the second scan serving as a reference scan to illustrate changes not caused by trypsin denaturation. Figure 1 As shown, when trypsin is immobilized on a solid support, T m The temperature changed from approximately 45°C to 80°C. The prototype based on silicon dioxide was used in T... m Significant improvements have also been made, particularly in the commercial product Smart Digest. TM Compared to that, it increased by at least 5°C.

[0106] Example 2. Thermal stability of modified immobilized trypsin obtained by NanoDSC

[0107] Preferably, two hydrophilic crosslinking agents with preferred lengths were selected. For example... Figure 2 As shown, compared with unmodified trypsin, cross-linked trypsin has a higher T value. m The temperature was increased by 5°C. Site-specific modification targeting specific amino acids also showed an effect on T... m Some improvements, though not so significant, such as Figure 2 As shown.

[0108] Example 3. Non-specific binding test of surface chemicals on immobilized carriers

[0109] 10 μg of NIST mAb standard digest (Waters) was mixed with 10 μL of resin and diluted to 200 μL with digestion buffer containing 50 mM Tris, 250 mM CaCl2, and 5% glycerol. The mixture was then incubated at 75 °C in an Eppendorf container. Incubate at 1400 rpm for 5 minutes on a C shaker (available from Eppendorf, Hamburg, Germany), then centrifuge at 3,000 g for 1 minute, and submit 100 μL of the supernatant for LC-MS analysis.

[0110] By mixing those with similar pore sizes to The nonspecific binding effect of NIST mAb digests on two different surface chemicals was evaluated. Peptides were collected for analysis after 5 minutes of incubation. The lowest recovery of modified PS-DVB among the selected hydrophobic peptides is shown here. Similarly, nonspecific binding effects were tested in different hydrophilic coatings immobilized with the same amount of trypsin. Figure 4 As shown, for all the peptides tested, SmartDigest... TM All tests showed poor recovery rates. However, this basic test only reflects the non-specific binding effect of peptides after digestion from proteins.

[0111] Table 1: Parameters of LC-MS analysis performed on BioAccord

[0112]

[0113]

[0114] Example 4. Quantification of released trypsin

[0115] Dilute 10 μL of resin to 200 μL using a digestion buffer containing 50 mM Tris, 250 mM CaCl2, and 5% glycerol. Incubate the mixture at 75 °C on an Eppendorf thermo mixer C at 1400 rpm for 30 min and 60 min, then centrifuge at 3,000 g for 1 min. Submit 100 μL of the supernatant for fluorescence analysis (excitation: 280 nm, emission: 370 nm). A calibration curve is generated using free trypsin dissolved in digestion buffer at concentrations ranging from 0.004 mg / mL to 0.4 mg / mL.

[0116] The released trypsin was quantified after incubation at 70°C for 30 and 60 minutes. Figure 5 As shown, after 30 minutes of incubation, Smart Digest was observed to... TM The loss of immobilized trypsin on the substrate was approximately 20%, while other prototypes (i.e., silica-based solid carriers with coatings of the present invention) showed a loss of less than 10%.

[0117] Example 5. Digestion of NIST mAb using immobilized enzymes

[0118] Approximately 50 μg of NIST mAb was denatured and reduced in 8 M guanidine buffer with 5 mM DTT for 1 h, followed by alkylation with 15 mM IAM in the dark for 30 min. The alkylated protein was then desalted using a NAP-5 column (GE Healthcare) and mixed with 15 μL of immobilized enzyme. Digestion was performed on a shaker at 70 °C for 10 min, and 100 μL of the supernatant was submitted for LC-MS analysis.

[0119] As shown in Figure 6, NIST mAb was used as a model protein to be digested by immobilized trypsin. The peptide map generated by standard LC-MS analysis showed overall characteristics similar to digestion in solution; however, Smart Digest... TM More severe incomplete digestion occurred, with more than 20% of the material not being digested, while the better prototype showed only about 8%. Figure 7 (and Figure 8). Incomplete digestion may be caused by unfavorable surface chemicals of the immobilized carrier that induce nonspecific binding and interactions, and may also be related to the unconjugation of trypsin as discussed in Examples 3 and 4. The prototypes tested utilized a hydrophilic coating with adjustable "thickness" to maximize digestion efficiency. Figure 7 As shown, the optimal digestion efficiency was achieved by using a preferred coating of the modifier labeled as modified prototype 3.

[0120] Example 6. Effect of temperature on digestion efficiency

[0121] According to Example 5, the samples were digested at 60°C, 70°C, and 80°C, as follows: Figure 8A , Figure 8B and Figure 8C As shown.

[0122] Example 7.Ca 2+ Effects on digestion efficiency and nonspecific binding

[0123] According to Example 5, the Ca content from 1 mM to 50 mM was evaluated. 2+ Effects on digestion, such as Figure 9A and Figure 9B As shown.

[0124] Example 8. Effect of pH on digestion efficiency and deamidation.

[0125] According to Example 5, the samples were digested at pH 7.6 and pH 6.6, as shown in Table 2 below.

[0126] Table 2. pH of buffer solution measured at different temperatures

[0127] temperature pH of 50 mM Tris solution pH for immobilizing trypsin 25℃ 7.6 6.6 70℃ 7.0 5.8

[0128] Example 9. Effect of additives on digestion efficiency

[0129] 5% polyol was added to the digestion buffer, and the sample was digested according to Example 5. The effect of 50 mM methionine on preventing artificial oxidation was evaluated. 5% and 10% acetonitrile were added to the digestion buffer to evaluate their effect on reducing artificial deamidation, such as... Figure 14 As shown.

[0130] Example 10. PCR tube screening

[0131] According to Example 5, the samples were digested in five different PCR tubes from Biologix, Axygen, Andwin Scientific, RPIScientific, and Applied Biosciences (see Example 5). Figure 15 and Figure 17 ).

[0132] Example 11. Tris concentration optimization

[0133] Using 50 mM, 100 mM, and 200 mM Tris as digestion buffers, and following Example 5, the protein sample was digested at 70°C. Figure 10A and Figure 10B As shown.

[0134] Example 12. Effects of arginine and dimethyl-arginine on the recovery rate of hydrophobic peptides

[0135] 5 mM and 20 mM dimethyl-arginine and 20 mM and 50 mM arginine were added to the digestion buffer, and the protein sample was digested at 70°C according to Example 5. Figure 3A and Figure 3B As shown. All protein digests are filtered through a PVDF membrane filter with a positive pressure manifold.

[0136] While this disclosure has been specifically shown and described with reference to exemplary embodiments thereof, those skilled in the art will understand that various changes in form and detail may be made without departing from the scope of the technology as covered by the appended claims. For example, other chromatographic or detection systems may be used.

Claims

1. A method for digesting a sample containing IgG1 antibodies, the method comprising: The sample is added to a device that contains a buffer solution and a solid carrier surface including a surface coating, wherein the surface coating comprises two parts: a first coating part having functional groups for bioconjugation, and a second coating part having functional groups that reduce nonspecific binding between the IgG1 antibody and the solid carrier surface. Trypsin is immobilized on the surface coating for digesting the IgG1 antibody in the sample, and wherein the immobilized surface coating includes a hydrophilic coating having immobilized trypsin attached to its surface. The pH of the solution within the device is between 5.0 and 9.0; and The sample is heated to complete the thermally assisted digestion of the IgG1 antibody, wherein the heating occurs in an elevated temperature range from above 45°C to no more than 85°C, and wherein complete digestion includes digesting the IgG1 antibody to less than 15% of uncut portions, and wherein complete digestion occurs within 10 minutes.

2. The method of claim 1, wherein the buffer solution is selected from the group consisting of Tris, BIS-Tris, MES, HEPES, triethanolamine, and trimethylamine.

3. The method of claim 1, wherein the buffer solution in the solution further comprises one or more additives selected from the group consisting of xylitol, methionine, and CaCl2.

4. The method of claim 1, wherein heating the sample to complete the digestion of the IgG1 antibody occurs within 5 minutes.

5. The method of claim 1, wherein heating is applied using a device selected from the group consisting of: an oven, an incubator, a shaker, and a heat mixer.

6. The method of claim 1, wherein the second coating portion makes the surface hydrophilic.

7. The method of claim 6, wherein the surface coating provides at least 5 µmol / m² on the surface of the solid carrier. 2 Surface coverage.

8. The method according to claim 1, wherein the trypsin is modified with hydrophilic crosslinking groups to increase chemical and thermal stability.

9. The method of claim 1, wherein the trypsin is modified with a hydrophobic modifier to increase its affinity for the IgG1 antibody.

10. The method of claim 1, wherein the solid carrier comprises a polymer-based material, a silica-based material, a hybrid material, agarose, or cellulose.

11. The method of claim 1, wherein the solid carrier on which the trypsin is immobilized is non-porous, porous, magnetic, and in the form of a membrane, particle, solid material, surface of a device, or surface of a microchip.

12. The method of claim 1, wherein the entire process of digesting the IgG1 antibody in the sample includes one or more pretreatment steps performed on the sample prior to heating the sample to complete the digestion.

13. The method of claim 12, wherein the one or more pretreatment steps comprise denaturing the IgG1 antibody in the sample, reducing the IgG1 antibody in the sample, alkylating the IgG1 antibody in the sample, or desalting the IgG1 antibody in the sample, or any combination thereof.

14. The method of claim 1, wherein heating the sample to complete digestion provides digestion of the IgG1 antibody to have less than 15% uncut and greater than 85% sequence coverage.

15. The method according to any one of claims 1 to 14, the method further comprising submitting the digested IgG1 antibody to downstream analysis, said downstream analysis comprising at least one of liquid chromatography-ultraviolet (LC-UV) or liquid chromatography-mass spectrometry (LC-MS).

16. The method of claim 1, wherein The pH of the sample solution within the device is between 5.0 and 7.0; and The second coating portion makes the surface hydrophilic, and the functional group comprises a diol obtained by hydrolysis of an epoxy-containing silane, wherein the epoxy-containing silane is selected from the group consisting of 3-glycidoxypropyl trifunctional silane and 2-(3,4-epoxycyclohexyl)ethyl trifunctional silane.

17. The method of claim 16, wherein the IgG1 antibody is digested within 5 minutes.

18. A method for processing a sample containing IgG1 antibodies, the method comprising: The sample is added to a device that contains a buffer solution and a solid carrier surface including a surface coating, wherein the surface coating comprises two parts: a first coating part having functional groups for bioconjugation; The second coating portion has functional groups that reduce non-specific binding between the IgG1 antibody and the surface of the solid carrier; Trypsin is immobilized on the surface coating for digesting the IgG1 antibody in the sample, wherein the immobilized surface coating includes a hydrophilic coating having immobilized trypsin attached to its surface. The pH of the solution within the device is between 5.0 and 9.0; The affinity ligand is immobilized on the surface coating for targeted capture of a portion of the sample; The IgG1 antibody in the sample was digested with immobilized trypsin on the surface coating; The sample was targeted and captured using immobilized affinity ligands on the surface coating, and The sample is heated to complete the thermally assisted digestion of the IgG1 antibody, wherein the heating occurs in an elevated temperature range from above 45°C to no more than 85°C, and wherein complete digestion includes digesting the IgG1 antibody to less than 15% of uncut portions, and wherein complete digestion occurs within 10 minutes.

19. The method of claim 18, wherein the step of targeting and capturing said portion of the sample occurs before, during, or after the step of digesting the IgG1 antibody in the sample.

20. The method of claim 18, wherein the surface of the solid carrier is a particle.

21. The method of claim 20, wherein the trypsin and the affinity ligand are immobilized on the same particle.

22. The method of claim 18, wherein heating is applied using a device selected from the group consisting of: an oven, an incubator, a shaker, and a heat mixer.

23. The method of claim 18, wherein the surface coating provides at least 5 µmol / m² on the surface of the solid carrier. 2 Surface coverage.

24. A kit for preparing IgG1 antibody samples, the kit comprising: A solid carrier surface, the solid carrier surface including a surface coating, wherein the surface coating includes two parts: a first coating part containing immobilized trypsin; The second coating portion has functional groups that reduce non-specific binding between the IgG1 antibody and the surface of the solid carrier; The surface coating provides at least 5 µmol / m 2 Surface coverage; A container for holding a buffer solution; A container for containing the surface of the solid carrier and the buffer solution; as well as A heat source, which is used to assist in the preparation of the IgG1 antibody sample.

25. The kit of claim 24, wherein the heat source is a device selected from the group consisting of: an oven, an incubator, a shaker, and a heat mixer.

26. The kit according to claim 24, wherein the heat source includes a microwave heat source, a convection heat source, or an infrared heat source.

27. The kit of claim 24, wherein the solid carrier is non-porous, porous, or surface-porous, and is in the form of a particle, a bulk material, a membrane, or the surface of a device, microchip, or glass slide.

28. The kit of claim 24, wherein the surface of the solid carrier comprises a plurality of particles, each having a particle size in the range of 10 nm to 200 micrometers.

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