METHOD FOR DIRECTLY DETECTING PATHOGENIC STRAIN HAVING RESISTANCE TO BETA-LACTAM ANTIBIOTICS

Abstract

The present invention relates to a method for detecting a pathogenic strain having resistance to β-lactam antibiotics in a biological sample, and a method for identifying a protein involved in resistance in β-lactam antibiotics, which is contained in a biological sample. According to the present invention, it is possible to quickly and accurately determine not only whether a pathogenic strain has resistance to antibiotics, but also the type of protein involved in the resistance, by directly identifying an extended spectrum β-lactamase (ESBL) protein with a truncated N-terminus through mass spectrometry. Accordingly, the present invention can be effectively utilized to quickly establish an appropriate antibiotic administration strategy at the initial stage of infection.

Claims

1. A method of detecting a pathogenic strain having resistance to β-lactam antibiotics in a biological sample, comprising: (a) isolating a protein expressed by a pathogenic strain in a biological sample isolated from a subject; and (b) performing top-down mass spectrometry on the isolated protein, wherein it is determined that the pathogenic strain having resistance to β-lactam antibiotics is present in the biological sample, when a protein having the same mass as β-lactamase from which 28 amino acid residues at the N-terminus thereof have been removed is detected as a result of the mass spectrometry.

2. The method of claim 1, further comprising performing ion exchange chromatography on the protein isolated in step (a).

3. The method of claim 2, wherein the ion exchange chromatography is anion exchange chromatography.

4. The method of claim 1, wherein the β-lactamase is CTX-M protein.

5. The method of claim 4, wherein the CTX-M protein is at least one protein selected from the group consisting of CTX-M1 to CTX-M7, CTX-M9, CTX-M10, CTX-M12 to CTX-M17, CTX-M19 to CTX-M24, CTX-M27 to CTX-M38, CTX-M40 to CTX-M44, CTX-M46 to CTX-M56, CTX-M58 to CTX-M69, CTX-M71 to CTX-M77, CTX-M79 to CTX-M88, CTX-M90, CTX-M92, CTX-M93, CTX-M95 to CTX-M105, CTX-M110 to CTX-M117, CTX-M121 to CTX-M127, CTX-M129 to CTX-M132, CTX-M134, CTX-M136 to CTX-M139, CTX-M141, CTX-M142, CTX-M144, CTX-M146 to CTX-M148, CTX-M150, CTX-M155 to CTX-M159, CTX-M161 to CTX-M184, CTX-M186 to CTX-M204, CTX-M206 to CTX-M210, CTX-M212 to CTX-M216, and CTX-M218 to CTX-M226.

6. The method of claim 5, wherein the CTX-M protein is at least one protein selected from the group consisting of CTX-M1, CTX-M3, CTX-M10, CTX-M15, CTX-M22, CTX-M23, CTX-M28, CTX-M32 to CTX-M34, CTX-M36, CTX-M42, CTX-M52 to CTX-M55, CTX-M58, CTX-M61, CTX-M62, CTX-M64, CTX-M69, CTX-M71, CTX-M72, CTX-M79, CTX-M80, CTX-M82, CTX-M88, CTX-M101, CTX-M103, CTX-M114, CTX-M116, CTX-M117, CTX-M123, CTX-M127, CTX-M132, CTX-M136, CTX-M138, CTX-M142, CTX-M144, CTX-M146, CTX-M150, CTX-M155 to CTX-M158, CTX-M166, CTX-M167, CTX-M169, CTX-M170, CTX-M172, CTX-M173, CTX-M175 to CTX-M184, CTX-M187 to CTX-M190, CTX-M193, CTX-M197, CTX-M199, CTX-M201 to CTX-M204, CTX-M206 to CTX-M209, CTX-M212, CTX-M216, CTX-M218, CTX-M220, CTX-M222, and CTX-M225.

7. The method of claim 5, wherein the CTX-M protein is at least one protein selected from the group consisting of CTX-M1, CTX-M14, CTX-M15, CTX-M27, CTX-M142 and CTX-M186.

8. The method of claim 1, wherein the step (a) is performed by adding a surfactant to the biological sample.

9. The method of claim 1, wherein the step (a) is performed by applying osmotic pressure to the biological sample.

10. The method of claim 1, wherein the step (b) is performed using a mass spectrometry method 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).

11. The method of claim 10, wherein the step (b) is performed using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry.

12. The method of claim 1, wherein it is determined that the pathogenic strain having resistance to beta-lactam antibiotics is present in the biological sample, when one or more mass values (m/z×z) selected from the group consisting of 28210, 28287, 28166, 28164, 28203, 28116, 27946, 28153, 28089, 27974, 28108, 27888, 27973, 27964, 28321, 27932, 28165, 28197, 28044, 27916, 28107, 28167, 28273, 28152, 28081, 28212, 28277, 28182, 28139, 28043, 27819, 27810, 28170, 28298, 28356, 28027, 28073, 28001, 28099, 28000, 27974, 28184, 28148, 28136, 28314, 28214, 28263, 28181, 28211, 28156, 28046, 28071, 27947, 28180, 28121, 28154, 28067, 28006, 28291, 28261, 28307, 28135, 28194, 27998, 28134, 27983, 27944, 27958, 28008, 28018, 28002, 28317, 27931, 28289, 28095, 28033, 27798, 28005, 28193, 28059, 28024, 27948, 28109, 28229, 28042, 27852, 28345, 28238, 27992, 28053, 28036, 28094, 28050, 27915, 28260, 28014, 28218, 28092, 28138, 28240, 28078, 28151, 28140, 28131, 28150, 27975, 27889, 27985, 28125, 28313, 28023, 28120, 28178, 28198, 28090, 27976, 28025, 28122, 28224, 27917, and values within the ranges of these values ±5 are detected as a result of the mass spectrometry.

13. The method of claim 12, wherein the mass values (m/z×z) additionally include a mass value that increased by 16 or 32 from each mass value.

14. A method for identifying a protein involved in resistance to β-lactam antibiotics in a biological sample, comprising: (a) isolating a protein expressed by a pathogenic strain in a biological sample isolated from a subject; (b) performing mass spectrometry on the isolated protein by a top-down method; and (c) determining the type of protein involved in resistance to beta-lactam antibiotics, which is contained in the biological sample, by comparing the result of the mass spectrometry with a mass value selected from the group consisting of the mass values (m/z×z) of β-lactamases listed in Table 1, from which 28 amino acid residues at the N-terminus thereof have been removed, values within the ranges of the mass values ±5, values that increased by 16 from the mass values, and values that increased by 32 from the mass values.

15. The method of claim 14, further comprising performing ion exchange chromatography on the protein isolated in the step (a).

16. The method of claim 15, wherein the ion exchange chromatography is anion exchange chromatography.

17. The method of claim 14, wherein the step (a) is performed by adding a surfactant to the biological sample.

18. The method of claim 14, wherein the step (a) is performed by applying osmotic pressure to the biological sample.

19. The method of claim 14, wherein the step (b) is performed using a mass spectrometry method 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, liquid chromatography-mass spectrometry (LC-MS), and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS).

20. The method of claim 19, wherein the step (b) is performed using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0057] FIGS. 1A and 1B show the results of SDS-PAGE analysis for the expression and sizes of CTX-M proteins derived from an antibiotic-resistant strain, and shows SDS-PAGE results for CTX-M1 protein (FIG. 1A) and CTX-M15 protein (FIG. 1B) as representative examples.

[0058] FIGS. 2A and 2B show the results of SDS-PAGE analysis for the expression and size of protein in a recombinant strain containing the CTX-M protein and gene derived from a clinical strain, and indicates the protein difference between a crude extract and a crude enzyme solution, obtained by addition of a nonionic surfactant (FIG. 2A) and an ionic surfactant (FIG. 2B), respectively.

[0059] FIG. 3 shows the results of SDS-PAGE analysis for the expression and size of a target protein in a sample pretreated with osmotic stimulation.

[0060] FIG. 4 shows the results of separating and purifying a target protein using ion chromatography.

[0061] FIGS. 5A and 5B indicate CTX-M protein peptides identified as MS-GF+, and shows a table (FIG. 5A) showing information on the position and sequence of each identified peptide and alignment results (FIG. 5B) that comparatively show the identification ranges (grey) of representative CTX-M proteins identified in the strain and the oxidized methionine residues (bold and underlined).

[0062] FIGS. 6A, 6B, 6C and 6D show exemplary tandem spectrum results for peptides identified as N-terminal and C-terminal peptides in representative subtype peptides of CTX-M, and shows the separation chromatogram of the CTX-M1 peptide (FIG. 6A), the results of identification of the C-terminal peptide of CTX-M1 (FIG. 6B), the results of identification of the N-terminal peptide of CTX-M1 (FIG. 6C), and the results of identification of the N-terminal peptide of CTX-M15 (FIG. 6D).

[0063] FIG. 7 shows examples of the results of multiple alignment analysis of the CTX-M protein amino acid sequences, performed using the Clustal Omega program, and the results of identification of the conservative amino acid sequences.

[0064] FIGS. 8A, 8B and 8C show the results of protein identification performed using a high-resolution mass spectrometer, and shows the elution chromatogram of the CTX-M protein (eluted at RT 23.63 min) (FIG. 8A), and the mass spectrum of the multi-charged CTX-M protein (monoisotopic mass −28,192 m/z×z, average molecular weight −28,210 m/z ×z) (FIG. 8B), and the tandem spectra of CTX-M protein ions with a charge state of 25 and an example of identification results of the 29-291 a.a. sequence (top/average molecular weight −28,210 m/z×z: E=1.3E-27, bottom/average molecular weight 28,210+32 m/z×z: E=2.36E-25) (FIG. 8C).

[0065] FIG. 9 shows an example of the mass spectrometry spectrum of CTX-M protein, obtained using a low-resolution mass spectrometer (MALDI-TOF).

MODE FOR INVENTION

[0066] Hereinafter, the present invention will be described in more detail with reference to examples. These examples serve merely to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention according to the subject matter of the present invention is not limited by these examples.

EXAMPLES

Example 1. Cloning of ESBL Gene and Construction of Antibiotic Resistant Strain

[0067] Based on information about the CTX-M gene sequence (European Molecular Biology Laboratory (EMBL) nucleotide sequence database accession number: CP008736.1, 876nt) (SEQ ID NO: 1) obtained from the gene for E. coli ESBL protein, the following oligonucleotides were prepared.

TABLE-US-00002 Primer 1:  5′-AACTGCAGGATGGTTAAAAAATCACTGCGTCAG-3′ (33 nt) Primer 2:  5′-GGAATTCTCACAAACCGTTGGTGACGATT-3′ (29 nt)

[0068] Restriction enzyme sites for cloning were added to the oligonucleotides, and an open reading frame (ORF) was created to induce expression directly in a cloning vector.

[0069] The target gene was amplified by PCR from the CTX-M template DNA. For PCR, a total of 50 μl of a PCR reaction solution was prepared using 3 μl of template DNA, 1.25 μl of 5′ primer, 1.25 μl of 3′ primer, 1 μl of dNTPs, and 10 μl of 5× buffer, and then PCR was performed under the following conditions: 1) denaturation—at 98° C. for 10 sec; 2) annealing-at 57° C. for 30 sec; 3) extension—at 72° C. for 30 sec. Cloning for construction of a recombinant plasmid containing the target gene was performed as follows: 1) For the insert gene and the vector, both ends of the DNA were cut into sticky ends using restriction enzymes, and 2) the insert gene was ligated into the vector using DNA ligase. 3) Thereafter, the vector was transformed into E. coli (E. coli Top10), and 4) recombinant E. coli was selected by the white/blue screening method. 5) A recombinant plasmid was extracted from the selected strain, and 6) the extracted plasmid was treated with restriction enzymes, and the DNA size was determined. 7) Finally, the inserted gene was confirmed through DNA sequencing.

Example 2. Analysis of Expression and Size of Target Protein

[0070] E. coli transformed with the plasmid containing the target gene was inoculated into Luria-bertani liquid medium containing 50 mg/L of ampicillin antibiotic, and cultured at 37° C. for 16 hours or more. In order to analyze the expression and size of the target protein, the culture was centrifuged at 4,000 rpm for 15 minutes, and the cells were harvested by removing the supernatant. The harvested cells were added to SDS-sample buffer, heated at 95° C. for 5 min, and centrifuged at 15,000 rpm for 5 min. Using the prepared sample, the expression and size of the target protein were analyzed through SDS-PAGE gel analysis (FIG. 1).

Example 3. Sample Pretreatment Method and Identification of Target Protein from Crude Enzyme Solution

[0071] (1) Sample Pretreatment with Nonionic Surfactant

[0072] For sample pretreatment, the culture was centrifuged at 4,000 rpm for 15 minutes, and the cells were harvested by removing the supernatant. To obtain a crude extract, the cells were treated with a buffer solution (0.25 mM Tris-HCl, 2% OG) and incubated at room temperature for 10 minutes. The prepared crude extract was separated into a supernatant (hereinafter referred to as crude enzyme solution) and a precipitate by centrifugation at 15,000 rpm at 4° C. for 10 minutes. From the crude enzyme solution, the expression and size of the target protein were analyzed by SDS-PAGE analysis from (FIG. 2A).

[0073] (2) Sample Pretreatment with Ionic Surfactant

[0074] Sample pretreatment was performed by the following steps: 1) 100 μl of the expressed cell culture was centrifuged to recover the cells, and 2) the supernatant was removed, and then a buffer solution (0.25 mM Tris-HCl, pH 8.0 and 2% DOC) containing 2% sodium deoxycholate (DOC) as a nonionic surfactant was added to the cells. 3) The suspension was incubated at room temperature for 10 minutes, and 4) centrifuged at 15,000 rpm at 4° C. for 10 minutes to obtain a crude enzyme solution.

[0075] From the crude extract and the crude enzyme solution, obtained by treatment with the nonionic surfactant (OG) and the ionic surfactant (DOC), respectively, the expression and size of the protein were analyzed by SDS-PAGE gel analysis (FIG. 2B).

[0076] (3) Cell Lysis by Osmotic Pressure

[0077] The strain culture was added to a washing buffer (pH 8.0 Tris-HCl+500 mM NaCl), and then incubated at room temperature for 10 minutes. Next, the supernatant was removed by centrifugation at 14,000 g for 10 minutes, and triple distilled water was added to the pellet, followed by incubation for 10 minutes. This pretreated sample was further separated into a supernatant (crude enzyme solution) and a precipitate by centrifugation. From the crude enzyme solution, the expression and size of the target protein were analyzed by SDS-PAGE analysis (FIG. 3).

Example 4. Confirmation of Genotype of CTX-M Derived from Clinical Strain and Confirmation of Protein

[0078] In order to confirm CTX-M derived from a clinical strain, a strain confirmed as positive for ESBL was collected. The collected strain was subjected to colony PCR using the primers used for CTX-M gene amplification. The resulting amplified PCR product was subjected to agar gel electrophoresis to confirm the size of the target gene. The PCR product whose size has been confirmed was subjected to DNA sequencing to confirm the exact genotype of the CTX-M gene.

[0079] The strain whose genotype has been confirmed was cultured using LB broth, and SDS-PAGE gel analysis was performed to examine whether CTX-M protein would be expressed. In addition, a recombinant strain containing the CTX-M gene derived from the clinical strain was constructed in the same manner as the existing recombinant strain comprising the vector containing the CTX-M gene, and the recombinant strain was also subjected to SDS-PAGE gel analysis in the same manner as the clinical strain to confirm the size of the actually expressed CTX-M protein (FIG. 3).

Example 5. Separation/Purification of Target Protein

[0080] Ion exchange chromatography was used to separate/purify the target protein. Q-resin was used as the ion exchange resin, 500 μl of the crude enzyme solution was loaded into a column containing Q-resin, and then the eluted solution was collected. The column was washed with 1 ml of 20 mM Tris-HCl (pH 8.0) buffer, and eluted with 1M NaCl, 20 mM Tris-HCl (pH 8.0) buffer. The sample collected in each step was subjected to SDS-PAGE gel electrophoresis to confirm the separated/purified zone (FIG. 4). Finally, high-purity CTX-M protein was separated/purified from the cell lysate using the same method as above.

Example 6. Analysis of Expression and Size of Target Protein

[0081] The protein whose expression and size were confirmed on the SDS-PAGE gel was identified using the in-gel digestion method and the nano-LC-MS/MS method, thereby confirming the type of antibiotic resistance protein actually expressed in the strain (FIG. 5A).

[0082] (1) In-Gel Digestion

[0083] Only the band portion corresponding to the size of the target protein on the SDS-PAGE gel was obtained, and the stained gel was destained. The destained gel was subjected to a reduction/alkylation process, and then the protein was selectively digested using a trypsin enzyme. The digested peptides were recovered and desalted using DK-Tip (C18 Tip).

[0084] (2) Nano-LC-MS/MS

[0085] In order to confirm the sequence and range of the active protein expressed in the strain, nano liquid chromatography and high-resolution mass spectrometry were performed (Q-Exactive HF-X mass spectrometry system). The desalted peptide sample was dissolved with 0.1% formic acid solution and then loaded into a column. The peptide sample was separated using a C18 column (75 μm×70 cm) and nanoflow liquid chromatography. The gradient conditions for sample loading and separation used at this time are as follows: [0086] buffer A: 0.1% formic acid in water/buffer B: 0.1% formic acid in acetonitrile [0087] sample loading: from 0 to 5 min, 5% (B), 5 μL/min flow rate [0088] concentration gradient for separation:

[0089] from 5 to 7 min, from 5% to 10% (B), 300 nL/min flow rate

[0090] from 7 to 38 min, from 10% to 40% (B), 300 nL/min flow rate

[0091] from 38 to 38.5 min, from 40% to 80% (B), 300 nL/min flow rate

[0092] from 38.5 to 39.5 min, 80% (B), 300 nL/min flow rate

[0093] from 39.5 to 40 min, from 80% to 5% (B), 300 nL/min flow rate

[0094] from 40 to 60 min, 5% (B), 300 nL/min flow rate

[0095] The parameters of the mass spectrometer used at this time are as follows: [0096] resolution: Full MS 60,000, MS2 30,000 [0097] Full MS: 350 to 2,000 m/z, 100 msec [0098] MS2: 50 msec, NCE 28, 1, ionized materials with a charge state of >6 were excluded from MS2 analysis

[0099] As software for identifying peptides and proteins based on bottom-up data, the ‘MS-GF+’ search engine developed at San Diego State University, USA was used, and protein/peptide identification was based on a FDR (false discovery rate) of 1%. Among proteins from E. coli, CTX-M protein was identified, and 143 peptides were identified (total coverage of 90.38%). In addition, peptides in which one or two methionine among six methionine residues (71, 78, 120, 138, 189, and 214) are in oxidized form were also identified. Specifically, the N-terminal sequence peptide consisting of residues 1 to 28 was not detected, and when the sequence except for residues 1 to 28 was considered, peptides spanning all sequence ranges were identified, suggesting that the coverage was 100% (gray background in FIG. 5B).

[0100] (3) Identification of N-Terminal and C-Terminal Sequences (FIG. 7)

TABLE-US-00003 -N-terminal sequence:  (A)/QTADVQQK (semi-tryptic) -C-terminal sequence:  (R)/RDVLASAAKIVTNGL-(semi-tryptic)

Example 7. Amino Acid Sequencing and Characterization of CTX-M Protein

[0101] Based on the MS2 results of Example 3, multiple alignment analysis was performed on all 205 CTX-M proteins known to date in the National Center for Biotechnology Information (NCBI) database (FIG. 7). As a result, 98 CTX-M proteins in which 97.6% or more of the full-length amino acid sequence is conserved were identified, and 82 CTX-M proteins containing the same N-terminal peptide (1 to 28 a.a.) as CTX-M-1 were identified. All 82 proteins identified were characterized by a sequence consisting of 291 amino acids.

Example 8. Target Protein Identification Using Mass Spectrometry (Top-Down Method)

[0102] To determine the mass value of the active protein expressed in the strain, top-down mass spectrometry was performed. To confirm the exact mass value of the protein, the crude protein extract derived from the strain was used, and a top-down LC-MS/MS system (Nano-LC and Q-Exactive HF-X mass spectrometry system) was used.

[0103] (1) Separation of Crude Protein Extract

[0104] About 0.1 μg of a protein sample was mixed with a 1% formic acid solution at a ratio of 1:1 and adjusted to approximately pH 3, and then the sample was loaded into a column. The crude protein extract was separated using a column (150 μm×20 cm) packed with PLRP-S resin and nanoflow liquid chromatography. The gradient conditions for sample loading and separation used at this time are as follows. [0105] Buffer A: 0.1% formic acid in water/buffer B: 0.1% formic acid in acetonitrile [0106] sample loading: from 0 to 10 min, 5% (B), 5 μL/min flow rate [0107] Concentration gradient for separation:

[0108] from 10 to 10.01 min, from 5% to 10% (B), 300 nL/min flow rate

[0109] from 10.01 to 40 min, from 10% to 40% (B), 300 nL/min flow rate

[0110] from 40 to 41 min, from 40% to 80% (B), 300 nL/min flow rate

[0111] from 41 to 42 min, 80% (B), 300 nL/min flow rate

[0112] from 42 to 43 min, from 80% to 5% (B), 300 nL/min flow rate

[0113] from 43 to 60 min, 5% (B), 300 nL/min flow rate

[0114] (2) Mass Spectrometry of CTX-M Protein Using High-Resolution Mass Spectrometry

[0115] Using the protein mode analysis method with the Q-Exactive HF-X mass spectrometer, the mass values of the intact proteins and the tandem mass spectra of the proteins were obtained and identified (FIGS. 8A, 8B and 8C). The parameters used in this case are as follows. [0116] Resolution: use of Full MS 240,000, MS2 120,000 [0117] Full MS: 620 to 2,400 m/z, 100 msec [0118] MS2: use of 1 microscan, 1,000 msec, NCE 50; ionized materials with a charge state of 1 to 8 were excluded from MS2 analysis.

[0119] Software for identifying proteins based on top-down data, ‘Informed Proteomics’ developed by US PNNL (Pacific Northwest National Laboratory) was used. Several multi-charged CTX-M protein peaks appeared at around 23.63 minutes (FIG. 5 FIG. 8A), and it was confirmed that a representative mass value obtained by deconvolution of the peaks was an average molecular weight of 28,209.69 m/z×z, and 28,192.69 m/z×z as a monoisotopic mass. In addition, other peaks resulting from methionine oxidation were observed (e.g., polypeptides with two oxidized methionines showed an average molecular weight of 28,210+32 m/z×z). They partially coincided with the positions of oxidized methionine residues (71, 78, 120, 138, 189, and 214) within the CTX protein range (29-291 a.a.) obtained through in-gel digestion (bottom-up method). Polypeptides including the N-terminal sequence consisting of residues 1-29 were not observed even in the analysis based on the top-down method.

[0120] (3) Mass Spectrometry of CTX-M Protein Using Low-Resolution Mass Spectrometer

[0121] The mass spectrometry spectrum of CTX-M protein was obtained using the Bruker Biotyper MALDI-TOF MS system. First, 1 μL of a sinapinic acid (SA, present at 10 mg/mL in 0.1% TFA/50% acetonitrile) matrix and about 100 ng of CTX-M protein were placed on the plate spot, dried completely, and subjected to mass spectrometry. At this time, the maximum energy used was 30%, random position acquisition was performed, a total of 2,000 laser shots (40 shots per time) were irradiated, and each spectral data was cumulatively obtained. Mass spectrometry spectra were obtained for the range of 10,000 to 40,000 m/z, and CTX-M protein with a charge state of +1 as well as CTX-M protein with a charge state of +2 were simultaneously detected (FIG. 9).

[0122] (4) Comparison of Mass Values of CTX-M Proteins

[0123] The exact mass values of the CTX-M proteins were confirmed through the above-described method, and the mass values of the active CTX-M proteins from which the N-terminal peptide has been removed could be confirmed based on the confirmed mass values. Thus, for all CTX-M proteins identified in NCBI, the exact mass value of the active protein can be confirmed through high-resolution or low-resolution mass spectrometry, and it is possible to rapidly and accurately identify various types of CTX-M proteins through mass spectrometry (Table 2).

TABLE-US-00004 TABLE 2 Mass data of CTX-M protein Full-length CTX-M protein Active protein Average Average N- molecular Monoisotopic molecular Monoisotopic terminus CTX-M Da weight mass PI Da weight mass PI removed 1 31246 31245.69 31226.29 9.25 28210 28209.95 28192.58 8.63 1-28aa 2 31378 31377.79 31358.23 8.91 28287 28287.02 28269.62 7.99 1-28aa 3 31202 31201.64 3118.26 9.25 28166 28165.90 28148.55 8.63 1-28aa 4 31255 31254.52 31235.00 7.86 28164 28163.75 28146.39 6.59 1-28aa 5 31294 31293.67 31274.17 9.12 28203 28202.90 28185.57 8.60 1-28aa 6 31207 31206.51 31187.07 8.61 28116 28115.74 28098.46 7.15 1-28aa 7 31202 31201.64 31182.25 9.10 28166 28165.90 28148.54 7.99 1-28aa 9 30951 30951.32 30932.03 9.09 27946 27945.57 27928.42 8.02 1-28aa 10 31202 31201.64 31182.25 9.10 28166 28165.90 28148.54 7.99 1-28aa 12 31159 31158.61 31139.26 9.25 28153 28152.90 28135.55 8.63 1-28aa 13 31127 31126.57 31107.10 9.07 28089 28088.76 28071.52 8.01 1-28aa 14 30979 30979.38 30960.06 9.09 27974 27973.62 27959.46 8.02 1-28aa 15 31144 31143.60 31124.26 9.38 28108 28107.86 28090.54 8.95 1-28aa 16 30893 30893.29 30874.28 9.27 27888 27887.53 27870.42 8.70 1-28aa 17 30978 30978.43 30959.11 9.39 27973 27972.68 27955.51 9.02 1-28aa 19 30969 30969.34 30950.04 9.09 27964 27963.58 27946.44 8.02 1-28aa 20 31412 31411.81 31392.21 8.91 28321 28321.04 28303.61 7.99 1-28aa 21 30897 30897.17 30877.98 9.21 27932 27931.54 27914.41 8.02 1-28aa 22 31201 31200.65 31181.28 9.38 28165 28164.91 28147.57 8.95 1-28aa 23 31233 31232.70 31213.30 9.38 28197 28196.96 28179.59 8.95 1-28aa 24 31048 31048.48 31029.13 9.27 28044 28043.73 28025.53 8.70 1-28aa 27 30921 30921.34 30902.06 9.27 27916 27915.59 27898.45 8.70 1-28aa 28 31143 31142.62 31123.27 9.49 28107 28106.88 28089.56 9.16 1-28aa 29 31114 31113.58 31094.25 9.38 28108 28107.86 28090.54 8.95 1-28aa 30 31173 31172.60 31153.24 9.10 28167 28166.88 28149.53 7.99 1-28aa 31 31364 31363.76 31344.21 8.91 28273 28272.99 28255.61 7.99 1-28aa 32 31188 31187.66 31168.28 9.38 28152 28151.92 28134.57 8.95 1-28aa 33 31117 31116.58 31097.25 9.28 28081 28080.84 28063.53 8.95 1-28aa 34 31248 31247.72 31228.24 9.00 28212 28211.98 28194.53 7.91 1-28aa 35 31368 31367.75 31348.21 8.91 28277 28276.98 28259.60 7.99 1-28aa 36 31218 31217.64 31198.26 9.25 28182 28181.90 28164.54 8.63 1-28aa 37 31149 31148.53 31129.20 8.90 28139 28138.83 28121.49 6.95 1-28aa 38 31048 31048.48 31029.13 9.27 28043 28042.73 28025.53 8.70 1-28aa 40 31065 31065.43 31046.01 8.45 27819 27819.40 27802.30 5.97 1-28aa 41 31046 31045.52 31025.98 8.96 27810 27810.46 27793.28 6.95 1-28aa 42 31206 31205.63 31186.26 9.25 28170 28169.89 28152.54 8.63 1-28aa 43 31389 31388.86 31369.29 9.27 28298 28298.09 28280.69 8.91 1-28aa 44 31447 31446.90 31427.30 9.12 28356 28356.13 28338.69 8.60 1-28aa 46 31032 31032.44 31013.09 9.09 28027 28026.69 28009.48 8.02 1-28aa 47 31079 31078.51 31059.14 9.27 28073 28072.76 28055.54 8.70 1-28aa 48 31006 31006.40 30987.07 9.09 28001 28000.65 27983.47 8.02 1-28aa 49 31105 31104.55 31085.16 9.27 28099 28098.80 28081.55 8.70 1-28aa 50 31005 31005.41 30986.08 9.09 28000 27999.66 27982.47 8.02 1-28aa 51 30979 30979.38 30960.06 9.09 27974 27973.62 27956.46 8.02 1-28aa 52 31220 31219.65 31200.27 9.25 28184 28183.91 28166.56 8.63 1-28aa 53 31184 31183.71 31164.31 9.25 28148 28147.97 28130.60 8.63 1-28aa 54 31233 31232.65 31213.27 9.25 28197 28196.91 28179.56 8.63 1-28aa 55 31172 31171.66 31152.29 9.38 28136 28135.92 28118.58 8.95 1-28aa 56 31405 31404.81 31385.24 8.91 28314 28314.05 28296.63 7.99 1-28aa 58 31250 31249.68 31230.28 9.25 28214 28213.94 28196.57 8.63 1-28aa 59 31354 31353.81 31334.25 8.91 28263 28263.04 28245.65 7.99 1-28aa 60 31187 31186.67 31167.29 9.25 28181 28180.95 28163.59 8.63 1-28aa 61 31247 31246.68 31227.27 9.10 28211 28210.94 28193.56 7.99 1-28aa 62 31192 31191.60 31172.24 9.25 28156 28155.86 28138.53 8.63 1-28aa 63 31092 31092.46 31073.02 8.45 28046 28045.63 28028.41 5.97 1-28aa 64 31117 31116.61 31097.31 9.30 28081 28080.86 28063.60 8.66 1-28aa 65 31077 31076.54 31057.16 9.27 28071 28070.79 28053.56 8.70 1-28aa 66 31229 31228.67 31209.27 9.25 28166 28165.90 28148.55 8.63 1-28aa 67 30952 30952.35 30933.05 9.09 27947 27946.60 27929.45 8.02 1-28aa 68 31190 31189.63 31170.27 9.27 28180 28179.93 28162.57 8.63 1-28aa 69 31157 31156.60 31137.25 9.40 28121 28120.86 28103.54 8.97 1-28aa 71 31190 31189.69 31170.24 9.30 28154 28153.95 28136.53 8.84 1-28aa 72 31103 31102.50 31083.18 9.10 28067 28066.76 28049.47 7.99 1-28aa 73 31011 31011.44 30992.03 9.09 28006 28005.68 27988.43 8.02 1-28aa 74 31382 31381.78 31362.22 8.91 28291 28291.01 28273.62 7.99 1-28aa 75 31368 31367.75 31348.21 8.91 28287 28287.02 28269.62 7.99 1-28aa 76 31352 31351.71 31332.18 8.91 28261 28260.94 28243.57 7.99 1-28aa 77 31398 31397.86 31378.29 9.25 28307 28307.09 28289.69 8.89 1-28aa 79 31171 31170.67 31151.30 9.49 28135 28134.93 28117.59 9.16 1-28aa 80 31230 31229.69 31210.29 9.25 28194 28193.95 28176.58 8.63 1-28aa 81 31003 31003.31 30983.99 7.77 27998 27997.56 27980.38 5.97 1-28aa 82 31170 31169.64 31150.27 9.38 28134 28133.90 28116.56 8.95 1-28aa 83 30988 30988.39 30969.06 9.09 27983 27982.63 27965.46 8.03 1-28aa 84 30949 30949.35 30930.05 9.09 27944 27943.60 27926.45 8.02 1-28aa 85 30963 30963.33 30944.03 9.09 27958 27957.58 27940.42 8.02 1-28aa 86 31013 31013.39 30994.05 9.09 28008 28007.64 27990.44 8.02 1-28aa 87 31023 31023.47 31004.12 9.09 28018 28017.72 28000.52 8.02 1-28aa 88 31125 31124.56 31105.21 9.25 28089 28088.82 28071.50 8.63 1-28aa 90 31007 31007.43 30988.09 9.09 28002 28001.68 27984.49 8.02 1-28aa 92 31408 31407.82 31388.24 8.91 28317 28317.05 28299.63 7.99 1-28aa 93 30936 30936.31 30917.03 9.27 27931 27930.56 27913.43 8.70 1-28aa 95 31380 31379.76 31360.20 8.58 28289 28288.99 28271.59 7.15 1-28aa 96 31101 31100.58 31081.25 9.38 28095 28094.86 28077.55 8.95 1-28aa 97 31279 31278.65 31259.15 8.58 28287 28287.02 28269.62 7.99 1-28aa 98 30949 30949.39 30930.09 9.27 27944 27943.64 27926.48 8.70 1-28aa 99 31038 31038.45 31019.11 9.27 28033 28032.69 28015.50 8.70 1-28aa 100 31033 31033.47 31013.94 8.96 27798 27798.40 27781.25 6.95 1-28aa 101 31170 31169.68 31150.31 9.38 28134 28133.94 28116.60 8.95 1-28aa 102 30979 30979.38 30960.06 9.09 27974 27973.62 27956.46 8.02 1-28aa 103 31171 31170.63 31151.27 9.38 28135 28134.89 28117.55 8.95 1-28aa 104 31006 31006.40 30987.07 9.09 28001 28000.65 27983.47 8.02 1-28aa 105 31007 31007.43 30988.09 9.09 28002 28001.68 27984.49 8.02 1-28aa 110 31094 31094.42 31075.05 8.53 28089 28088.67 28071.45 6.20 1-28aa 111 31010 31010.39 30991.07 9.09 28005 28004.64 27987.46 8.02 1-28aa 112 30949 30949.35 30930.05 9.09 27944 27943.60 27926.45 8.02 1-28aa 113 31007 31007.43 30988.10 9.27 28002 28001.68 27984.50 8.70 1-28aa 114 31144 31143.60 31124.26 9.38 28108 28107.86 28090.54 8.95 1-28aa 115 31408 31407.82 31388.24 8.91 28317 28317.05 28299.63 7.99 1-28aa 116 31229 31228.71 31209.31 9.38 28193 28192.97 28175.60 8.95 1-28aa 117 31175 31174.62 31155.26 9.38 28139 28138.88 28121.55 8.95 1-28aa 121 30951 30951.37 30932.07 9.27 27946 27945.61 27928.46 8.70 1-28aa 122 31064 31064.48 31045.12 9.27 28059 28058.73 28041.52 8.70 1-28aa 123 31157 31156.58 31137.31 9.40 28121 28120.84 28103.59 8.97 1-28aa 124 31378 31377.79 31358.23 8.91 28287 28287.02 28269.62 7.99 1-28aa 125 31029 31029.44 31010.09 9.09 28024 28023.69 28006.48 8.03 1-28aa 126 30953 30953.34 30934.05 9.09 27948 27947.59 27930.44 8.02 1-28aa 127 31145 31144.59 31125.24 9.25 28109 20108.85 28091.53 8.63 1-28aa 129 30949 30949.35 30930.06 9.30 27944 27943.60 27926.46 8.72 1-28aa 130 31029 31029.44 31010.09 9.09 28024 28023.69 28006.48 8.03 1-28aa 131 31320 31319.75 31300.22 9.12 28229 28228.98 28211.62 8.60 1-28aa 132 31078 31077.54 31058.23 9.38 28042 28041.80 28024.52 8.95 1-28aa 132 30951 30951.37 30932.07 9.27 27946 27945.61 27928.46 8.70 1-28aa 136 31230 31229.69 31210.29 9.25 28194 28193.95 28176.58 8.63 1-28aa 137 30857 30857.39 30838.05 9.32 27852 27851.64 27834.44 8.95 1-28aa 138 31220 31219.61 31200.24 9.25 28184 28183.87 28166.52 8.63 1-28aa 139 31128 31127.60 31108.26 9.40 28108 28107.86 28090.54 8.95 1-28aa 141 31436 31435.83 31416.24 8.58 28345 28345.06 28327.63 7.15 1-28aa 142 31143 31142.62 31123.27 9.49 28107 28106.88 28089.56 9.16 1-28aa 144 31188 31187.70 31168.32 9.38 28152 28151.96 28134.61 8.95 1-28aa 146 31274 31273.71 31254.29 9.27 28238 28237.97 28220.58 8.64 1-28aa 147 30997 30997.39 30978.07 9.09 27992 27991.64 27974.47 8.02 1-28aa 148 31059 31058.51 31039.20 9.27 28053 28052.75 28035.60 8.70 1-28aa 150 31203 31202.68 31183.28 9.38 28167 28166.94 28149.57 8.95 1-28aa 155 31072 31071.54 31052.24 9.49 28036 28035.80 28018.52 9.16 1-28aa 156 31130 31129.53 31110.20 9.27 28094 28093.79 28076.49 8.64 1-28aa 157 31086 31085.57 31066.25 9.49 28050 28049.83 28032.54 9.16 1-28aa 158 31274 31273.75 31254.32 9.25 28238 28238.01 28220.61 8.63 1-28aa 159 30920 30920.40 30901.11 9.51 27915 27914.64 27897.50 9.24 1-28aa 161 31051 31051.44 31032.08 8.86 28046 28045.69 28028.48 6.96 1-28aa 162 31133 31132.53 3113.19 9.10 28166 28165.90 28148.55 8.63 1-28aa 163 31074 31074.49 31055.19 9.25 28108 28107.86 28090.54 8.95 1-28aa 164 31172 31171.70 31152.32 9.47 28136 28135.92 28118.58 8.95 1-28aa 165 31351 31350.76 31331.22 8.91 28260 28259.99 28242.61 7.99 1-28aa 166 31274 31273.75 31254.32 9.25 28238 28238.01 28220.61 8.63 1-28aa 167 31248 31247.73 31228.25 9.16 28212 28211.99 28194.54 8.51 1-28aa 168 31019 31019.40 31000.07 9.09 28014 28013.65 27996.46 8.03 1-28aa 169 31117 31116.58 31097.25 9.38 28081 28080.84 28063.53 8.95 1-28aa 170 31143 31142.62 31123.27 9.49 28107 28106.88 28089.56 9.16 1-28aa 171 31309 31308.68 31289.16 8.58 28218 28217.91 28200.55 7.15 1-28aa 172 31128 31127.56 31108.22 9.38 28092 28091.82 28074.51 8.95 1-28aa 173 31174 31173.63 31154.27 9.38 28138 28137.89 28120.55 8.95 1-28aa 174 30906 30906.37 30887.08 9.27 27916 27915.59 27898.45 8.70 1-28aa 175 31276 31275.72 31256.30 9.25 28240 28239.98 28222.59 8.63 1-28aa 176 31114 31113.58 31094.25 9.38 28078 28077.84 28060.53 8.95 1-28aa 177 31187 31186.67 31167.29 9.25 28151 28150.93 28133.57 8.63 1-28aa 178 31174 31173.63 31154.27 9.38 28138 28137.89 28120.55 8.95 1-28aa 179 31176 31175.65 31156.28 9.38 28140 28139.90 28122.57 8.95 1-28aa 180 31174 31173.63 31154.27 9.38 28138 28137.89 28120.55 8.95 1-28aa 181 31167 31166.64 31147.27 9.38 28131 28130.90 28113.56 8.95 1-28aa 182 31172 31171.62 31152.26 9.40 28136 28135.88 28118.55 8.97 1-28aa 183 31171 31170.67 31151.30 9.47 28135 28134.93 28117.59 9.14 1-28aa 184 31131 31130.60 31111.26 9.38 28095 28094.86 28077.55 8.95 1-28aa 186 31118 31117.52 31098.20 9.38 28108 28107.86 28090.54 8.95 1-28aa 187 31206 31205.58 31186.22 9.25 28170 28169.84 28152.51 8.63 1-28aa 188 31174 31173.63 31154.27 9.38 28138 28137.89 28120.55 8.95 1-28aa 189 31114 31113.58 31094.25 9.38 28078 28077.84 28060.53 8.95 1-28aa 190 31186 31185.68 31166.30 9.38 28150 28149.94 28132.59 8.95 1-28aa 191 30980 30980.36 30961.04 8.86 27975 27974.61 27957.44 6.95 1-28aa 192 30949 30949.35 30930.06 9.30 27944 27943.60 27926.46 8.72 1-28aa 193 31174 31173.63 31154.27 9.38 28138 28137.89 28120.55 8.95 1-28aa 194 31125 31124.56 31105.21 9.25 28108 28107.86 28090.54 8.95 1-28aa 195 30894 30894.31 30875.04 9.27 27889 27888.56 27871.44 8.70 1-28aa 196 30990 30990.45 30971.12 9.42 27985 27984.70 27967.52 9.04 1-28aa 197 31130 31129.58 31110.24 9.38 28094 28093.84 28076.53 8.95 1-28aa 198 30953 30953.34 30934.05 9.09 27974 27973.62 27956.46 8.02 1-28aa 199 31161 31160.66 31141.34 9.30 28125 28124.92 28107.63 8.66 1-28aa 200 31404 31403.87 31384.28 8.91 28313 28313.10 28295.68 7.99 1-28aa 201 31208 31028.45 31009.08 9.07 28023 28022.70 28005.48 8.01 1-28aa 202 31156 31155.66 31136.29 9.38 28120 28119.92 28102.58 8.95 1-28aa 203 31230 31229.65 31210.27 9.27 28194 28193.91 28176.56 8.64 1-28aa 204 31214 31213.69 31194.30 9.25 28178 28177.95 28160.59 8.63 1-28aa 206 31175 31174.61 31155.25 9.25 28139 28138.87 28121.54 8.63 1-28aa 207 31202 31201.68 31182.30 9.38 28166 28165.94 28148.59 8.95 1-28aa 208 31202 31201.64 31182.26 9.25 28166 28165.90 28148.55 8.63 1-28aa 209 31156 31155.66 31136.29 9.38 28120 28119.92 28102.58 8.95 1-28aa 210 31174 31173.63 31154.27 9.38 28138 28137.89 28120.55 8.95 1-28aa 212 31234 31233.68 31214.29 9.25 28198 28197.94 28180.58 8.63 1-28aa 213 31128 31127.51 31108.04 8.51 28090 28089.70 28072.47 6.21 1-28aa 214 30981 30981.35 30962.04 9.09 27976 27975.60 27958.44 8.02 1-28aa 215 31030 31030.42 31011.07 8.86 28025 28024.67 28007.47 7.04 1-28aa 216 31174 31173.63 31154.27 9.38 28138 28137.89 28120.55 8.95 1-28aa 218 31158 31157.63 31138.27 9.38 28122 28121.89 28104.56 8.95 1-28aa 219 30981 30981.39 30962.08 9.09 27976 27975.64 27958.47 8.02 1-28aa 220 31175 31174.61 31155.25 9.25 28139 28138.87 28121.54 8.63 1-28aa 221 30905 30905.39 30886.03 9.18 27900 27899.64 27882.43 8.63 1-28aa 222 31260 31259.72 31240.30 9.25 28224 28223.98 28206.59 8.63 1-28aa 223 30922 30922.37 30903.08 9.27 27917 27916.61 27899.47 8.70 1-28aa 224 31015 31015.47 30996.20 9.38 28108 28107.86 28090.54 8.95 1-28aa 225 31174 31173.63 31154.27 9.38 28138 28137.89 28120.55 8.95 1-28aa 226 31176 31175.65 31156.28 9.38 28136 28135.92 28118.58 8.95 1-28aa

[0124] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto.