Novel internal standard substance for mass calibration

Abstract

The present invention relates to an internal standard substance for determining the quality of a protein and a method for detecting a protein in a biological sample using the internal standard substance. The present invention enables the measurement quality and quantitative value of a target protein to be quickly calibrated with a high degree of confidence by a simple process of adding a recombinant protein in which a labeled peptide is coupled to the target protein as an internal standard to a sample. Therefore, even if a low-resolution mass spectrometer is used, the high-molecular-weight target protein and other proteins with small mass difference in the sample can be clearly distinguished, so that the method can be effectively applied to high-sensitivity analysis of environmental samples, diagnosis of infectious pathogens and the like.

Description

Technical Field

[0001] The present invention relates to a method for accurately and effectively correcting the measured mass value of an analyte protein by using a recombinant protein as an internal standard, wherein the recombinant protein is formed by connecting a specific peptide segment with known mass information with a protein identical to the analyte protein. Background Art

[0002] Matrix desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry is an instrument that measures the molecular weight of an analyte by drying a mixture of sample and matrix to form a crystalline structure, irradiating it with a laser to desorb and ionize it, and measuring the time of flight to the detector. MALDI-TOF has the advantage of being able to quickly perform mass spectrometry on large molecules such as proteins because it does not fragment the analyte, but it also has the limitation of low resolution, making it difficult to accurately identify proteins with small mass differences. It is also important to note that drying a mixture of sample and matrix will produce an uneven crystal structure, resulting in low reproducibility.

[0003] At present, in order to improve the accuracy of MALDI-TOF mass spectrometry, the external standard method is usually used to calibrate the mass, but this method requires measuring the standard material at one point or multiple points to obtain the calibration parameters, which are then used for sample calibration at another point. When using external standards to calibrate the mass values ​​of high molecular weight proteins of 20 kDa or more in low-resolution MALDI-TOF instruments, it is difficult to expect accurate mass calibration due to the large error range of mass values ​​caused by different protein sizes, and there are limitations in separating / identifying proteins of similar sizes. In addition, although internal standards based on mass spectrometers have been reported, most of them are mainly used for quantitative methods at the peptide level, and it is also a challenge to develop quantitative methods for protein levels. Therefore, it is necessary to develop efficient mass spectrometry calibration and quantitative calibration methods to achieve more accurate mass analysis at the protein level.

[0004] Throughout this specification, many publications and patent documents are referenced and cited. In order to more clearly describe the state of the art and the content of the present invention, the disclosures of the cited publications and patent documents are fully incorporated herein by reference.

[0005] invention

[0006] Technical issues

[0007] The present inventors have conducted intensive research to develop an efficient mass spectrometry method for accurately and quickly separating and identifying high molecular weight target proteins in samples when using low-resolution mass spectrometry equipment. Therefore, the present inventors have found that when a recombinant protein formed by a tag peptide of any amino acid sequence with a known molecular weight connected to the N-terminus or C-terminus of a protein having the same amino acid sequence as the target protein is used as an internal standard, the mass and quantitative value of the target protein can be simply and accurately calibrated according to the molecular weight of the tag peptide corresponding to the molecular weight difference between the target protein and the internal standard, thereby completing the present invention.

[0008] Therefore, an object of the present invention is to provide an internal standard for determining protein quality.

[0009] Another object of the present invention is to provide a method for detecting proteins in a biological sample using the internal standard.

[0010] Other objects and advantages of the present invention will become more apparent from the following detailed description, the appended claims and the accompanying drawings.

[0011] Technical Solution

[0012] In one aspect of the present invention, there is provided an internal standard for determining the quality of a protein, comprising:

[0013] (a) a protein to be analyzed; and

[0014] (b) A tag peptide containing 1 to 100 consecutive random amino acids bound to the N-terminus or C-terminus of the protein to be analyzed.

[0015] The present inventors have conducted intensive research to develop an efficient mass spectrometry method for accurately and quickly separating and identifying high molecular weight target proteins in samples when using low-resolution mass spectrometry equipment. Therefore, the present inventors have found that when a recombinant protein formed by a tag peptide of any amino acid sequence with a known molecular weight bound to the N-terminus or C-terminus of a protein having the same amino acid sequence as the target protein is used as an internal standard, the mass and quantitative value of the target protein can be simply and accurately calibrated according to the molecular weight of the tag peptide corresponding to the molecular weight difference between the target protein and the internal standard.

[0016] As used herein, the term "protein" refers to a linear molecule formed by amino acid residues linked together by peptide bonds. In the present invention, the protein detected by mass spectrometry can be a protein used as a biomarker, for example, to confirm the presence of a pathogenic strain in a sample (i.e., diagnosis of pathogen infection) or to identify its type and phenotype.

[0017] As used herein, the term "pathogenic strain" refers to any bacteria that is the cause of infection or disease, including, but not limited to, for example, Staphylococcus aureus, Streptococcus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas otitidis, Micrococcus luteus, Citrobacter koseri, Proteus mirabilis, and Mycobacterium ulcerans.

[0018] The target proteins to be analyzed in the present invention include, for example, marker proteins that can predict the presence of pathogenic strains and their phenotypes (such as antibiotic resistance).

[0019] As used herein, the term "antibiotic resistance" means that a particular pathogenic microorganism can grow even in an environment containing a high concentration or effective amount of an antibiotic targeting the microorganism. Whether a pathogenic microorganism has antibiotic resistance can be determined by detecting the presence of an enzyme protein that is secreted by the pathogenic microorganism and removes or reduces the activity of the antibiotic by degrading the antibiotic. For example, β-lactam antibiotics that inhibit bacterial cell wall synthesis, such as penicillins, cephalosporins, monocyclic-β-lactams and carbapenems, are inactivated by β-lactamases, so they cannot inhibit pathogens that express β-lactamases. Therefore, the term "resistance" is used interchangeably with the term "low therapeutic responsiveness".

[0020] As used herein, the term "internal standard" refers to a compound with a known concentration or mass that is added to a sample to be analyzed in an indicated amount. It corrects quantitative errors in the mass of the target analyte (i.e., target protein) by comparing the signal from the target analyte with the signal from the internal standard and evaluating their difference or ratio.

[0021] As used herein, the term "tag peptide" refers to an amino acid sequence that is gene-transferred into a recombinant protein for various purposes, such as facilitating separation, purification, solubilization, visual labeling, etc. of a target protein. The tag peptide of the present invention can be used as a component of an internal standard to calibrate the mass value of the target protein by coupling it to the N-terminus or C-terminus of a recombinant protein having the same amino acid sequence as the target protein to be detected, thereby generating a mass difference between the target protein and the internal standard equal to the molecular weight of the tag peptide during mass spectrometry analysis. Therefore, the tag peptide of the present invention does not need to be removed by a protease or the like after analysis, because it only constitutes a part of the internal standard, unlike the case where it is directly bound to the target protein to be analyzed.

[0022] According to a specific embodiment of the present invention, the tag peptide contains 2 to 80 consecutive random amino acids bound to the C-terminus of the protein to be analyzed. More specifically, the tag peptide contains 2 to 70 consecutive random amino acids bound to the C-terminus, more specifically, the tag peptide contains 2 to 60 consecutive random amino acids bound to the C-terminus, more specifically, the tag peptide contains 2 to 50 consecutive random amino acids bound to the C-terminus, more specifically, the tag peptide contains 2 to 40 consecutive random amino acids bound to the C-terminus, more specifically, the tag peptide contains 2 to 30 consecutive random amino acids bound to the C-terminus, more specifically, the tag peptide contains 2 to 20 consecutive random amino acids bound to the C-terminus, more specifically, the tag peptide contains 2 to 10 consecutive random amino acids bound to the C-terminus, and most specifically, the tag peptide contains 2 to 8 consecutive random amino acids bound to the C-terminus.

[0023] According to a specific embodiment, the tag peptide is selected from the group consisting of a repeating sequence of identical amino acid residues, a consecutive sequence of different amino acid residues, a repeat of a consecutive sequence of different amino acid residues, or a combination thereof.

[0024] In a more specific embodiment, the identical amino acid is histidine (His).

[0025] According to a more specific embodiment, the continuous sequence of different amino acid residues includes the amino acids shown in SEQ ID NO: 7. According to the present invention, SEQ ID NO: 7 is the amino acid sequence of a Strep-tag peptide (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) having affinity for streptavidin.

[0026] The tag peptide of the present invention is not limited to the above examples, but may be any peptide that generates a molecular weight difference between the target protein and the internal standard by a tag peptide mass value calculated based on a known amino acid sequence.

[0027] According to a specific embodiment, the mass spectrometer is selected from the group consisting of matrix-assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometer, surface-enhanced laser desorption / ionization time of flight (SELDI-TOF) mass spectrometer, electrospray ionization time of flight (ESI-TOF) mass spectrometer, liquid chromatography-mass spectrometer (LC-MS) and liquid chromatography-mass spectrometer / mass spectrometer (LC-MS / MS). More specifically, the mass spectrometer is matrix desorption / ionization time of flight (MALDI-TOF) mass spectrometer.

[0028] MALDI-TOF mass spectrometry is a method in which a sample supported by a matrix is ​​desorbed and ionized by laser irradiation, and then the molecular weight of the generated ions is analyzed by measuring the time it takes for the ions to reach the detector (time of flight). According to this method, the mass of large biomolecules such as proteins can be measured quickly and accurately because the target substance does not fragment. When the ionized molecules are accelerated by an electric field and the flight time is measured, a mass-to-charge ratio (m / z) is generated, and the molecular weight of the target material can be determined from this m / z value.

[0029] At present, in order to improve the accuracy of MALDI-TOF mass spectrometry, external standards are mainly used for mass calibration. However, for high molecular weight proteins of 20 kDa or more, low-resolution MALDI-TOF instrument measurement using external standards will result in excessive mass value errors and make reliable analysis impossible. The present invention uses a recombinant protein with a labeled peptide coupled to the target protein as an internal standard, so that a high molecular weight target protein with a small mass difference from other proteins can be clearly identified from a sample even when a low-resolution MALDI-TOF device is used.

[0030] In another aspect of the present invention, a nucleic acid molecule encoding the internal standard of the present invention is provided.

[0031] As used herein, the term "nucleic acid molecule" is intended to include DNA (gDNA and cDNA) and RNA molecules. Nucleotides are the basic structural units of nucleic acid molecules, including not only natural nucleotides, but also analogs with modified sugar or base moieties (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)). It will be clear to those skilled in the art that the nucleotide sequence encoding the amino acid sequence of the internal standard of the present invention, i.e., a recombinant protein having a tag peptide bound to a target protein, is not limited to the nucleotide sequences listed in the attached sequence table or directly cloning the nucleotide sequences in the embodiments of the present invention. The modifications of nucleotides included in the embodiments include modifications that do not produce changes in protein levels, i.e., nucleic acid molecules with functionally equivalent codons, codons encoding the same amino acids due to codon degeneracy, or codons encoding biologically equivalent amino acids.

[0032] Considering the above-mentioned variants with biologically equivalent activity, it can be interpreted as the nucleotides listed in the sequence list or directly cloned in the examples, as well as the sequences showing substantial identity with the sequences. Substantial identity refers to sequences having at least 70% homology, specifically at least 75% homology, more specifically at least 80% homology, more specifically at least 85% homology, more specifically at least 90% homology, and more specifically at least 95% homology, determined by aligning the sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. The alignment method for sequence comparison is disclosed in Huang et al., Computer Applications in Biological Sciences (Comp. Appl. BioSci.) 8: 155-65 (1992) and Pearson et al., Methods of Molecular Biology (Meth. Mol. Biol.). 24: 37-31 (1994).

[0033] According to one embodiment, the internal standard of the present invention can be obtained recombinantly by expressing a nucleic acid molecule encoding it in a host cell.

[0034] As used herein, the term "expression" refers to artificial replication in target cells by chromosome integration as an extrachromosomal factor or through a gene delivery system, so that the target cells express exogenous genes or overexpress endogenous genes. Therefore, "expression" can be used interchangeably with "transformation", "transfection" or "transduction". More specifically, "expression" in the present invention refers to artificially expressing exogenous genes in target cells.

[0035] The term "gene delivery system" or "gene delivery vector" as used herein refers to any means for delivering genes into cells, and the term "gene delivery" has the same meaning as the intracellular transduction of genes. At the cellular or tissue level, gene delivery has the same meaning as gene propagation. Therefore, the gene delivery system of the present invention may be referred to as a gene transduction system or a gene propagation system.

[0036] In order to construct the gene delivery system of the present invention, the nucleotide sequence is effectively connected to a suitable expression control sequence in a suitable expression construct. As used herein, the term "effective connection" refers to the functional connection between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or a transcriptional regulatory factor binding site group) and a target nucleic acid sequence. Through the connection, the regulatory sequence regulates the transcription and / or translation of the target nucleic acid sequence.

[0037] The gene delivery system of the present invention can be produced in a variety of forms, including (i) naked recombinant DNA molecules, (ii) plasmids, (iii) viral vectors, and (iv) liposomes or liposomes containing naked recombinant DNA molecules or plasmids.

[0038] In another aspect of the present invention, a method for detecting a protein in a biological sample is provided, comprising:

[0039] (a) adding the internal standard substance described in any one of claims 1 to 7 to a biological sample containing a protein to be analyzed;

[0040] (b) measuring the mass values ​​of the protein to be analyzed and the internal standard in the biological sample; and

[0041] (c) The measured mass value of the protein to be analyzed is calibrated according to the measured mass value of the internal standard.

[0042] As used herein, the term "biological sample" refers to any material that may contain the target protein to be analyzed or cells expressing the target protein and their cultures, including samples isolated from organisms (such as blood, plasma, serum, saliva, tissues, organs, etc.), materials obtained from the environment (such as water, air, soil, etc.), or artificially mixed samples.

[0043] According to one embodiment, step (b) is implemented using a mass spectrometry method selected from the group consisting of MALDI-TOF mass spectrometry, SELDI-TOF mass spectrometry, ESI-TOF mass spectrometry, LC-MS and LC-MS / MS. More specifically, step (b) is implemented by MALDI-TOF mass spectrometry.

[0044] The mass spectrometry method used in the present invention has been described in detail above and is therefore omitted to avoid unnecessary redundancy.

[0045] In the method of the present invention, an internal standard is added to the biological sample to be analyzed, and then protein separation and purification and mass spectrometry analysis are performed. If a molecular weight difference of a protein is detected that corresponds to the mass of the tag peptide relative to the internal standard, it can be determined that the target protein is present in the sample. By calibrating the mass value of the target protein according to the mass of the internal standard, the reproducibility of each mass measurement is improved, making it closer to the theoretical mass value, thereby significantly improving the reliability of the mass measurement.

[0046] In another aspect of the present invention, an internal standard for quantifying protein is provided, comprising:

[0047] (a) a protein to be analyzed; and

[0048] (b) A tag peptide containing 1 to 100 consecutive random amino acids bound to the N-terminus or C-terminus of the protein to be analyzed.

[0049] In another aspect of the present invention, a method for quantifying protein in a biological sample is provided, comprising:

[0050] (a) adding the internal standard substance as claimed in claim 12 to a biological sample containing a protein to be analyzed;

[0051] (b) measuring the mass values ​​of the protein to be analyzed and the internal standard in the biological sample; and

[0052] (c) The quantitative value of the analyzed protein is calibrated based on the signal intensity of the measured mass value of the internal standard.

[0053] The internal standard of the present invention, in which a tag peptide is coupled to the terminus of a protein having the same amino acid sequence as the protein to be analyzed, has been described in detail above and is therefore omitted to avoid unnecessary redundancy.

[0054] In the method of the present invention, an internal standard is added to the biological sample to be analyzed, and then protein separation, purification and mass spectrometry analysis are performed. The method is based on the mass spectrum peak intensity value that objectively reflects the concentration of the internal standard in the sample to calibrate the target protein intensity value, and the method can not only significantly improve the reproducibility and reliability of the target protein mass measurement, but also significantly improve the reproducibility and reliability of the target protein quantification.

[0055] In another aspect of the present invention, there is provided an internal standard for determining the quality of a protein, comprising:

[0056] (a) any protein with a known mass value; and

[0057] (b) Tag peptides containing 1 to 100 consecutive random amino acids bound to the N-terminus or C-terminus of a protein.

[0058] In another aspect of the present invention, a method for detecting a protein in a biological sample is provided, comprising:

[0059] (a) adding the internal standard substance as claimed in claim 14 to a biological sample containing a protein to be analyzed;

[0060] (b) measuring the mass values ​​of the protein to be analyzed and the internal standard in the biological sample; and

[0061] (c) Calibrate the measured mass value of the protein to be analyzed based on the measured mass value of the internal standard.

[0062] In another aspect of the present invention, an internal standard for quantifying protein is provided, comprising:

[0063] (a) any protein with a known mass value; and

[0064] (b) Tag peptides containing 1 to 100 consecutive random amino acids bound to the N-terminus or C-terminus of a protein.

[0065] In another aspect of the present invention, a method for quantifying protein in a biological sample is provided, comprising:

[0066] (a) adding the internal standard substance as claimed in claim 17 to a biological sample containing a protein to be analyzed;

[0067] (b) measuring the mass values ​​of the protein to be analyzed and the internal standard in the biological sample; and

[0068] (c) The quantitative value of the analyzed protein is calibrated based on the signal intensity of the measured mass value of the internal standard.

[0069] In another aspect of the present invention, a nucleic acid molecule encoding the internal standard of the present invention is provided.

[0070] The tag peptides, nucleic acid molecules, mass spectrometry methods, and detection and quantification methods of the proteins to be analyzed have been described in detail above and are therefore omitted to avoid unnecessary redundancy.

[0071] According to the present invention, the internal standard of the present invention is not limited to the recombinant protein obtained by coupling the tag peptide to a protein having the same amino acid sequence as the target protein to be analyzed. It can also include a recombinant protein obtained by coupling the tag peptide to any protein, wherein the arbitrary protein is different from the target protein but has known mass value information, thereby allowing the mass difference between it and the target protein to be clearly deduced. In this case, the molecular weight difference between the target protein and the internal standard is the sum of the molecular weight difference between the two proteins and the tag peptide. In this way, multiple target proteins with different mass values ​​in the sample can be detected or quantified simultaneously.

[0072] According to a specific embodiment, the mass value difference between any (arbitrary) protein of the present invention and the target protein to be analyzed is ±5,000Da or less, more specifically ±4,000Da or less, even more specifically ±3,000Da or less, most specifically ±2,000Da or less.

[0073] Beneficial Effects

[0074] The features and advantages of the present invention are summarized as follows:

[0075] (a) The present invention provides an internal standard for determining the quality of a protein and a method for detecting a protein in a biological sample using the internal standard.

[0076] (b) The present invention makes it possible to quickly calibrate the measured mass and quantitative value of the target protein with high confidence by means of a simple process of adding a recombinant protein formed by coupling a labeled peptide to the target protein as an internal standard to the sample.

[0077] (c) The present invention can be applied to high-sensitivity analysis of environmental samples and diagnosis of infectious pathogens. Even when using a low-resolution mass spectrometer, it can clearly distinguish high-molecular-weight target proteins from other proteins with smaller mass differences in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The results of SDS-PAGE analysis are shown, confirming the expression and size of each tagged protein (KPC-2_6xhis-tag, KPC-2_2xhis-tag, KPC-2_strep-tag).

[0079] FIG2 shows the results of separation and purification of the tag protein (KPC-2_6xhis-tag) by chromatography. Figure 2a is the result of metal affinity chromatography. Figure 2b The results are obtained by separation and purification using an anion exchange resin column.

[0080] Figure 3 Shown is the mass spectrum of the tagged protein (KPC-2_6xhis-tag) obtained by bottom-up mass spectrometry on a high-resolution mass spectrometer.

[0081] Figure 4 The results of MALDI-TOF analysis using KPC-2_6xhis-tag as an internal standard and mass calibration based on the difference in mass values ​​between the target protein and the internal standard are shown.

[0082] Figure 5 The results of MALDI-TOF analysis using KPC-2_2xhis-tag as an internal standard and mass calibration based on the difference in mass values ​​between the target protein and the internal standard are shown.

[0083] Figure 6 The results of MALDI-TOF analysis using KPC-2_strep-tag as an internal standard and mass calibration based on the difference in mass values ​​between the target protein and the internal standard are shown.

[0084] Figure 7 The MALDI-TOF analysis results of different concentrations of tagged protein (KPC-2_6xHis-tag) and the corresponding mass calibration effects are shown.

[0085] Figure 8 The MALDI-TOF analysis results of different concentrations of tagged protein (KPC-2_6xHis-tag) and the corresponding quantitative calibration effects are shown.

[0086] Fig. 9 The results of using the tagged protein to identify the target protein in 43 clinical strains are shown. The mass difference of the target protein was compared in three cases: without using the tagged protein as an internal standard, using the tagged protein as an internal standard material, and performing mass calibration after applying the tagged protein.

[0087] FIG10 shows the mass error generated when hKPC-2 is used as an internal standard based on the distance to the target pathogenic protein. Fig.10a is the mass spectrum of the target protein, showing the distance between each target protein and the internal standard (hKPC-2). Each arrow indicates the peak of the respective pathogenic protein. Fig.10b The mass error of the target protein is plotted against the molecular weight distance between the internal standard (hKPC-2) and the target protein. All data are expressed as standard deviation (n=3).

[0088] Figure 11 shows the mass error of KPC-type proteins derivatized using an internal standard (KPC-2). Fig.11a Shown is the mass spectrum of a KPC-type protein calibrated with an external standard (Ex-Cal, KPC-2) and an internal standard (In-Cal, hKPC-2). Fig.11b The average mass error of Ex-Cal (KPC-2) and In-Cal (hKPC-2) for each protein with standard deviation (n=3) is shown.

[0089] FIG. 12 is a graphical representation of ADLC mass errors calculated based on distance from an internal standard after mass calibration. Fig.12ais an example of a mass spectrum showing the mass distance from the internal standard. (1) to (6) indicate the mass distance between each internal standard [(1) nhALDC, (2) KPC-2, (3) trypsinogen, (4) myoglobin, (5) BSA, (6) ubiquitin] and the target protein ALDC. The theoretical mass differences (ΔM) from the singly charged ALDC ion are (1) 2119.7, (2) 10758.1, (3) 15495.2, (4) 22524.9, (5) 26920.9, and (6) 30911.4 Da, respectively. Figure 12b The ALDC mass errors for Ex-Cal (KPC-2) and In-Cal (h) measurements are shown.

[0090] Invention method

[0091] The present invention will be described in more detail below in conjunction with specific examples. These examples are only used to illustrate the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is based on the subject matter of the present invention and is not limited by these examples. Example

[0092] Cloning of tag genes and preparation of strains.

[0093] According to the target gene sequence information, the required tags are marked and synthesized.

[0094] The following primers were constructed to synthesize genes encoding: KPC-2 protein tagged with 6 histidine repeats at the C-terminus, KPC-2 protein tagged with 2 histidine repeats at the C-terminus, and KPC-2 protein tagged with 2 Strep repeats at the C-terminus:

[0095] 1) KPC-2 with 6 histidine tagged at the C-terminus (KPC-2_6xhis-tag)

[0096] Primer 1: 5'-AACTGCAGGATGTCACTGTATCGCCGTCTA-3' (30 base units (30mer))

[0097] Primer 2: 5′-GGAATTCTCAGTGGTGGTGGTGGTGGTGGTGCTGCCCGTTGACGCCCA-3′ (45 base units (45mer))

[0098] 2) KPC-2 with 2 histidine residues tagged at the C-terminus (KPC-2_2xhis-tag)

[0099] Primer 1: 5′-AACTGCAGGATGTCACTGTATCGCCGTCTA-3′ (30 base units (30mer))

[0100] Primer 2: 5′-GGAATTCTCAGTGGTGCTGCCCGTTGACGCCCA-3′ (33 base units (33mer))

[0101] 3) KPC-2 with two Streps tagged at the C-terminus

[0102] Primer 1: 5′-AACTGCAGGATGTCACTGTATCGCCGTCTA-3′ (30 base units (30mer))

[0103] Primer 2: 5′-GGAATTCTCATTTTTCGAACTGCGGGTGGCTCCACTGCCCGTTGACGCCCA-3′ (51 base units (51mer))

[0104] Primers are designed with restriction endonuclease sites for cloning and aligned with the open reading frame (ORF) to drive direct expression of the cloning vector. For PCR reaction, 50 μl of reaction solution is prepared with 1 μl of template DNA, 1.25 μl of 5' primer, 1.25 μl of 3' primer, 1 μl of dNTPs, 10 μl of 5X buffer, and 5X GC enhancement buffer. PCR is performed under the following conditions:

[0105] 1) Denaturation: -98°C, 10 seconds;

[0106] 2) Annealing: -57°C, 30 seconds;

[0107] 3) Extension: -72°C, 30 seconds.

[0108] The cloning method for constructing the recombinant expression vector is as follows:

[0109] 1) Use restriction endonucleases to cut the inserted gene and vector into the sticky ends of the DNA; 2) Use DNA ligase to connect the inserted gene to the vector; 3) Transform the vector into E. coli top10; 4) Use the white / blue screening method to screen the recombinant E. coli. Then, 5) Extract the recombinant plasmid from the selected strain; 6) Finally confirm the inserted gene by DNA sequencing

[0110] Expression and identification of marker proteins

[0111] The transformed E. coli was inoculated into LB liquid medium containing 50 mg / L ampicillin and incubated at 37°C for 16 hours. To determine the expression and size of the tagged protein, the culture was centrifuged at 4000 rpm for 15 minutes, the supernatant was removed, and the cells were collected. The collected cells were added to SDS buffer, heated at 95°C for 10 minutes, and centrifuged at 14000g for 10 minutes. The prepared samples were analyzed by SDS-PAGE gel to confirm the expression and size of the tagged protein ( Figure 1 ).

[0112] Sample preparation

[0113] (1) Sample preparation using ultrasound

[0114] The culture of the expression strain was centrifuged at 4000rpm for 15min, the supernatant was taken, and the cells were collected. The collected cells were added to 500mM NaCl, 25mM Tris-HCl, pH8.0 and resuspended. The cells were destroyed using an ultrasonic processor (VC-505, Sonic&Materials, USA). The cells were centrifuged at 4°C, 14000g for 10min, the supernatant (hereinafter referred to as the crude enzyme solution) and the precipitate were separated, and the crude enzyme solution was recovered to separate and purify the tag protein. Ultrasonic treatment can be performed using a probe type or a sonic bath, and the above method is an example of pretreatment using a probe type ultrasonic treatment.

[0115] (2) Sample preparation using osmotic lysis method

[0116] The sample preparation steps are as follows: 1) Centrifuge the culture of the expressed strain, remove the supernatant, and collect the cells. 2) Add the collected cells to a hypertonic solution (500mM NaCl, 25mM Tris-HCl, pH8.0) and resuspend them, and react at room temperature for 10 minutes. 3) Centrifuge at 14000g, 4℃ for 10 minutes to remove the supernatant, add third-grade distilled water to resuspend, and react at room temperature for 10 minutes. 4) Centrifuge at 14000g for 10 minutes at 4℃ to recover the crude enzyme solution.

[0117] (3) Preparation of surfactant samples

[0118] The pretreatment method is as follows: 1) centrifuge the expressed bacterial culture, remove the supernatant, and collect the cells. 2) add BugBuster reagent to the collected cells to resuspend them and react at room temperature for about 20 minutes. 3) centrifuge at 14000g for 10 minutes at 4°C to recover the crude enzyme solution.

[0119] Isolation and purification of tagged proteins

[0120] Metal affinity chromatography and ion exchange chromatography were used to separate / purify the tagged protein. Ni-NTA resin was used for metal affinity chromatography, and Q-resin column was used for anion exchange chromatography.

[0121] (1) Anion exchange resin chromatography

[0122] The crude enzyme solution was loaded onto the Q-resin column, and the eluate was collected. The column was washed with 20 mM tri-HCl, pH 8.0 buffer, and the eluate containing 200 mM NaCl, 400 mM NaCl and 600 mM NaCl was loaded in sequence, and the eluate from each chamber was collected.

[0123] (2) Metal affinity chromatography

[0124] The crude enzyme solution was loaded onto a Ni-NTA resin column, and the eluate was collected. The column was washed with 20 mM tri-HCl, pH 8.0 buffer, and elution buffers containing 50 mM imidazole, 100 mM imidazole, 300 mM imidazole, and 500 mM imidazole were sequentially loaded, and the eluate from each chamber was collected.

[0125] Finally, the above two chromatography steps are performed sequentially to separate / purify the tagged protein ( FIG. 2 ).

[0126] Protein desalting and buffer exchange

[0127] The concentration filter was washed with water at 3500 rpm for 5 min, the purified protein and water were loaded onto the concentration filter, 3500 rpm, 5 min, and the remaining solution was collected. The same method was repeated 4 times to obtain the protein through desalting and buffer exchange.

[0128] Identification of tagged proteins

[0129] The marker proteins whose expression and size were confirmed by SDS-PAGE were confirmed by in-gel digestion (bottom-up method) and LC-MS / MS. Figure 3 ).

[0130] (1) In-gel enzymatic hydrolysis

[0131] Only the bands corresponding to the tagged proteins were extracted from the SDS-PAGE gel and then separated. The destained gel was reduced / alkylated and cleaved with trypsin, and the cleaved peptides were desalted and recovered using a C18 pipette tip.

[0132] (2) LC-MS / MS

[0133] To determine the sequence and range of the marker protein expressed in the strain, Evosep liquid chromatography and high-resolution mass spectrometry (Q-Exactive HF-X mass spectrometry system) were used. The desalted peptide samples were dissolved in 0.1% formic acid solution and loaded. The peptide samples were separated using a C18 column (8 cm × 100 μm, 3 μm: EV1064) and nanoflow liquid chromatography. EVOSEP PD100 (11.5 min) was used.

[0134] - Buffer A: 0.1% formic acid in water; Buffer B: 0.1% formic acid in acetonitrile water.

[0135] The mass spectrometer parameters used in this example are:

[0136] - Resolution: Full MS 60,000, Applied MS 215,000

[0137] -Full MS: 300 to 2000 m / z, 100 msec

[0138] -MS2: 28msec, NCE 27,1,>6 Gas ionizers are not subject to MS2

[0139] Thermo's Proteome Discoverer (v2.4) search engine was used as software to identify peptides and proteins using bottom-up data. Protein / peptide identification was performed based on an FDR of 1%. From the analyzed data, six additional histidine amino acids that bind to the C-terminus of the target protein were determined by comparing the theoretical mass values ​​of the precursor ions and fragment ions.

[0140] Mass spectrometry analysis of target and tagged proteins using MALDI-TOF

[0141] In order to confirm the mass values ​​and peak intensities of the target protein and the tagged protein, a mixture of the target protein and the tagged protein ( Figure 4-6 ) for MALDI-TOF analysis. Using a Bruker Biotyper MALDI-TOF mass spectrometer, 1 μL of the mixed solution of the target protein and the labeled protein and 1 μL of sinapinic acid (SA) substrate (20 mg / mL, in 0.1% TFA / 50% acetonitrile) were placed on a spot plate, mixed, and dried thoroughly for later use.

[0142] The pulsed ion extraction time was 450 ns, and a total of 2000 laser irradiations were performed to collect random positions, 40 times at each position. The spectral data were accumulated to obtain the final result.

[0143] In positive ion and linear modes, the laser frequency was 100 Hz, and mass spectra were obtained by detecting both +1 and +2 ions simultaneously in the range of 12,000 to 32,000 m / z.

[0144] Mass spectrometry data were collected using Bruker's Flex Control version 4.1 software, processed using Lexa Analysis version 4.1 software, smoothed using the Savitzky Golay method, and baseline subtracted using the OpHat method. The intensity of each peak was measured using the centroid peak detection algorithm.

[0145] The mass value of the target protein was calibrated based on the mass value of the internal standard (labeled protein) at all concentration combinations, and the mass reproducibility was significantly improved, and the mass value was close to the theoretical mass ( Figure 7 ). In addition, the target protein intensity was calibrated according to the intensity of the tag protein, and the quality reproducibility of the target protein intensity was also significantly improved ( Figure 8 ).

[0146] Calibrating the quality of target proteins derived from clinical strains using tagged proteins

[0147] In order to verify the mass calibration results of the target protein (KPC) derived from clinical strains using the tag protein of the present invention, the clinical strain genotyped as KPC was cultured on blood agar plate medium. The identification method of the target protein is the same as that of the clinical strain cultured above. Specifically, the tag protein (MW = 29,541Da) was used as an internal standard to calibrate the mass spectrometry mass value containing the KPC target protein (MW = 28,718Da), and the calibrated mass value was used to identify the target protein ( Fig. 9 ).

[0148] Confirmation of calibration mass range using IS proteins

[0149] In order to evaluate the appropriate mass range of the internal standard (label protein), the present invention determines the mass error values ​​of three carbapenemases (IMP-6, VIM-2 and GES-5) and a β-lactamase (CTX-M-1), whose masses differ from the internal standard by hundreds to 5000Da. As shown in Figure 10, the mass errors of all proteins are less than 3.2Da. Among them, CTX-M-1 and VIM-2 have very small errors (<1.6Da) compared with other proteins, and the distances from the internal standard are 1331 and 4026Da, respectively. IMP-6 has the largest mass difference with the internal standard hKPC-2, and the mass error is also the largest (Figure 10). The average mass error of all proteins obtained using the external standard (Ex-Cal, KPC-2) and the internal standard (In-Cal, hKPC-2) is ~83.5ppm, and the mass error distribution range is -3.0 to +3.2Da.

[0150] Identification of KPC subtypes with calibrated quality using hKPC-2

[0151] Ex-Cal (KPC-2) and In-Cal (hKPC-2) were used to identify KPC isoform proteins (KPC-2, KPC-3, KPC-4, and KPC-17). The MALDI mass spectra and average mass errors (-0.9 to 0.9 Da) of these proteins are shown in Table 1 and Figure 11. Although the mass difference of KPC isoforms ranges from -34 to +26 Da (Table 1 and Fig.11b ), but all KPC subtypes were correctly identified with an error of less than 1Da for both standard and clinical strains.

[0152] Table 1

[0153]

[0154] Mass calibration range determined using internal fluorine standard

[0155] In order to evaluate whether the method of using 6x HIS-tagged proteins as internal standards in the present invention can be generally applied to other mass ranges, the inventors used hALDC to check the mass error of another target protein ALDC. As an internal standard, the mass of hALDC labeled with 6x His is 40.6 kDa, which is more than 12 kDa higher than hKPC-2. The present invention uses six proteins (Ubi, Myo, Try, KPC-2, BSA and hALDC) as candidate internal standards for internal calibration (In-Cal) ( Fig.12a ). In order to evaluate how the mass distance between the internal standard and the target protein affects the mass error, the mass error value of ALDC after In-Cal calibration was calculated. The mass distance between the six candidate internal standard proteins and ALDC was approximately 2100-31000Da (Figure 12). The results show that, like hKPC-2, hALDC as an internal standard of ALDC protein again produces a very low mass error (<3.4Da, <85ppm). However, other candidate internal standards that differed from the target protein by more than 10kDa could not be used as standards at all (error was 3000ppm).

[0156] After describing the specific embodiments of the present invention in detail above, it should be understood that various changes or modifications falling within the scope of the present invention will be obvious to those skilled in the art. The scope of the present invention is determined by the appended claims and their equivalents.

Claims

1. An internal standard for determining protein quality, characterized in that: include: (a) the protein to be analyzed; and (b) A tag peptide containing 1 to 100 consecutive random amino acids bound to the N-terminus or C-terminus of the protein to be analyzed.

2. The internal standard substance according to claim 1, characterized in that The tag peptide contains 2 to 80 consecutive random amino acids bound to the C-terminus of the protein to be analyzed.

3. The internal standard substance according to claim 2, characterized in that The tag peptide is selected from the following group: a repeated sequence of identical amino acid residues, a continuous sequence of different amino acid residues, a repeat of a continuous sequence of different amino acid residues, or a combination thereof.

4. The internal standard substance according to claim 3, characterized in that The identical amino acid is histidine (His).

5. The internal standard substance according to claim 3, characterized in that The continuous sequence of different amino acid residues includes the amino acids shown in SEQ ID NO:

7.

6. The internal standard substance according to claim 3, characterized in that The mass spectrometry is selected from the group consisting of matrix-assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometry, surface-enhanced laser desorption / ionization time of flight (SELDI-TOF) mass spectrometry, electrospray ionization time of flight (ESI-TOF) mass spectrometry, liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS).

7. The internal standard substance according to claim 6, characterized in that The mass spectrometer is a MALDI-TOF (matrix desorption / ionization time of flight) mass spectrometer.

8. A nucleic acid molecule encoding the internal standard substance according to any one of claims 1 to 7.

9. A method for detecting protein in a biological sample, comprising: (a) adding the internal standard substance described in any one of claims 1 to 7 to a biological sample containing a protein to be analyzed; (b) measuring the mass values ​​of the protein to be analyzed and the internal standard in the biological sample; and (c) The measured mass value of the protein to be analyzed is calibrated according to the measured mass value of the internal standard.

10. The method according to claim 9, characterized in that The step (b) is implemented using a mass spectrometry method selected from the following group: matrix-assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometry, surface-enhanced laser desorption / ionization time of flight (SELDI-TOF) mass spectrometry, electrospray ionization time of flight (ESI-TOF) mass spectrometry, liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS).

11. The method according to claim 9, characterized in that The step (b) is performed using matrix desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry.

12. An internal standard for quantifying protein, characterized in that: include: (a) the protein to be analyzed; and (b) A tag peptide containing 1 to 100 consecutive random amino acids bound to the N-terminus or C-terminus of the protein to be analyzed.

13. A method for quantifying protein in a biological sample, characterized in that: include: (a) adding the internal standard substance as claimed in claim 12 to a biological sample containing a protein to be analyzed; (b) measuring the mass values ​​of the protein to be analyzed and the internal standard in the biological sample; and (c) The quantitative value of the analyzed protein is calibrated based on the signal intensity of the measured mass value of the internal standard.

14. An internal standard for determining protein quality, characterized in that: include: (a) Any protein with a known mass value; and (b) Tag peptides containing 1 to 100 consecutive random amino acids bound to the N-terminus or C-terminus of a protein.

15. A method for detecting protein in a biological sample, characterized in that: include: (a) adding the internal standard substance as claimed in claim 14 to a biological sample containing a protein to be analyzed; (b) measuring the mass values ​​of the protein to be analyzed and the internal standard in the biological sample; and (c) Calibrate the measured mass value of the protein to be analyzed based on the measured mass value of the internal standard.

16. An internal standard for quantifying protein, characterized in that: include: (a) Any protein with a known mass value; and (b) Tag peptides containing 1 to 100 consecutive random amino acids bound to the N-terminus or C-terminus of a protein.

17. A method for quantifying protein in a biological sample, characterized in that: include: (a) adding the internal standard substance as claimed in claim 17 to a biological sample containing a protein to be analyzed; (b) measuring the mass values ​​of the protein to be analyzed and the internal standard in the biological sample; and (c) The quantitative value of the analyzed protein is calibrated based on the signal intensity of the measured mass value of the internal standard.

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