Method and apparatus for protein sequence analysis

Abstract

The present disclosure relates to methods and devices for protein sequence analysis, and the use of droplets for improving protein sequencing by accelerating enzyme digestion. The method comprises the following steps: a) forming a solution containing protein into droplets, the size of which is small enough to accelerate protein digestion; b) introducing the droplets into a mass spectrometer (MS) for real-time detection; c) obtaining the analysis results of the protein from the mass spectrometer (MS); the protein has been completely digested in the droplets before entering the mass spectrometer (MS). The method and device can achieve simple and almost complete protein digestion in a very short time and obtain a high sequence coverage.

Description

Technical Field

[0001] The present invention relates to methods and devices for protein sequence analysis. More specifically, the present invention relates to methods for protein sequence analysis and droplet-MS (droplet-mass spectrometer) devices for protein sequence analysis. The present invention also relates to the use of droplets for improving protein sequencing by accelerating enzymatic digestion, wherein the droplets are formed from a solution containing protein and the size of the droplets is small enough to result in accelerated protein digestion. Background Art

[0002] In bottom-up proteomics, enzymatic digestion of proteins is an essential and critical method to break down proteins into smaller peptides before their structure is analyzed by mass spectrometry (MS). 1 . In a typical enzymatic digestion process, a protein solution is mixed with an appropriate amount of an enzyme (e.g., trypsin) and incubated overnight at 37°C. However, such a process is time-consuming. To facilitate digestion, protein denaturation is usually performed before digestion to disrupt the dense globular structure and expose more proteolytic cleavage sites. Methods commonly used for protein denaturation include applying external pressure or additives, such as heat, radiation, or urea. In addition, reductive alkylation is often used to remove disulfide bonds. In order to further accelerate protein digestion, various attempts have been made to reduce the digestion time from overnight to a few minutes, including increasing the digestion temperature, using chromatographic columns or porous materials for immobilizing trypsin, adding organic solvents, applying microwave energy or focused ultrasound fields, or any combination of these. 2 However, traditional methods for protein sequence analysis still require a pretreatment step before enzymatic digestion, which is relatively time-consuming and inconvenient.

[0003] The optimal situation for enzymatic digestion in proteomics studies is achieved when all cleavage sites are digested, but in reality, enzymes often fail to cleave all scissile bonds even with sufficiently long reaction times. The main reason for failure to achieve complete coverage is the neighboring amino acids surrounding the cleavage site. It has been reported that the presence of acidic residues glutamic acid (E) or aspartic acid (D) near the cleavage site can significantly reduce the rate of protein hydrolysis by forming salt bridges with basic arginine (R) and lysine (K) and inhibiting R or K from approaching the complementary aspartate at the bottom of the trypsin active site.

[0004] In the past few years, it has been widely reported that microdroplets significantly accelerate various single-phase or two-phase organic reactions with slow kinetics or assisted by specific catalytic effects in the bulk phase. Many explanations for this behavior have been proposed, some of the earliest involving the concentration of reagents, which is increased by evaporation and fragmentation of the droplets. When combined with online MS analysis, droplet chemistry has also been used to capture and identify transient reaction intermediates, to study fast reaction kinetics by droplet fusion MS, to perform preparative synthesis, and also to aid materials synthesis. Many means have been constructed to generate micrometer-sized droplets (microdroplets), including microfluidics, surface drop coating, or different spray-based ionization methods. 3 . The exact reason for the acceleration of reaction rates in microdroplets has not yet been clearly determined, but it is generally believed to be mainly caused by the significant differences between the microdroplet environment and the corresponding bulk phase. Various factors (such as droplet size, surface charge, reagent limitation, solvent composition, and droplet evaporation) may affect this acceleration. In addition to achievements in organic synthesis, microdroplets have also attracted attention in promoting biochemical reactions due to the mildness of the process, especially because aqueous microdroplets provide a benign environment compatible with life. However, few studies have addressed the application of microdroplets in biochemical analysis.

[0005] References

[0006] 1.Shan, B., Yates, JR, Baek, M.-C., Zhang, Y. & Fonslow, BR Protein Analysis by Shotgun / Bottom-up Proteomics. Chem. Rev. 113, 2343–2394 (2013).

[0007] 2. Basile, F. & Hauser, N. Rapid online nonenzymatic protein digestion combining microwave heating acid hydrolysis and electrochemical oxidation. Anal. Chem. 83, 359–367 (2011).

[0008] 3. Yan, Summary of the invention

[0009] Technical issues

[0010] Conventional methods or devices for protein sequence analysis require a pretreatment step of enzymatic digestion, which is relatively time-consuming and inconvenient. Moreover, even if the reaction time is long enough, the enzyme often cannot cleave all scissile bonds. Therefore, there is a need in the art to reduce the digestion time of proteins and improve the sequence coverage for protein identification.

[0011] The present invention aims to solve the above problems and provides a method and device for protein sequence analysis.

[0012] Means of solving the problem

[0013] The inventors surprisingly found that despite the negative effects of acidic droplets on enzymatic digestion, droplets can still significantly accelerate the rate of protein hydrolysis, and the digestion rate increases significantly as the size of the aqueous droplets decreases. Based on this, when used directly in conjunction with a mass spectrometer (MS), droplets can be used as a simple, ultra-fast and powerful tool for protein analysis.

[0014] Therefore, the present invention relates to a method for protein sequence analysis, comprising the following steps: a) forming a solution containing protein into droplets, wherein the size of the droplets is small enough to accelerate protein digestion; b) introducing the droplets into a mass spectrometer (MS) for real-time detection; c) obtaining analysis results of the protein from the mass spectrometer (MS); the protein has been completely digested in the droplets before entering the mass spectrometer (MS).

[0015] Furthermore, the method of the present invention does not require any pretreatment steps for enzymatic digestion.

[0016] Furthermore, according to the method of the present invention, the protein is refractory to trypsin digestion.

[0017] Furthermore, according to the method of the present invention, the pH value of the solution containing the protein is 4 to 11.

[0018] Furthermore, according to the method of the present invention, the pH value of the solution containing the protein is about 8.

[0019] Furthermore, according to the method of the present invention, in step a), the droplets are formed by a spray-based ionization method.

[0020] Furthermore, according to the method of the present invention, in step a), the droplets are formed by a capillary of the nebulizer.

[0021] Furthermore, according to the method of the present invention, pressurized atomizing N2 gas is supplied to the nebulizer at a pressure above 120 psi.

[0022] Furthermore, according to the method of the present invention, a positive voltage or a negative voltage of 3 kV or more is applied to the nebulizer.

[0023] Furthermore, according to the method of the present invention, the distance travelled by the droplet before entering the mass spectrometer is 10 mm to 10 cm.

[0024] Furthermore, according to the method of the present invention, the distance travelled by the droplets before entering the mass spectrometer is 50 mm.

[0025] Furthermore, according to the method of the present invention, the distance travelled by the droplets before entering the mass spectrometer is 2 cm.

[0026] On the other hand, the present invention relates to a droplet-MS device for protein sequence analysis, comprising: a droplet generating unit and a mass spectrometer (MS) unit, wherein the mass spectrometer (MS) unit is directly connected to the droplet generating unit, and the droplet generating unit causes a solution containing proteins to form droplets, wherein the size of the droplets is small enough to result in accelerated protein digestion.

[0027] Furthermore, according to the droplet-MS apparatus of the present invention, the droplet generating unit includes a nebulizer.

[0028] Furthermore, according to the droplet-MS apparatus of the present invention, the sprayer has a capillary having an inner diameter of 50-100 μm.

[0029] Furthermore, according to the droplet-MS apparatus of the present invention, a positive voltage or a negative voltage of 3 kV or more is applied to the nebulizer.

[0030] Furthermore, according to the droplet-MS apparatus of the present invention, the travel distance of the droplet from the nebulizer to the mass spectrometer is 10 mm to 10 cm.

[0031] Furthermore, according to the droplet-MS apparatus of the present invention, the travel distance of the droplets from the nebulizer to the mass spectrometer is 50 mm.

[0032] Furthermore, according to the droplet-MS apparatus of the present invention, the travel distance of the droplets from the nebulizer to the mass spectrometer is 2 cm.

[0033] In another aspect, the present invention relates to the use of droplets for improving protein sequencing by accelerating enzymatic digestion, wherein the droplets are formed from a solution containing the protein and the size of the droplets is small enough to result in accelerated protein digestion.

[0034] Beneficial effects of the present invention

[0035] The present invention relates to a method for protein sequence analysis, comprising the following steps: a) forming a solution containing protein into droplets, the size of the droplets being small enough to accelerate protein digestion; b) introducing the droplets into a mass spectrometer (MS) for real-time detection; c) obtaining protein analysis results from the mass spectrometer (MS); the protein has been completely digested in the droplets before entering the mass spectrometer (MS). By using room temperature droplet chemistry, the method of the present invention achieves simple and almost complete protein digestion in a very short time (e.g., less than 1 ms).

[0036] The present invention also relates to a droplet-MS device for protein sequence analysis, comprising: a droplet generation unit and a mass spectrometer (MS) unit, wherein the mass spectrometer (MS) unit is directly connected to the droplet generation unit, wherein the droplet generation unit forms droplets from a solution containing proteins, wherein the size of the droplets is small enough to accelerate protein digestion. The droplet-MS device of the present invention provides a suitable interface to directly connect sample separation with MS to perform continuous digestion and online analysis of trace protein mixtures, and the droplet generation unit in the device also acts as an MS emitter.

[0037] The method and apparatus of the present invention achieve simple and nearly complete protein digestion in a very short time and obtain high sequence coverage. Unexpectedly, the droplets generated during electroacoustic spray ionization (ESSI) and directly coupled to a mass spectrometer (droplet-MS) can achieve online digestion of relatively large peptides. Therefore, the method and apparatus of the present invention for protein sequence analysis do not require any pretreatment steps for enzymatic digestion.

[0038] It is demonstrated herein that droplets are a practical and nearly universal technology for protein sequencing. In particular, droplet MS is able to significantly accelerate the digestion of proteins, even those that have proven particularly refractory to trypsin digestion. Thus, the methods and apparatus of the present invention are applicable to sequence analysis of a wide variety of proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : Schematic diagram of the experimental setup for online proteolysis by microdroplet chemistry coupled to mass spectrometry (microdroplet-MS). The inner capillary has an inner diameter of 50 μm and an outer diameter of 148 μm, to which a high-pressure source is connected.

[0040] Figure 2: Mass spectra of 10 μM human ACTH (1-24) in 5 mM NH4HCO3 sprayed by a homemade nebulizer: (a1) undigested (no trypsin); (a2-a5) digested with 5 μg / mL trypsin, with travel distances between the nebulizer tip and the MS injection port of 2 mm, 10 mm, 20 mm, and 50 mm, respectively. * indicates a peptide, and # indicates an undigested ACTH peak. (b) 26 peptides obtained by digestion of human ACTH (1-24). The sequence is SYSMEHFR|WGKPVGK|K|R|RPVK|VYP, with vertical lines added where trypsin digestion is expected to occur. (c) Intensity of the selected peptide at m / z 632.4 ( Figure 2 The ratio of the peak intensity of the intact multiply charged ACTH (pink marker in the middle) to the sum of all peak intensities varies with the droplet travel distance between the nebulizer tip and the MS inlet.

[0041] Figure 3 : MS / MS-CID spectrum of a peptide at m / z 632.4 from a 10 μM ACTH digest in 5 mM NH4HCO3 obtained by droplet-MS with a normalized energy of 25 and an isolation width of 1 m / z.

[0042] Figure 4 : Comparison of various methods for digestion of ACTH: (a) standard ESI-MS, (b) bulk phase digestion at 37°C for 3 h followed by standard ESI-MS analysis, (c) 16 peptide peaks found after bulk phase digestion of human ACTH (1-24) at 37°C for 3 h. * indicates peptides, # indicates undigested ACTH peaks.

[0043] Figure 5 : Mass spectra of myoglobin digested by various methods: (a) standard ESI-MS, (b) bulk phase digestion at 37°C for 14 h, followed by standard ESI-MS analysis and droplet-MS analysis, applying (c) a positive high voltage of +3 kV, and (d) a negative high voltage of -3 kV.

[0044] Figure 6 : Peptides identified from myoglobin digests by droplet-MS and standard MS with positive voltage applied.

[0045] Figure 7 : Peptides identified from myoglobin digest by droplet-MS with negative voltage applied.

[0046] Figure 8 : Peptides identified from a digest of cytochrome c by droplet-MS with application of positive voltage.

[0047] Fig. 9: (a) Peptide sequence, (b) mass spectra of pure peptides, and mass spectra of peptides digested by various methods: (c) standard ESI-MS, (d) bulk phase digestion at 37°C for 1 h followed by standard ESI-MS analysis, and (e) droplet-MS with a high voltage of +3 kV applied.

[0048] Fig.10 : Mass spectra of digests of synthetic peptides in 5 mM NH4HCO3 at different pH values ​​obtained by droplet-MS with applied positive voltage.

[0049] Fig.11 : Mass spectra of (a) α-casein and (b) cytochrome c trypsin microdroplet digestion at a positive voltage of +3 kV. (c) PAGE gel showing two protein bands stained with Coomassie Brilliant Blue. Red asterisks indicate peptides.

[0050] Fig.12 : Peptides identified from cytochrome c and α-casein digests extracted from SDS-PAGE gels by droplet-MS with applied positive voltage. DETAILED DESCRIPTION

[0051] Before explaining any embodiment of the present disclosure in detail, it should be understood that the application of the present invention is not limited to the construction details and the arrangement of parts set forth in the following description or shown in the drawings and examples. The section headings used herein are only for organizational purposes and should not be interpreted as limiting the subject matter described. For all purposes, all references cited in this application are expressly incorporated herein by reference.

[0052] The disclosure encompasses other embodiments and is practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein are for the purpose of description and should not be regarded as limiting.

[0053] As used herein, the term "electrospray ionization (ESSI)" refers to an ionization technique that combines electrospray ionization (ESI) and sonic spray ionization (SSI).

[0054] As used herein, the term "collision induced dissociation (CID)" refers to a tandem mass spectrometry technique that induces fragmentation of selected ions by collision with the gas phase.

[0055] As used herein, the term "adrenocorticotropic hormone (ACTH)" refers to a polypeptide consisting of 39 amino acids and produced by the anterior pituitary gland. The function of ACTH is to regulate the level of the steroid hormone Cortisol released by the adrenal glands.

[0056] The term "myoglobin" as used herein refers to a protein containing 153 amino acid residues and having an iron heme group at its center. Myoglobin (symbol Mb or MB) is an iron and oxygen binding protein that is normally found in the muscle tissue of vertebrates and is present in almost all mammals. Myoglobin has been shown to be difficult to digest with trypsin.

[0057] The protein sequence analysis method of the present invention comprises the following steps: a) forming a solution containing protein into droplets, wherein the size of the droplets is small enough to accelerate protein digestion; b) introducing the droplets into a mass spectrometer (MS) for real-time detection; c) obtaining analysis results of the protein from the mass spectrometer (MS); the protein has been completely digested in the droplets before entering the mass spectrometer (MS).

[0058] The droplet-MS device for protein sequence analysis of the present invention comprises: a droplet generation unit and a mass spectrometer (MS) unit, wherein the mass spectrometer (MS) unit is directly connected to the droplet generation unit, wherein the droplet generation unit forms droplets from a solution containing proteins, wherein the size of the droplets is small enough to accelerate protein digestion. The droplet-MS device of the present invention is a useful tool for protein sequence analysis. In particular, droplet-MS is a powerful proteolytic tool that can easily cleave most theoretically cleavable bonds and produce fewer missed cleavage peptides.

[0059] In bottom-up proteomics, sample preparation including protein digestion is a lengthy step and still encounters a bottleneck in terms of time. Table 1 lists various methods to accelerate protein digestion.

[0060] Table 1. Comparison of various techniques for accelerating protein digestion.

[0061]

[0062]

[0063] The inventors surprisingly found that digestibility increased significantly as the size of the aqueous droplets decreased. This finding suggests that as the droplet diameter decreases, the ratio of droplet surface area to droplet volume increases and protein bonds are cleaved at or near the air-water interface.

[0064] As shown in Table 1, according to the present invention, the shortest digestion time (less than 1 ms) was achieved by combining droplets with a mass spectrometer (MS). In addition, digestion in droplets consumes only a very small amount of sample (0.16 μL / s) and can be easily extended to other devices that generate droplets.

[0065] Microdroplets can be produced by various spray-based ionization methods.

[0066] In a preferred embodiment, in order to achieve better acceleration of enzymatic digestion while ensuring good MS signal, capillaries with a range of diameters are used to obtain the desired droplets.

[0067] In a preferred embodiment, the droplets are generated by a nebulizer having a capillary with an inner diameter of 50-100 μm. If the size of the capillary is too large, the droplets generated are too large and the acceleration effect is reduced. If the size of the capillary is too small, the sample may not be injected continuously, which may deteriorate the MS signal. In one embodiment, the capillary has an inner diameter of 60 μm, 70 μm, 80 μm or 90 μm.

[0068] Furthermore, to achieve better acceleration of enzymatic digestion, the pressure of the N2 nebulizing gas applied to the nebulizer was increased to obtain smaller droplets.

[0069] In a particularly preferred embodiment, the pressure of the N2 pressurized atomizing gas applied to the nebulizer is above 120 psi, preferably above 130 psi, more preferably above 140 psi, even more preferably above 150 psi, even more preferably above 200 psi.

[0070] It will be appreciated that, although negative potentials are preferred over positive potentials, the temperature of the mass spectrometer inlet and the polarity of the voltage applied to the spray capillary have little effect on the digestion process. Even without any voltage applied it is effective, but the effect is reduced.

[0071] In a preferred embodiment, the droplets containing proteins and enzymes are generated by electroacoustic spraying. In electroacoustic spraying, a sheath stream of fast-flowing dry N2 gas surrounds a capillary that is typically maintained at -3 kV.

[0072] In a preferred embodiment, collision induced dissociation (CID) is used for fragmentation of isolated precursor ions for sequencing of the peptide of interest, in full scan mode, with an isolation width of 1 m / z and an optimized collision energy of 25.

[0073] In a preferred embodiment, protein sequences and their specific accession numbers are obtained from the UniprotKB database. The target protein is digested in silico using the MS-digest program of Protein Prospector version 5.19.1 (University of California, San Francisco, CA, USA), and the peptide sequence is identified by comparing the observed peptide molecular weight with the theoretical molecular weight.

[0074] Once the droplets enter the heated injection port, they evaporate and the reaction stops. Therefore, the digestion time in the droplets can be determined by the travel distance between the nebulizer tip and the MS injection port. And, the digestion efficiency is directly related to the reaction time in the droplets. Therefore, the digestion yield can be increased by increasing the travel distance.

[0075] In a preferred embodiment, the distance traveled by the droplets from the nebulizer to the mass spectrometer is at least 10 mm, preferably in the range of 10 mm to 10 cm, more preferably 20 mm, even more preferably 50 mm, even more preferably 2 cm. When the travel distance is 10 mm or more, the method of the present invention can successfully identify fragments covering the entire sequence of a polypeptide. When the travel distance is 50 mm or more, the method of the present invention can achieve almost complete digestion of relatively large polypeptides.

[0076] In a preferred embodiment, the pH value of the protein-containing solution used for droplet-MS analysis is greater than 4, preferably in the range of 4-11, and more preferably about 8. The pH value in the range of 4-11 allows droplet-MS to maintain an excellent effect of accelerating protein hydrolysis.

[0077] In a preferred embodiment, the method and apparatus of the present invention are used for the digestion and analysis of proteins, particularly proteins that have been shown to be particularly difficult to digest by trypsin. By using the method and apparatus of the present invention, the digestion time of proteins that have been shown to be particularly difficult to digest by trypsin can be significantly reduced, for example, from overnight to less than 1 ms.

[0078] In certain embodiments, proteins include, but are not limited to, adrenocorticotropic hormone (ACTH), myoglobin, cytochrome c, BSA, hemoglobin, β-lactoglobulin, casein, and other common proteins known in the art, as well as synthetic peptides.

[0079] When used directly in conjunction with MS, aqueous droplets can be used as a simple, ultrafast, and powerful tool for protein analysis. Proteolysis-resistant proteins such as myoglobin can be fully digested to obtain high sequence coverage, and theoretically cleavable peptide bonds can be fully cleaved, indicating that droplets provide a unique environment for great advances in sample mixing and protein structural alterations.

[0080] Therefore, the present invention also relates to the use of droplets for improving protein sequencing by accelerating enzymatic digestion, wherein the droplets are formed from a solution containing proteins and the size of the droplets is small enough to result in accelerated protein digestion.

[0081] Example

[0082] The present invention will be specifically described with reference to Examples, but the present invention is not limited to the Examples.

[0083] Ammonium bicarbonate and all protein reagents used were purchased from Sigma-Aldrich (Shanghai, China). Deionized water (18.2 MΩ cm) was prepared with a Milli Q purification system (Millipore Advantage A10) and used in all aqueous solutions.

[0084] Example 1

[0085] Optimizing the performance of droplet-MS.

[0086] To optimize the performance of droplet-MS, trypsin-digested ACTH was used as a simple model system.

[0087] A 5 mM ammonium bicarbonate (NH4HCO3, pH 8) aqueous solution containing 10 μM human adrenocorticotropic hormone (ACTH, 1-24, Genscript, China) and 5 μg / mL trypsin was injected into a homemade nebulizer (capillary inner diameter 50 μm, outer diameter 148 μm) at a flow rate of 10 μL / min using a syringe to generate a microdroplet flow (e.g. Figure 1 shown).

[0088] The sample solution was sprayed from the tip of a fused quartz capillary (148 μm OD, 50 μm ID, Polymicro Technologies, China) with the aid of dry N2 nebulizer gas at a pressure of 120 psi. The droplets were introduced into the MS for real-time detection by placing the nebulizer in an appropriate position in front of a high-resolution mass spectrometer (LTQ Orbitrap Elite, Thermo Scientific, San Jose, CA). The MS inlet capillary was maintained at 275 ° C and the capillary voltage was maintained at 0 V. No other source of gas was used when digesting in the droplets.

[0089] The droplet size was estimated to be about 6 μm in diameter. The droplets traveled in the air at a speed of 84 ± 18 m / s. The digestion time in the droplets depended on the travel distance between the nebulizer tip and the MS injection port.

[0090] like Figure 2 As shown, digestion progresses with increasing travel time into the mass spectrometer inlet. Digestion efficiency is directly related to the reaction time in the droplet, which can be determined by examining Figure 2 As shown in (a2-a5), the digestion yield of ACTH increased dramatically by increasing the travel distance from 2 mm to 20 mm. Figure 2As shown in b, 26 peptide peaks were successfully identified, completely covering the entire sequence of ACTH. When the distance was increased to 50 mm, corresponding to a digestion time of 0.6 ms (based on the previously reported droplet speed of 80 m / s), ACTH was almost completely digested. Figure 2 In (a5), only a small peak of ACTH appeared. Figure 2 The peak intensity of the most abundant peptide at m / z 632.4 in (a2)-2(a5) is comparable to all peaks of intact multiply charged ACTH (including (ACTH+3H) 3+ 、(ACTH+4H) 4+ 、(ACTH+5H) 5+ 、(ACTH+6H) 6+ and (ACTH+7H) 7+ ) and the dependence of the digestion yield on the distance traveled by the droplets is also clearly shown in Figure 2 c. The amount of selected peptide increases with the distance traveled. Figure 3 As shown, the sequence of the peptide was confirmed by tandem MS using collision induced dissociation (CID).

[0091] Comparative Example 1

[0092] ACTH digestion was performed using standard ESI-MS.

[0093] ACTH was mixed with a trypsin solution and injected into the MS for digestion and detection using a standard ESI source. During this process, only very small amounts of ACTH digestion were observed.

[0094] like Figure 4 As shown in a, only a small amount of digestion was observed due to the larger droplet size produced by the commercial ESI source (larger capillary inner diameter).

[0095] The acceleration of digestion due to standard ESI-MS is negligible (e.g. Figure 4 a), firstly, the same bulk solution was digested at 37°C for 3 hours using conventional methods: 10 μM human adrenocorticotropic hormone (ACTH, 1-24, Genscript, China) or 100 μg / mL protein was denatured by heating at 95°C for 5 minutes, and then incubated with 5 μg / mL trypsin in 5 mM NH4HCO3 buffer (pH 8) at 37°C. Aliquots of 100 μL were taken at different reaction times and frozen at -20°C to terminate the reaction. Then, the digestion products were recorded using standard ESI-MS.

[0096] For analysis using MS, samples were injected directly with a syringe at a flow rate of 10 μL / min and sprayed using a commercial electrospray ionization (ESI) source with a needle of 500 μm in diameter, supplemented by a sheath gas flow of 10 arbitrary units and a gas flow of 5 arbitrary units. The temperature of the MS inlet capillary was set to 275 °C and the ESI voltage was set to ±3 kV.

[0097] Compared with the 26 peptides that completely covered the entire sequence of ACTH detected by droplet-MS in Example 1 ( Figure 2 b), using standard ESI-MS, only 16 peptides were detected after 3 h of bulk phase digestion ( Figure 4 b and Figure 4 c).

[0098] Example 2

[0099] Digestion and analysis of proteins that are particularly refractory to trypsin using droplet-MS.

[0100] A 5 mM ammonium bicarbonate (NH4HCO3, pH 8) aqueous solution containing myoglobin (10 μM) and trypsin (5 μg / mL) was injected into a homemade nebulizer (capillary inner diameter 50 μm, outer diameter 148 μm) at a flow rate of 10 μL / min using a syringe to generate a microdroplet flow (e.g. Figure 1 shown).

[0101] The sample solution was sprayed from the tip of a fused quartz capillary (148 μm outer diameter, 50 μm inner diameter, Polymicro Technologies, China) with the aid of a dry N2 nebulizer gas at a pressure of 120 psi. The nebulizer was placed in an appropriate position in front of a high-resolution mass spectrometer (LTQ Orbitrap Elite, Thermo Scientific, San Jose, California), and a positive high voltage of +3 kV (BOHER HV, Genvolt, UK) was applied to the nebulizer to import the droplets into the MS for real-time detection. The MS inlet capillary was maintained at 275 ° C and the capillary voltage was maintained at 0 V. No other source of gas was used when digesting in the droplets.

[0102] like Figure 5 c and Figure 6 As shown, 31 peaks corresponding to 19 peptides were identified, and a high sequence coverage of 86% was obtained.

[0103] To find the missing sequences, a negative high voltage of -3 kV was applied instead of a positive high voltage due to being more compatible with the pH of trypsin digestion.

[0104] like Figure 5 d and Figure 7 As shown, surprisingly, 55 peaks corresponding to 38 peptides were identified. This corresponds to 100% sequence coverage. By matching the experimental results with the results of computer simulation digestion, it was found that all theoretically cleavable peptide bonds after K and R were destroyed, except when proline was followed by K and R, which was not destroyed due to steric hindrance. In addition, it was shown that trypsin digested almost all proteins in the starting solution under the action of droplet-MS.

[0105] Comparative Example 2

[0106] Myoglobin digestion was performed using standard ESI-MS.

[0107] Myoglobin was mixed with trypsin solution and injected into MS for digestion and detection with a standard ESI source. During this process, only very small amounts of myoglobin digestion were observed.

[0108] like Figure 5 As shown in a, only a small amount of digestion was observed.

[0109] The acceleration of digestion due to standard ESI-MS is negligible (e.g. Figure 5 a), first the same bulk solution was digested at 37°C for 14 hours using a conventional method: 10 μM human adrenocorticotropic hormone (ACTH, 1-24, Genscript, China) or 100 μg / mL protein was denatured by heating at 95°C for 5 minutes, and then incubated with 5 μg / mL trypsin in 5 mM NH4HCO3 buffer (pH 8) at 37°C. Aliquots of 100 μL were taken at different reaction times and frozen at -20°C to terminate the reaction. Then, the digestion products were recorded using standard ESI-MS.

[0110] For analysis using MS, samples were injected directly with a syringe at a flow rate of 10 μL / min and sprayed using a commercial electrospray ionization (ESI) source with a needle of 500 μm in diameter, supplemented by a sheath gas flow of 10 arbitrary units and a gas flow of 5 arbitrary units. The temperature of the MS inlet capillary was set to 275 °C and the ESI voltage was set to ±3 kV.

[0111] Compared with the 38 peptides corresponding to 100% sequence coverage of myoglobin detected by droplet-MS in Example 2 ( Figure 5 d), using standard ESI-MS, only 13 peptides corresponding to 60% sequence coverage were detected after 14 h of bulk phase digestion ( Figure 5 b), demonstrating that the digestion time of myoglobin can be significantly reduced from overnight to less than 1 millisecond by droplet-MS.

[0112] Example 3

[0113] Digestion and analysis of cytochrome c by droplet-MS.

[0114] Droplet-MS was further used for digestion and analysis of cytochrome c following the same method as described in Example 1 and Example 2 at a positive voltage of +3 kV.

[0115] like Figure 8 As shown, 33 peptides of cytochrome c were successfully identified (corresponding to 83% sequence coverage). The results show that droplet-MS is a versatile tool for protein digestion.

[0116] Example 4

[0117] Following the same method as described in Examples 1 and 2, the droplet-MS was used at +3 kV to measure the kinetic constant of the molecule with a very low kinetic constant (0.24×10 -3 s -1 ) were subjected to enzymatic digestion with a synthetic peptide sample (LYAA-[DTR]-LYAVR, 10 μM in 5 mM NH 4 HCO 3 ).

[0118] like Fig. 9 As shown in Figure e, the droplets can completely cleave the synthetic peptide sample (100% digestion), which is mainly because the droplets greatly accelerate the rate of protein hydrolysis (although the acidic droplets have an adverse effect on enzymatic digestion).

[0119] Comparative Example 3

[0120] The synthetic peptide sample (LYAA-[DTR]-LYAVR) was analyzed by standard ESI-MS.

[0121] The synthetic peptide sample (LYAA-[DTR]-LYAVR) was mixed with trypsin solution and injected into the MS for digestion and detection using a standard ESI source. During this process, only a small amount of peptide digestion was observed.

[0122] like Fig. 9 As shown in c, only about 5% digestion was observed by standard ESI-MS alone.

[0123] Therefore, the same bulk solution was digested at 37°C for 1 hour using the conventional method as described in Comparative Examples 1 and 2. The digestion products were then recorded using standard ESI-MS.

[0124] like Fig. 9 As shown in d, after 1 hour of digestion at 37°C, about 80% digestion was observed by standard ESI-MS, which is still better than the 100% digestion obtained by droplet-MS in Example 4 ( Fig. 9 e) is much lower.

[0125] Example 5

[0126] The pH range in which droplet-MS maintains excellent proteolysis acceleration.

[0127] It is known that higher H + Concentration can promote acid hydrolysis of protein, while higher OH - concentration can promote trypsin activity.

[0128] The inventors also studied the appropriate pH range in which droplet-MS can maintain excellent proteolysis acceleration. Synthetic peptides (LYAA-[DTR]-LYAVR) with different pH values ​​(i.e., pH 3-4, 5, and 11) in 5 mM NH4HCO3 were subjected to positive voltage droplet-MS analysis.

[0129] like Fig.10 As shown, it was found that when the pH was below 4, the accelerated digestion by droplet-MS was inhibited and only 40% of the synthetic peptides were digested. When the pH value reached 11, the droplet-MS could still completely digest the synthetic peptides. Therefore, the appropriate pH range can be determined to be 4-11.

[0130] Example 6

[0131] Utility in proteomics

[0132] To further demonstrate the utility in proteomics research, a protein mixture containing cytochrome c and α-casein was first separated by 15.5% SDS polyacrylamide gel electrophoresis (PAGE), as shown in Fig.11 Then, the Coomassie blue-stained protein bands were excised from the gel and digested by droplet-MS following the processing procedure described below.

[0133] To separate proteins by gel electrophoresis, 10 μL of sample solution containing cytochrome c (1 mg / mL) and α-casein (1 mg / mL) was loaded onto a 15.5% SDS-PAGE gel. All devices and reagents for gel electrophoresis were purchased from Sangon (Shanghai, China). Electrophoresis was performed at 200 V for 1 h at room temperature. A low-range protein ladder was used as a size marker. The gel was stained with Coomassie blue and then destained with an aqueous solution containing 30% ethanol and 12.5% ​​acetic acid. The stained protein bands were cut from the gel and ground into small pieces for efficient protein extraction using a commercial kit (Sangon, Shanghai). The samples were sonicated in an ultrasonic water bath for 30 minutes until the gel pieces became opaque. The extracted proteins were further purified by precipitating 3 times at -20 ° C in pure acetone (99.9%, Adamas, China) and then desalting with a centrifugal filter (Amicon Ultra-0.5, Millipore, USA) with a nominal molecular weight limit of 10 kDa. The purified protein samples were diluted in 5 mM NH 4 HCO 3 buffer, pH 8, and further digested by microdroplet-MS.

[0134] As a result, Fig.11 (a)-(b) and Fig.12 The mass spectra gave high sequence coverage of 90.3% and 99% for αS1-casein and cytochrome c, respectively. The time required to extract proteins from the gel and perform off-gel digestion using droplet-MS was significantly reduced from overnight in-gel digestion to less than 1 hour.

[0135] The above illustrates the tremendous advances and potential significance of droplet-MS in proteomics, including a significant reduction in digestion time (from overnight to less than one millisecond), complete cleavage of peptide bonds on the C-terminal side of lysine or arginine residues (except when followed by proline), and an increase in sequence coverage from 60% to 100%.

[0136] The present invention has been described in terms of specific embodiments that have been discovered or proposed, including specific modes for carrying out the invention. Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in conjunction with specific embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, it will be apparent to those skilled in the relevant art that various modifications of the described modes for carrying out the invention are within the scope of the appended claims. Sequence Listing <110> Fudan University <120> Method and apparatus for protein sequence analysis <160> 5 <170> PatentIn version 3.5 <210> 1 <211> twenty four <212> PRT <213> Artificial Sequence <220> <223> ACTH <400> 1 SYSMEHFRWG KPVGKKRRPV KVYP <210> 2 <211> 154 <212> PRT <213> Artificial Sequence <220> <223> Myoglobin <400> 2 MGLSDGEWQQ VLNVWGKVEA DIAGHGQEVL IRLFTGHPET LEKFDKFKHL KTEAEMKASEDLKKHGTVVL TALGGILKKK GHHEAELKPL AQSHATKHKI PIKYLEFISD AIIHVLHSKH PGDFGADAQGAMTKALELFR NDIAAKYKEL GFQG <210> 3 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> Cytochrome c <400> 3 MGDVEKGKKI FVQKCAQCHT VEKGGKHKTG PNLHGLFGRK TGQAPGFSYT DANKNKGITWGEETLMEYLE NPKKYIPGTK MIFAGIKKKG EREDLIAYLK KATNE <210> 4 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> αS1-Casein <400> 4 MKLLILTCLVAVALARPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELSKDIGSESTEDQAMEDIKQMEAESISSSEEIVPNSVEQKHIQKEDVPSERYLGYLEQLLRLKKYKVPQLEIVPNSAEERLHSMKEGIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEKTTMPLW <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 5 LYAADTRLYA VR

Claims

1. A protein sequence analysis method comprising the following steps: a) forming a protein-containing solution into droplets by means of a nebulizer, wherein the droplets are small enough to result in accelerated protein digestion; b) introducing the droplets into a mass spectrometer MS for real-time detection; c) Obtain protein analysis results from mass spectrometry MS; The pH value of the protein-containing solution is 4 to 11, The droplets travel a distance of 50 mm to 10 cm from the nebulizer to the mass spectrometer MS; The protein is completely digested in the droplets before entering the mass spectrometer MS. The method does not require any pretreatment steps for enzymatic digestion.

2. The method according to claim 1, wherein: The protein is refractory to trypsin digestion.

3. The method according to claim 1, wherein: The pH value of the protein-containing solution is 8.

4. The method according to claim 1, wherein: In step a), the droplets are formed by a spray-based ionization method.

5. The method according to claim 1, wherein: In step a), the droplets are formed by the capillary tube of the nebulizer.

6. The method according to claim 1, wherein: The nebulizer was supplied with pressurized nebulizing N2 gas at a pressure above 120 psi.

7. The method according to claim 1, wherein: A positive voltage or a negative voltage of 3 kV or more is applied to the nebulizer.

8. The method according to claim 1, wherein: The droplets travel a distance from the nebulizer to the mass spectrometer MS of 50 mm to 2 cm.

9. The method according to any one of claims 1 to 8, wherein: The diameter of the droplets was 6 μm.

10. A droplet-MS device for protein sequence analysis, comprising: a droplet generating unit, and a mass spectrometer MS unit, the mass spectrometer MS unit being directly connected to the droplet generation unit, The droplet generating unit comprises a sprayer, which forms droplets from a solution containing proteins, the size of the droplets being small enough to accelerate protein digestion, the distance traveled by the droplets from the sprayer to the mass spectrometer MS being 50 mm to 10 cm, and the pH value of the solution containing proteins being 4 to 11.

11. The droplet-MS device according to claim 10, wherein: The nebulizer has a capillary with an inner diameter of 50-100 μm.

12. The droplet-MS device according to claim 10, wherein: A positive voltage or a negative voltage of 3 kV or more is applied to the nebulizer.

13. The droplet-MS device according to claim 10, wherein: The droplets travel a distance from the nebulizer to the mass spectrometer MS of 50 mm to 2 cm.

14. The droplet-MS device according to any one of claims 10 to 13, wherein The diameter of the droplets was 6 μm.

15. Use of droplets for improving protein sequencing by accelerating enzymatic digestion, wherein: The droplets are formed by the method of any one of claims 1 to 9 or the device of any one of claims 10 to 14.

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