A new internal standard for mass calibration

Abstract

The present invention relates to an internal standard for measuring protein mass and a method for detecting proteins in biological samples using the same. The present invention enables rapid and reliable calibration of the measured mass and quantitative values ​​of a target protein through a simple process of adding a recombinant protein in which a tag peptide is conjugated to the target protein to a sample as an internal standard. Therefore, the present invention can clearly distinguish a high-molecular-weight target protein from other proteins with slight mass differences in the sample, even using a low-resolution mass spectrometer, making it useful for high-sensitivity analyses such as the analysis of environmental samples and the diagnosis of pathogenic bacterial infections.

Description

[Technical Field]

[0001] The present invention relates to a method for accurately and efficiently correcting the measured mass value of a target protein by using, as an internal standard, a recombinant protein in which a specific peptide with known mass information is conjugated to the same protein as the target protein. [Background technology]

[0002] A MALDI-TOF (Matrix Desorption / Ionization Time of Flight) mass spectrometer measures the molecular weight of an analyte by drying a mixture of a sample and a matrix to form a crystalline structure, irradiating it with a laser to desorb and ionize it, and then measuring the time-of-flight (TFO) required for the analyte to reach a detector. MALDI-TOF has the advantage of being able to rapidly perform mass analysis of macromolecules such as proteins without fragmentation. However, its low resolution makes it difficult to accurately distinguish proteins with minute mass differences. Another limitation is that drying a mixture of a sample and a matrix can result in an inhomogeneous crystalline structure, resulting in poor reproducibility.

[0003] Currently, mass calibration using external standards is commonly used to improve the accuracy of MALDI-TOF mass spectrometry. This method involves measuring the standard at one or more spots to obtain calibration parameters, which are then used to calibrate samples at other spots. When using external standards to calibrate the mass values ​​of high-molecular-weight proteins (>20 kDa) using low-resolution MALDI-TOF instruments, the wide error range of mass values ​​due to protein size makes accurate mass calibration difficult, limiting the ability to separate and identify proteins of similar size. While examples using internal standards based on mass spectrometry have been reported, most methods are primarily used for peptide-level quantification, and research into protein-level quantification remains a challenge. Therefore, the development of efficient mass calibration and quantification methods that enable more accurate mass analysis at the protein level is essential.

[0004] Numerous papers and patent documents are referenced throughout this specification and citations are provided, the disclosures of which are incorporated herein by reference in their entirety to more clearly describe the state of the art and the content of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have conducted extensive research to develop an efficient mass spectrometry method that can accurately and quickly separate and identify high-molecular-weight target proteins in a sample to be analyzed, even when using a low-resolution mass spectrometer. As a result, they have discovered that when a recombinant protein in which a tag peptide of a known molecular weight is attached to the N- or C-terminus of a protein having the same amino acid sequence as the target protein is used as an internal standard, the mass value and quantification value of the target protein can be easily and accurately corrected based on the molecular weight of the tag peptide, which corresponds to the molecular weight difference between the target protein and the internal standard. This discovery led to the completion of the present invention.

[0006] It is therefore an object of the present invention to provide an internal standard for measuring the mass of proteins.

[0007] Another object of the present invention is to provide a method for detecting a protein in a biological sample using the internal standard.

[0008] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the drawings. [Means for solving the problem]

[0009] According to one aspect of the present invention, there is provided an internal standard for mass determination of a protein, comprising: (a) the analyte protein; and (b) A tag peptide consisting of any 1 to 100 consecutive amino acids bound to the N-terminus or C-terminus of the target protein.

[0010] The present inventors have conducted extensive research to develop an efficient mass spectrometry method that can accurately and quickly separate and identify high-molecular-weight target proteins in a sample to be analyzed, even when using a low-resolution mass spectrometer. As a result, they discovered that when a recombinant protein in which a tag peptide with a known molecular weight is attached to the N- or C-terminus of a protein with the same amino acid sequence as the target protein is used as an internal standard, the mass value and quantification value of the target protein can be easily and accurately corrected based on the molecular weight of the tag peptide, which corresponds to the molecular weight difference between the target protein and the internal standard.

[0011] 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 to be detected by mass spectrometry may be a protein that serves as a biological marker for, for example, determining the presence or absence of a pathogenic strain in a sample (i.e., diagnosing the presence or absence of pathogenic bacterial infection), or identifying the type and phenotype of the pathogenic strain.

[0012] As used herein, "pathogenic bacterial strain" includes all bacteria that act as a cause of infection or disease, including, but not limited to, Staphylococcus aureus, Streptococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas otitidis, Micrococcus luteus, Citrobacter koseri, Proteus mirabilis, and Mycobacterium ulcerans.

[0013] The target proteins to be analyzed in the present invention include, for example, marker proteins that can predict not only the presence or absence of a pathogenic strain, but also the phenotype of the strain, such as antibiotic resistance.

[0014] As used herein, "antibiotic resistance" refers to the ability of a microorganism to grow in an environment where a high concentration or effective amount of an antibiotic is present against that microorganism. Antibiotic resistance can be determined by detecting the presence of enzymes, proteins secreted by pathogenic microorganisms, that degrade the antibiotic and eliminate or reduce its activity. For example, beta-lactam antibiotics, such as penicillin, cephalosporin, monobactam, and carbapenem, which inhibit bacterial cell wall synthesis, are neutralized by beta-lactamase and cannot inhibit pathogens that express them. Therefore, the term "resistance" is used interchangeably with "resistance" or "poor therapeutic response."

[0015] As used herein, the term "internal reference material" refers to a compound with a known concentration or mass that is added in a fixed amount to an analyte sample and used to correct quantitative errors in the concentration or mass value of the analyte (i.e., target protein) by comparing the signal from the analyte with the signal from the internal standard and determining the difference or ratio between them.

[0016] As used herein, the term "tag peptide" refers to an amino acid sequence genetically grafted onto a recombinant protein for various purposes, such as easy isolation, purification, solubilization, and visual labeling of the target protein. The tag peptide used in the present invention is a component of an internal standard and is attached to the N- or C-terminus of a recombinant protein having the same amino acid sequence as the target protein to be detected. This tag peptide can be used to correct the mass value of the target protein during mass spectrometry by generating a mass difference between the target protein and the internal standard equal to the molecular weight of the tag peptide. Therefore, unlike when the tag peptide is directly bound to the target protein, the tag peptide of the present invention merely constitutes part of the internal standard and does not need to be removed by protease or the like after analysis.

[0017] According to a specific embodiment of the present invention, the tag peptide consists of any 2 to 80 consecutive amino acids attached to the C-terminus of the analyte protein. More specifically, it may be any 2 to 70 consecutive amino acids attached to the C-terminus, even more specifically, any 2 to 60 consecutive amino acids attached to the C-terminus, even more specifically, any 2 to 50 consecutive amino acids attached to the C-terminus, even more specifically, any 2 to 40 consecutive amino acids attached to the C-terminus, even more specifically, any 2 to 30 consecutive amino acids attached to the C-terminus, even more specifically, any 2 to 20 consecutive amino acids attached to the C-terminus, even more specifically, any 2 to 10 consecutive amino acids attached to the C-terminus, and most specifically, any 2 to 8 consecutive amino acids attached to the C-terminus.

[0018] According to a specific embodiment of the present invention, the tag peptide is selected from the group consisting of: a repeated sequence of identical amino acid residues; a consecutive sequence of different amino acid residues; a repeated consecutive sequence of different amino acid residues; and combinations thereof.

[0019] According to a more specific embodiment, said identical amino acid may be histidine (His).

[0020] According to a more specific embodiment, the consecutive sequence of different amino acid residues may be a sequence comprising the amino acids of Sequence Listing No. 7. According to the present invention, Sequence Listing No. 7 is the amino acid sequence of Strep-tag peptide (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys), which has affinity for streptavidin.

[0021] The tag peptide is not limited to the above examples, and any form of peptide can be used without limitation as long as it causes a difference in molecular weight between the target protein and the internal standard by a mass value calculated based on the already known amino acid sequence information.

[0022] According to a specific embodiment of the present invention, the mass spectrometry is selected from the group consisting of MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization Time of Flight) mass spectrometry, SELDI-TOF (Surface Enhanced Laser Desorption / Ionization Time of Flight) mass spectrometry, ESI-TOF (Electrospray ionization time-of-flight) mass spectrometry, liquid chromatography-mass spectrometry (LC-MS) and LC-MS / MS (Liquid chromatography-Mass Spectrometry / Mass Spectrometry), more specifically MALDI-TOF (Matrix Desorption / Ionization Time of Flight) mass spectrometry.

[0023] MALDI-TOF mass spectrometry is a method of analyzing the molecular weight of ions by irradiating a matrix-supported sample with a laser to desorb and ionize them, and then measuring the time it takes for the generated ions to reach a detector (time-of-flight). Because no fragmentation of the target material occurs, it can quickly and accurately measure the mass of large biomolecules such as proteins. When ionized molecules are accelerated by an electric field and their time-of-flight 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.

[0024] Currently, mass calibration using external standards is commonly performed to improve the accuracy of MALDI-TOF mass spectrometry. However, for proteins with high molecular weights of 20 kDa or greater, measurement using low-resolution MALDI-TOF instruments using external standards results in excessively large mass error, making reliable analysis difficult. The present invention uses a recombinant protein conjugated with a tag peptide as an internal standard, enabling clear discrimination of high-molecular-weight target proteins from other proteins with slight mass differences in a sample, even when using a low-resolution MALDI-TOF instrument.

[0025] According to another aspect of the present invention, there is provided a nucleic acid molecule encoding the internal standard of the present invention as described above.

[0026] As used herein, the term "nucleic acid molecule" encompasses DNA (gDNA and cDNA) and RNA molecules. Nucleotides, the basic building blocks of nucleic acid molecules, include 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 internal standard, i.e., the amino acid sequence of a recombinant protein in which a tag peptide is bound to a target protein to be analyzed, in the present invention is not limited to the nucleotide sequences listed in the attached sequence listing or directly cloned in the examples of the present invention. Nucleotide modifications included in the embodiments of the present invention encompass all modifications that do not result in changes at the protein level, i.e., nucleic acid molecules having functionally equivalent codons, codons encoding the same amino acid due to codon degeneracy, or codons encoding biologically equivalent amino acids.

[0027] Considering the above-mentioned biologically equivalent variants, the nucleic acid molecules used in the present invention are also understood to include sequences that show substantial identity to the nucleotide sequences set forth in the sequence listing or directly cloned in the Examples. The term "substantial identity" refers to a sequence that shows at least 70% homology, specifically 80% homology, more specifically 90% homology, and most specifically 95% homology, when the sequences of the present invention are aligned with any other sequence for maximum correspondence and the aligned sequences are analyzed using algorithms commonly used in the art. Alignment methods for sequence comparison are well known in the art. Various alignment methods and algorithms are disclosed in Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994).

[0028] According to the present invention, the internal standards of the present invention are obtained recombinantly by expressing nucleic acid molecules encoding them in host cells.

[0029] As used herein, the term "express" refers to artificially introducing an exogenous gene into a target cell using a gene carrier to express the exogenous gene or increase the natural expression level of an endogenous gene, thereby making the gene replicable in the target cell as an extrachromosomal element or by chromosomal integration. Therefore, the term "expression" has the same meaning as "transformation," "transfection," or "transduction." More specifically, in the present invention, "expression" refers to artificially expressing an exogenous gene in a target cell.

[0030] As used herein, the term "gene carrier" or "gene delivery system" refers to any means for transporting a gene into a cell, and gene delivery is synonymous with intracellular gene transduction. At the tissue or cellular level, gene delivery is synonymous with gene spread. Therefore, the gene delivery system of the present invention can be described as a gene penetration system and a gene spread system.

[0031] To prepare the gene delivery vector of the present invention, the nucleotide sequence of the present invention is present in a suitable expression construct, and the nucleotide sequence of the present invention in the expression construct is preferably operably linked to an expression control sequence. As used herein, the term "operably linked" refers to the functional connection between a nucleic acid expression control sequence, such as a promoter, a signal sequence, or an array of transcriptional regulator binding sites, and another nucleic acid sequence, whereby the expression control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0032] The gene delivery vehicle of the present invention can be prepared in various forms, including (I) a naked recombinant DNA molecule, (II) a plasmid, (III) a viral vector, and (IV) a liposome or niosome containing the naked recombinant DNA molecule or plasmid.

[0033] According to yet another aspect of the present invention, there is provided a method for detecting a protein in a biological sample, comprising: (a) adding the internal standard of the present invention to a biological sample containing a target protein; (b) measuring the mass values ​​of the analyte protein and the internal standard in the biological sample; (c) correcting the measured mass value of the analyte protein based on the measured mass value of the internal standard.

[0034] In the present invention, the term "biological sample" is used to collectively refer to any substance that may contain the target protein to be analyzed, or cells or microorganisms that express the protein, or culture media thereof, including samples isolated from living organisms (e.g., blood, plasma, urine, saliva, tissues, organs), substances collected from the environment (e.g., water, air, soil, etc.), and artificially mixed samples.

[0035] According to a specific embodiment of the present invention, step (b) is carried out using a mass spectrometry method selected from the group consisting of MALDI-TOF mass spectrometry, SELDI-TOF mass spectrometry, ESI-TOF mass spectrometry, liquid chromatography mass spectrometry and LC-MS / MS, more specifically, using a MALDI-TOF mass spectrometry method.

[0036] The mass spectrometry method used in the present invention has already been described above, and therefore its description will be omitted to avoid excessive duplication.

[0037] In the method of the present invention, the internal standard of the present invention is added to a biological sample to be analyzed, and then proteins in the sample are separated and purified and subjected to mass spectrometry. If a protein with a molecular weight different from that of the internal standard by the mass value of the tag peptide is detected, it can be determined that the target protein is present in the sample. As a result of correcting the mass value of the target protein based on the mass value of the internal standard, the reproducibility of each measured mass is improved, bringing it closer to the theoretical mass value, and the reliability of the mass measurement can be greatly improved.

[0038] According to yet another aspect of the present invention, there is provided an internal standard for protein quantification comprising: (a) the analyte protein; and (b) A tag peptide consisting of any 1 to 100 consecutive amino acids bound to the N-terminus or C-terminus of the target protein.

[0039] According to yet another aspect of the present invention, there is provided a method for quantifying a protein in a biological sample, comprising: (a) adding the internal standard of claim 12 to a biological sample containing a target protein; (b) measuring the mass values ​​of the analyte protein and the internal standard in the biological sample; (c) correcting the quantitative value of the analyte protein based on the signal intensity of the measured mass value of the internal standard.

[0040] The internal standard of the present invention, which comprises a protein having the same amino acid sequence as the analyte protein and a tag peptide attached to the end thereof, has already been described above, and therefore will not be described again to avoid excessive duplication.

[0041] In the method of the present invention, the internal standard of the present invention is added to a biological sample to be analyzed, and then proteins in the sample are separated and purified and subjected to mass spectrometry. The intensity value of the target protein is corrected based on the peak intensity value of the mass spectrum, which objectively reflects the concentration of the internal standard in the sample, thereby significantly improving the reproducibility and reliability of not only the mass of the target protein but also the quantitative value.

[0042] According to yet another aspect of the present invention, there is provided an internal standard for mass determination of a protein, comprising: (a) any protein with a known mass value; and (b) A tag peptide consisting of 1 to 100 consecutive amino acids attached to the N-terminus or C-terminus of any of the proteins.

[0043] According to yet another aspect of the present invention, there is provided a method for detecting a protein in a biological sample, comprising: (a) adding the internal standard of claim 14 to a biological sample containing a target protein; (b) measuring the mass values ​​of the analyte protein and the internal standard in the biological sample; (c) correcting the measured mass value of the analyte protein based on the measured mass value of the internal standard.

[0044] According to yet another aspect of the present invention, there is provided an internal standard for protein quantification comprising: (a) any protein with a known mass value; and (b) A tag peptide consisting of 1 to 100 consecutive amino acids attached to the N-terminus or C-terminus of any of the proteins.

[0045] According to yet another aspect of the present invention, there is provided a method for quantifying a protein in a biological sample, comprising: (a) adding the internal standard of claim 16 to a biological sample containing a target protein; (b) measuring the mass values ​​of the analyte protein and the internal standard in the biological sample; (c) correcting the quantitative value of the analyte protein based on the signal intensity of the measured mass value of the internal standard.

[0046] According to yet another aspect, the present invention provides a nucleic acid molecule encoding the internal standard of the present invention described above.

[0047] The tag peptide, nucleic acid molecule, mass spectrometry method, and method for detecting and quantifying a target protein using the same used in the present invention have already been described above, so a description thereof will be omitted to avoid excessive duplication.

[0048] According to the present invention, the internal standard of the present invention can be not only a recombinant protein in which a tag peptide is conjugated to a protein with the same amino acid sequence as the target protein to be analyzed, but also a recombinant protein in which a tag peptide is conjugated to any protein different from the target protein that already has information about its mass value and from which the mass difference between the target protein and the internal standard can be trivially derived. In this case, the molecular weight difference between the target protein and the internal standard is the sum of the inherent molecular weight difference between the two proteins and the molecular weight of the tag peptide, thereby enabling simultaneous detection or quantification of multiple target proteins with various mass values ​​in a sample.

[0049] According to a specific embodiment of the present invention, any of the proteins of the present invention has a mass difference of ±5,000 Da or less from the analyte protein, more specifically a mass difference of ±4,000 Da or less, even more specifically a mass difference of ±3,000 Da or less, and most specifically a mass difference of ±2,000 Da or less. [Effects of the Invention]

[0050] The features and advantages of the present invention can be summarized as follows: (a) The present invention provides an internal standard for measuring the mass of a protein and a method for detecting a protein in a biological sample using the same. (b) The present invention enables rapid and reliable calibration of the measured mass and quantitative values ​​of a target protein through the simple process of adding a recombinant protein in which a tag peptide is conjugated to the target protein to a sample as an internal standard. (c) The present invention can be usefully applied to high-sensitivity analyses such as the analysis of environmental samples and the diagnosis of pathogenic bacterial infections, by clearly distinguishing high-molecular-weight target proteins from other proteins with minute mass differences in the sample, even when using a low-resolution mass spectrometer. [Brief explanation of the drawings]

[0051] [Figure 1]FIG. 1 shows the results of SDS-PAGE analysis confirming the expression and size of each tagged protein (KPC-2_6xhis-tag, KPC-2_2xhis-tag, KPC-2_strep-tag). [Figure 2] Figure 2 shows the results of separation and purification of a tagged protein (KPC-2_6xhis-tag) using chromatography. Figure 2A shows the results of separation and purification using metal affinity chromatography, and Figure 2B shows the results of separation and purification using an anion exchange resin column. [Figure 3] FIG. 3 shows the mass spectrum of a tagged protein (KPC-2_6xhis-tag) obtained by bottom-up mass spectrometry using a high-resolution mass spectrometer. [Figure 4] FIG. 4 shows the results of mass correction based on the difference in mass value between the target protein and MALDI-TOF analysis using KPC-2_6xhis-tag as an internal standard. [Figure 5] FIG. 5 shows the results of mass correction based on the difference in mass value between the target protein and MALDI-TOF analysis using KPC-2_2xhis-tag as an internal standard. [Figure 6] FIG. 6 shows the results of mass correction based on the difference in mass value between the target protein and MALDI-TOF analysis using KPC-2_strep-tag as an internal standard. [Figure 7] FIG. 7 shows the results of MALDI-TOF analysis and the effect of mass correction for various concentrations of tagged protein (KPC-2_6xhis-tag). [Figure 8] FIG. 8 shows the MALDI-TOF analysis results and quantitative correction effects for the application of tagged protein (KPC-2_6xhis-tag) at different concentrations. [Figure 9]Figure 9 shows the results of identifying target proteins using tagged proteins in 43 clinical strains. The mass values ​​of the target proteins were compared between when tagged proteins were used as an internal standard, when they were not used, and when mass correction was performed after using tagged proteins. [Figure 10] Figure 10 shows the mass error depending on the distance from the target pathogenic protein when hKPC-2 is used as an internal standard. Figure 10A shows 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 each pathogenic protein. Figure 10B shows a graph showing the mass error of the target protein versus the molecular weight distance between the internal standard (hKPC-2) and the target protein. All data are shown with standard deviation (n=3). [Figure 11] Figure 11 shows the results of measuring the mass error of KPC-subtype proteins derived using an internal standard (KPC-2). Figure 11A shows the mass spectrum of a KPC-subtype protein after calibration with an external standard (Ex-Cal, KPC-2) and an internal standard (In-Cal, hKPC-2). Figure 11B is a graph showing the average mass error value of each protein relative to Ex-Cal (KPC-2) and In-Cal (hKPC-2) with standard deviation (n=3). [Figure 12]Figure 12 shows the mass error of ADLC as a function of the distance from the internal standard after mass calibration. Figure 12A shows 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 shows the mass error of ALDC after Ex-Cal (KPC-2) and In-Cal (h). DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of explaining the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention. [Example]

[0053] Cloning of tagged genes and strain generation The target gene was synthesized and labeled with the desired tag based on the sequence information of the target gene.

[0054] The following primers were prepared to synthesize genes encoding KPC-2 protein tagged with six repeated histidines at the C-terminus; KPC-2 protein tagged with two repeated histidines at the C-terminus; and KPC-2 protein tagged with two repeated Strep residues at the C-terminus: 1)C-terminal His6 tag KPC-2(KPC-2_6xhis-tag) Primer 1: 5'-AACTGCAGGATGTCACTGTATCGCCGTCTA-3' (30mer) Primer 2: 5'-GGAATTCTCAGTGGTGGTGGTGGTGGTGCTGCCCGTTGACGCCCA-3' (45mer) 2)C-terminal His2 tag KPC-2(KPC-2_2xhis-tag) Primer 1: 5'-AACTGCAGGATGTCACTGTATCGCCGTCTA-3' (30mer) Primer 2: 5'-GGAATTCTCAGTGGTGCTGCCCGTTGACGCCCA-3' (33mer) 3)C-terminal Strep2 tag KPC-2 Primer 1: 5'-AACTGCAGGATGTCACTGTATCGCCGTCTA-3' (30mer) Primer 2: 5'-GGAATTCTCATTTTTCGAACTGCGGGTGGCTCCACTGCCCGTTGACGCCCA-3' (51mer) The primers were designed to add restriction enzyme sites for cloning and to incorporate the open reading frame (ORF) for direct expression in the cloning vector. PCR was performed using 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 enhancer buffer to create a total of 50 μl of PCR reaction mixture. PCR was performed under the following conditions: 1) Denaturation - 98°C for 10 seconds: 2) Annealing - 57°C for 30 seconds; 3) Extension - 72°C for 30 seconds.

[0055] Cloning to prepare the recombinant expression vector was performed as follows: 1) The insert gene and vector were cut at both ends of the DNA with restriction enzymes to form sticky ends; 2) the insert gene was joined to the vector using DNA ligase; 3) it was transformed into E. coli (E. coli top10); 4) recombinant E. coli was selected using the White / Blue screening method; 5) recombinant plasmids were extracted from the selected strains; and 6) the insert gene was finally confirmed by DNA sequence analysis.

[0056] Expression and characterization of tagged proteins The transformed E. coli was inoculated into Luria-Bertani (LB) liquid medium containing 50 mg / L ampicillin antibiotic and cultured at 37°C for approximately 16 hours. To confirm the expression and size of the tagged protein, the culture was centrifuged at 4,000 rpm for 15 minutes, the supernatant was removed, and the cells were harvested. SDS buffer was added to the harvested cells, which were then heated at 95°C for 10 minutes and centrifuged at 14,000 g for 10 minutes. The expression and size of the tagged protein were confirmed by SDS-PAGE gel analysis using the prepared sample (Figure 1).

[0057] Sample pretreatment (1) Sample pretreatment using ultrasonic disruption (sonication) The culture medium containing the expressed strain was centrifuged at 4,000 rpm for 15 minutes, and the supernatant was removed to harvest the cells. The harvested cells were resuspended in 500 mM NaCl, 25 mM Tris-HCl, pH 8.0, and then disrupted using an Ultrasonic Processor (VC-505, Sonics & Materials, USA). The supernatant (hereinafter referred to as the crude enzyme solution) was separated from the precipitate by centrifugation at 14,000 g for 10 minutes at 4°C, and the crude enzyme solution was collected for isolation and purification of the tagged protein. Ultrasonic disruption can be performed using either a probe or an ultrasonic bath; the above method is an example of pretreatment using a probe-type ultrasonic disruption method.

[0058] (2) Sample pretreatment using osmotic lysis The sample was pretreated in the following steps: 1) The culture medium of the expressed strain was centrifuged, the supernatant was removed, and the cells were harvested. 2) The harvested cells were resuspended in a hypertonic solution (500 mM NaCl, 25 mM Tris-HCl, pH 8.0) and incubated at room temperature for 10 minutes. 3) The cells were centrifuged at 14,000 g for 10 minutes at 4°C and the supernatant was removed. After that, the cells were resuspended in triple-distilled water and incubated at room temperature for 10 minutes. 4) After centrifugation at 14,000 g for 10 minutes at 4°C, the crude enzyme solution was collected.

[0059] (3) Pretreatment of samples using surfactants The pretreatment method was as follows: 1) The culture medium of the expressed strain was centrifuged, the supernatant was removed, and the cells were harvested. 2) BugBuster reagent was added to the harvested cells, and the cells were resuspended and incubated at room temperature for approximately 20 minutes. 3) After centrifugation at 14,000 g for 10 minutes at 4°C, the crude enzyme solution was collected.

[0060] Isolation and purification of tagged proteins For the separation and purification of tagged proteins, metal affinity chromatography and ion exchange chromatography were used. Metal affinity chromatography was performed using a column containing Ni-NTA resin, and anion exchange chromatography was performed using a column containing Q resin.

[0061] (1) Anion exchange resin chromatography The crude enzyme solution was loaded onto a column containing Q-resin, and the eluate was collected. After washing with 20 mM Tris-HCl, pH 8.0, the column was sequentially loaded with elution solutions containing 200 mM NaCl, 400 mM NaCl, and 600 mM NaCl, and the eluate was collected in each section.

[0062] (2) Metal affinity chromatography The crude enzyme solution was loaded onto a column containing Ni-NTA resin, and the eluate was collected. After washing with 20 mM Tris-HCl, pH 8.0, the column was sequentially loaded with elution solutions containing 50 mM imidazole, 100 mM imidazole, 300 mM imidazole, and 500 mM imidazole, and the eluate was collected in each section.

[0063] Finally, the two chromatographic steps were carried out sequentially to separate and purify the tagged protein (Figure 2).

[0064] Desalting and buffer exchange of purified proteins The concentrated filter was washed with water for 5 minutes at 3500 rpm, and the purified protein and water were loaded onto the concentrated filter. The remaining solution was collected at 3500 rpm for 30 minutes. The same procedure was repeated four times to obtain the protein through the destaining and buffer exchange process.

[0065] Confirmation of tagged proteins The expression and size of the tagged protein were confirmed by SDS-PAGE, and the results were confirmed using in-gel digestion (bottom-up method) and LC-MS / MS (Figure 3).

[0066] (1) In-gel digestion The band corresponding to the tagged protein was isolated from the SDS-PAGE gel and then destained. The destained gel was then reduced and alkylated, then digested with trypsin. The digested peptides were recovered by destaining using a C18 tip.

[0067] (2) LC-MS / MS To confirm the sequence and extent of the tagged proteins expressed in the strains, Evosep liquid chromatography and high-resolution mass spectrometry were performed (Q-Exactive HF-X mass spectrometer system). The destained peptide samples were dissolved in 0.1% formic acid solution and then injected onto the column. The peptide samples were separated using a C18 column (8 cm x 100 μm, 3 μm: EV1064) and nano-flow liquid chromatography. The method used was the Evosep SPD100 method (11.5 min).

[0068] -Buffer A: 0.1% formic acid in water / Buffer B: 0.1% formic acid in acetonitrile The mass spectrometer parameters used were as follows: -Resolution: Full MS 60,000, MS2 15,000 -Full MS: 300~2,000m / z, 100msec -MS2: 28msec, NCE27, ionized substances with 1 or more than 6 charges are excluded from MS2

[0069] The software used to identify peptides and proteins from bottom-up data was Thermo's Proteome Discoverer (v2.4) search engine. Protein / peptide identification was performed using an FDR of 1%. Six histidine amino acids attached to the C-terminus of the target protein were additionally identified by comparing the theoretical mass values ​​of precursor and fragment ions in the analyzed data.

[0070] Mass spectrometry of target proteins and tagged proteins using MALDI-TOF To confirm the mass values ​​and peak intensities of the target protein and tagged protein, the target protein and tagged protein were mixed and measured by MALDI-TOF (Figures 4-6). A Bruker Biotyper MALDI-TOF MS system was used. For the measurements, 1 μL of the target protein and tagged protein mixture and 1 μL of sinapinic acid (SA) 20 mg / mL in 0.1% TFA / 50% acetonitrile substrate were placed on a plate spot, mixed, and then completely dried.

[0071] The pulse ion extraction time was 450 ns, and random position acquisition was performed to irradiate the laser for 40 shots each, a total of 2,000 shots, and the spectral data were accumulated to obtain the data.

[0072] Mass spectra were obtained in positive ion and linear mode with a laser frequency of 100 Hz for the 12,000 to 32,000 m / z interval, and +1 and +2 charged ions were detected simultaneously.

[0073] Mass spectral data were acquired using Bruker's Flex Control version 4.1 software and processed using Flexa Analysis version 4.1 software with Savitzky-Golay smoothing and TopHat baseline subtraction. Peak intensity measurements were confirmed using a centroid peak detection algorithm.

[0074] Correcting the mass values ​​of the target proteins using the mass values ​​of the tagged proteins as the internal standard as a reference showed that the reproducibility of mass values ​​improved for all concentration combinations, bringing them closer to the theoretical mass values ​​(Figure 7). In addition, correcting the intensity values ​​of the target proteins using the intensity values ​​of the tagged proteins as a reference also confirmed a significant improvement in the reproducibility of the intensity of the target proteins (Figure 8).

[0075] Mass correction of target proteins derived from clinical strains using tagged proteins The mass values ​​of target proteins (KPC) derived from clinical strains were calibrated using the tagged protein of the present invention. To confirm this, clinical strains whose genotypes were confirmed using KPC were cultured on blood agar plates. The target proteins in the cultured clinical strains were identified using the same method as described above. Specifically, the mass values ​​of mass spectrometry spectra containing the KPC target protein (MW = 28,718 Da) were calibrated using the tagged protein (MW = 29,541 Da) as an internal standard, and the target proteins were identified from a total of 43 clinical strains using the calibrated mass values ​​(Figure 9).

[0076] Determination of calibration mass range using IS proteins To evaluate the appropriate mass range for the internal standard (tagged protein), we examined the mass error values ​​of three carbapenem-degrading enzymes (IMP-6, VIM-2, and GES-5) and one beta-lactam-degrading enzyme (CTX-M-1), which have mass differences ranging from several hundred to 5,000 Da relative to the internal standard. As shown in Figure 10, all proteins showed mass errors of less than 3.2 Da. Among these, CTX-M-1 and VIM-2 showed very low mass errors (<1.6 Da) compared to the other proteins, but their distances from the internal standard were 1,331 and 4,026 Da, respectively. IMP-6 had the largest mass difference from the internal standard, hKPC-2, and also the largest mass error of all proteins (Figure 10). The average mass error of all proteins derived using external standards (Ex-Cal, KPC-2) and internal standards (In-Cal, hKPC-2) was ~83.5 ppm, and the distribution range of mass error was -3.0 to +3.2 Da.

[0077] Identification of mass-corrected KPC subtypes using hKPC-2 Analysis using Ex-Cal (KPC-2) and In-Cal (hKPC-2) was performed to identify KPC subtype proteins (KPC-2, KPC-3, KPC-4, and KPC-17). The MALDI mass spectra and average mass errors (-0.9 to 0.9 Da) for these proteins are shown in Table 1 and Figure 11. All KPC subtypes were accurately identified with less than 1 Da error, regardless of whether they were reference strains or clinical strains, despite the mass difference ranging from -34 to +26 Da (Table 1 and Figure 11B).

[0078] [Table 1]

[0079] Determination of mass correction range using internal standards To assess whether the method of the present invention, which uses 6x HIS-tagged proteins as internal standards, can be generally applied to other mass ranges, we confirmed the mass error of another target protein, ALDC, using hALDC. 6x HIS-tagged hALDC as an internal standard has a mass of 40.6 kDa, more than 12 kDa higher than hKPC-2. We performed internal calibration (In-Cal) using six proteins (Ubi, Myo, Tryptophan, KPC-2, BSA, and hALDC) as internal standard candidates (Figure 12A). To evaluate the effect of the mass distance between the internal standard and the target protein on the mass error, we calculated the mass error value of ALDC after In-Cal. The mass distance range of the six internal standard candidate proteins to ALDC is approximately 2100–31,000 Da (Figure 12). The analysis results showed that when hALDC was used as an internal standard for ALDC, the mass error was very low (<3.4 Da, <85 ppm), as was the case for hKPC-2. However, other internal standard candidates with a mass difference of 10 kDa or more from the target protein were completely unable to function as standards (errors of >3000 ppm).

[0080] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention, and therefore the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Protein mass measurement internal standards including: (a) an analyte protein; and (b) a tag peptide consisting of any sequence of 1 to 100 consecutive amino acids attached to the N-terminus or C-terminus of the protein to be analyzed;

2. 2. The internal standard according to claim 1, wherein the tag peptide consists of any 2 to 80 consecutive amino acids bound to the C-terminus of the target protein.

3. The internal standard substance described in claim 2, characterized in that the tag peptide is selected from the group consisting of: a repeated sequence of the same amino acid residues; a consecutive sequence of different amino acid residues; a repeated consecutive sequence of different amino acid residues; and combinations thereof.

4. 4. The internal standard according to claim 3, wherein the identical amino acid is histidine (His).

5. 4. The internal standard of claim 3, wherein the consecutive sequence of different amino acid residues includes the amino acid sequence of sequence number 7 in the sequence listing.

6. The mass spectrometry includes MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization Time of Flight) mass spectrometry, SELDI-TOF (Surface Enhanced Laser Desorption / Ionization Time of Flight) mass spectrometry, ESI-TOF (Electrospray ionization time-of-flight) mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), and LC-MS / MS (Liquid Chromatography Mass Spectrometry).

2. The internal standard according to claim 1, wherein the internal standard is selected from the group consisting of: chromatographic-mass spectrometry / mass spectrometry.

7. 9. The internal standard of claim 8, wherein the mass spectrometry in the step is MALDI-TOF (Matrix Desorption / Ionization Time of Flight) mass spectrometry.

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

9. A method for detecting a protein in a biological sample, comprising: (a) adding an internal standard according to any one of paragraphs 1 to 7 to a biological sample containing a protein to be analyzed; (b) measuring the mass values ​​of the analyte protein and the internal standard in the biological sample; (c) correcting the measured mass value of the analyte protein based on the measured mass value of the internal standard.

10. The step (b) may be carried out by any of a variety of mass spectrometry techniques, including MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization Time of Flight) mass spectrometry, SELDI-TOF (Surface Enhanced Laser Desorption / Ionization Time of Flight) mass spectrometry, ESI-TOF (Electrospray ionization time-of-flight) mass spectrometry, liquid chromatography-mass spectrometry (LC-MS) and LC-MS / MS (Liquid Chromatography Mass Spectrometry).

10. The method of claim 9, wherein the method is carried out using a mass spectrometry method selected from the group consisting of mass spectrometry (MS / MS chromatography / mass spectrometry).

11. 11. The method of claim 10, wherein step (b) is performed using MALDI-TOF (Matrix Desorption / Ionization Time of Flight) mass spectrometry.

12. Protein quantitation internal standards including: (a) an analyte protein; and (b) a tag peptide consisting of any sequence of 1 to 100 consecutive amino acids attached to the N-terminus or C-terminus of the protein to be analyzed;

13. 1. A method for quantifying protein in a biological sample, comprising: (a) adding the internal standard substance according to claim 12 to a biological sample containing a target protein; (b) measuring the mass values ​​of the analyte protein and the internal standard in the biological sample; (c) correcting the quantitative value of the analyte protein based on the signal intensity of the measured mass value of the internal standard.

14. Protein mass measurement internal standards including: (a) any protein for which the mass value is known; and (b) A tag peptide consisting of 1 to 100 consecutive amino acids attached to the N-terminus or C-terminus of any of the above proteins.

15. A method for detecting a protein in a biological sample, comprising: (a) adding the internal standard according to claim 14 to a biological sample containing a target protein; (b) measuring the mass values ​​of the analyte protein and the internal standard in the biological sample; (c) correcting the measured mass value of the analyte protein based on the measured mass value of the internal standard.

16. Protein quantitation internal standards including: (a) any protein for which the mass value is known; and (b) A tag peptide consisting of 1 to 100 consecutive amino acids attached to the N-terminus or C-terminus of any of the above proteins.

17. 1. A method for quantifying protein in a biological sample, comprising: (a) adding the internal standard according to claim 16 to a biological sample containing a target protein; (b) measuring the mass values ​​of the analyte protein and the internal standard in the biological sample; (c) correcting the quantitative value of the analyte protein based on the signal intensity of the measured mass value of the internal standard.

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