Method for increasing sensitivity of a mass spectrometer using a reference protein

Abstract

The present invention relates to a method for optimizing mass spectrometer parameters for efficient detection of proteins. By pre-screening the pulsed ion extraction (PIE) value, laser power and / or detector gain value that maximizes the sensitivity and accuracy of the detection signal, the present invention can establish optimal parameters for a target protein using a reference protein having a similar molecular weight to the target protein. Even when a low-resolution mass spectrometry device (e.g., MALDI-TOF) is used, the present invention can detect a trace amount of a target protein with high sensitivity, accuracy and reproducibility, thereby providing a high-sensitivity analysis result that is not affected by changes in the environment at the time of detection. Furthermore, by establishing a linear proportional relationship between the molecular weight of the target protein and the optimal PIE value, and deriving a linear equation therefrom, the present invention can simply calculate the optimal PIE value that maximizes the performance of the mass spectrometer using only the mass information of the target protein, without the need for a reference protein.

Description

Technical Field

[0001] This invention relates to a method for significantly improving the analytical reliability and sensitivity of mass spectrometers (especially MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight) instruments by pre-optimizing key parameters using a reference protein with a molecular weight similar to that of the target protein. Background Technology

[0002] Compared to gene sequencing methods (such as PCR), mass spectrometry is a low-cost, high-efficiency identification system and an important tool for the rapid identification of biomolecules in samples. In particular, protein analysis using mass spectrometry is suitable for the rapid and accurate detection and identification of microorganisms in samples. A mass spectrometer basically consists of three parts: an ionization unit, an analyzer unit, and a detection unit, and is classified according to the structure and operating principle of the ionization and analyzer units used.

[0003] Matrix desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry is a device that measures the molecular weight of a target substance by drying a mixture of sample and matrix to form a crystal structure, irradiating it with a laser to desorb and ionize it, and then measuring the time of flight (TOF) to the detector. Microbial identification using MALDI-TOF mass spectrometry involves direct analysis of bacterial cells without complex pretreatment, taking only about 5 minutes per species. In particular, it has the advantage of rapid mass spectrometric analysis of macromolecules (e.g., proteins) because no lysis occurs in the target substance, and therefore, it is becoming a widely used method for microbial identification in clinical testing. However, MALDI-TOF has limited sensitivity in detecting low concentrations of proteins. Specifically, MALDI-TOF suffers from variability in results due to environmental factors (e.g., temperature and humidity) during repeated testing of the same sample. Therefore, to improve the sensitivity, reproducibility, and reliability of data interpretation for trace protein detection, it is crucial to set optimal parameters for the MALDI-TOF mass spectrometer to maximize the signal intensity of trace target proteins.

[0004] This specification references and cites numerous publications and patent documents. The disclosures of all cited publications and patent documents are incorporated herein by reference in their entirety to more clearly describe the state of the art to which this invention pertains and the content of this invention.

[0005] Public content Technical issues The inventors have conducted in-depth research and developed a reliable mass spectrometry method that can detect and identify target proteins with high sensitivity and reliability, even when the target protein is present in trace amounts in the sample. Therefore, they discovered that by pre-injecting a reference protein with a molecular weight similar to the target protein into the mass spectrometer to determine the optimal pulsed ion extraction (PIE) value that maximizes signal intensity, reduces full width at half maximum (FWHM), and increases peak-to-valley ratio, and using this value as a customized optimal parameter for the target protein, the sensitivity, accuracy, and reproducibility of the analysis can be significantly improved.

[0006] Therefore, the object of the present invention is to provide a method for optimizing proteomic parameters using a reference protein.

[0007] Another object of the present invention is to provide a method for obtaining pulsed ion extraction (PIE) values ​​based on the amount of target protein.

[0008] Other objects and advantages of the invention will become more apparent from the following detailed description, the appended claims and the accompanying drawings.

[0009] Technical solutions In one aspect of the present invention, a method for optimizing protein mass spectrometry parameters is provided, comprising: (a) Inject a reference protein with a mass of 80% to 120% of the target protein mass, or a reference protein with a mass difference of ±5000 Da or less relative to the target protein, into the mass spectrometer; and (b) Select a pulsed ion extraction (PIE) value that induces at least one of the following: an increase in signal intensity, a decrease in full width at half maximum (FWHM), and an increase in peak-to-valley ratio.

[0010] The inventors have conducted in-depth research to develop a reliable mass spectrometry method that can detect and identify target proteins with high sensitivity and reliability, even when the target protein is present in trace amounts in the sample during analysis. Therefore, they discovered that by pre-injecting a reference protein with a molecular weight similar to the target protein into the mass spectrometer to determine the optimal pulsed ion extraction (PIE) value that maximizes signal intensity, reduces full width at half maximum (FWHM), and increases peak-to-valley ratio, and using this value as a customized optimal parameter for the target protein, the sensitivity, accuracy, and reproducibility of the analysis can be significantly improved.

[0011] As used herein, the term "protein" refers to a linear molecule consisting of amino acid residues linked together by peptide bonds. In this invention, the protein detected by mass spectrometry can be a protein that serves as a biomarker, for example, for the presence of a pathogenic strain in a sample (i.e., diagnosis of pathogen infection) or for the identification of its type and phenotype, or it can be a protein in blood, such as hemoglobin, antibody, or M protein.

[0012] As used herein, the term "pathogenic strain" refers to any bacteria that is the cause of infection or disease, including but not limited to Staphylococcus aureus, Streptococcus, 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 this invention include, for example, marker proteins that can predict the presence of pathogenic strains and their phenotypes (such as antibiotic resistance).

[0014] As used herein, the term "antibiotic resistance" refers to the ability of a specific pathogenic microorganism to grow even in environments where antibiotics targeting the microorganism are present at high concentrations or effective levels. Whether a pathogenic microorganism exhibits antibiotic resistance can be determined by detecting the presence of enzyme proteins secreted by the microorganism, which remove or reduce the activity of the antibiotic by degrading it. For example, β-lactam antibiotics that inhibit bacterial cell wall synthesis, such as penicillins, cephalosporins, monotypicillins, and carbapenems, are inactivated by β-lactamases and therefore cannot inhibit pathogens expressing β-lactamases. Therefore, the term "resistance" and the term "low therapeutic responsiveness" are used interchangeably.

[0015] As used herein, the term "mass spectrometry" refers to a series of analytical techniques used to determine the mass and structural information of any particle, such as neutral atoms, molecules, clusters (aggregates of atoms or molecules), or polymers. Typically, analyte molecules are bombarded by a high-energy beam to form ions. These ions are accelerated by a magnetic, electrostatic, or electric field and separated according to their mass-to-charge ratio (m / z). An ion detector within the mass spectrometer then measures the relative abundance of each ion to produce a spectrum of signal intensity corresponding to the m / z.

[0016] As used herein, the term "reference protein" refers to a standard protein with a known quality value that is similar in quality to the target protein to be detected and exhibits similar detection characteristics, such as signal intensity, full width at half maximum (FWHM), and peak-to-valley ratio, under the same mass spectrometry conditions.

[0017] As used herein, the term "signal intensity" refers to the peak intensity (i.e., height) corresponding to the mass value of the target protein. Signal intensity is proportional to the amount of target protein in the sample, thus providing quantitative information about the target protein. Since the peak values ​​of trace proteins in a sample may be weak and difficult to detect, increasing signal intensity is crucial for the accurate detection and identification of trace proteins.

[0018] As used herein, the term "full width at half maximum (FWHM)" refers to a parameter representing the width of a given function, specifically the width of the function measured at half its maximum amplitude. More specifically, it is defined as the difference between two values ​​of the independent variable at which the function reaches half its maximum value. A lower FWHM value indicates a narrower peak and a more pronounced apex, thus providing a clearer indication of quality.

[0019] As used herein, the term "peak-to-trough ratio" refers to an indicator of the difference between the highest and lowest peaks in a mass spectrum; specifically, it is a quantitative value obtained by dividing the difference between the baseline and the peak by the difference between the baseline and the trough. The peak-to-trough ratio is used to measure the difference between a specific peak indicating the mass value (m / z) of a target protein and peaks indicating other substances.

[0020] As used herein, the term "pulsed ion extraction (PIE)" refers to a delayed ion extraction method to counteract the initial velocity diffusion of ions generated in a mass spectrometer and improve mass resolution, thereby ensuring that all ions with the same m / z value are aggregated temporally and spatially along the flight (z) axis in the detector plane. Therefore, the term "PIE value" refers to the time delay of ion extraction from the ion source to the flight tube. The PIE value is typically measured in nanoseconds (ns), and the optimal PIE value for each target protein is a key parameter for improving mass accuracy and resolution.

[0021] As used in this article, the term "laser power" refers to the intensity of laser energy used to irradiate a target protein in a mass spectrometer.

[0022] As used in this article, the term "detector gain" refers to the parameter used to measure and amplify the ion signal generated by the detector in a mass spectrometer.

[0023] According to the present invention, by pre-injecting a reference protein with a mass within a similar range to the target protein into the mass spectrometer and changing the PIE value (a key parameter determining analytical accuracy), the method of the present invention can maximize mass spectrometry efficiency by searching for an optimal PIE tailored to the target protein, which increases signal intensity and peak-to-valley ratio while reducing FWHM.

[0024] According to one specific embodiment, after step (b), the method of the present invention further includes step (c) of selecting a primary laser power value, or a primary detector gain value, or a combination thereof, which triggers at least one selected from the group consisting of: an increase in signal intensity at the selected PIE value, a decrease in full width at half maximum (FWHM), and an increase in peak-to-valley ratio.

[0025] According to the present invention, after determining the optimal PIE value through the above steps, the present invention can further improve the analytical sensitivity and accuracy of the target protein by additionally performing steps to optimize the laser energy range and / or detector gain range, so as to ionize the target protein under the same standards (increased signal intensity, decreased full width at half maximum, and increased peak-to-valley ratio).

[0026] As used herein, the terms "primary laser intensity" and "primary detector gain" refer to the optimal laser intensity and detector gain, respectively, which were initially selected based on changes in signal intensity, FWHM, and peak-to-valley ratio after the optimal PIE value set by the method of this invention was fixed. The primary laser intensity and primary detector gain can serve as final parameter values ​​for direct analysis of the target protein, or as preliminary values ​​for establishing cutoff values ​​for signal intensity and full width at half maximum (FWHM). These values ​​are used as criteria for resetting the optimal laser intensity and detector gain (i.e., setting the "secondary laser intensity" and "secondary detector gain") to analyze the same target protein in subsequent independent experiments.

[0027] According to a more specific embodiment, after step (c), the method of the present invention further includes step (d), selecting the signal intensity and full width at half maximum (FWHM) obtained at the selected primary laser power value, or primary detector gain value, or a combination thereof, as the cutoff value for analyzing the target protein.

[0028] According to a more specific embodiment, the method of the present invention further includes step (e) after step (d), selecting a secondary laser power value, a secondary detector gain value, or a combination thereof that satisfies the selected cutoff value, and applying it to the analysis of the target protein.

[0029] According to the present invention, after establishing the primary laser intensity value, the signal intensity and FWHM obtained at the established primary laser intensity / detector gain can be used as cutoff values ​​in subsequent independent experiments involving the same target protein. Then, laser intensity and detector gain values ​​that satisfy these cutoff values ​​can be selected as the actual parameters applied in subsequent experiments (i.e., "secondary laser intensity" and "secondary detector gain"). In this way, the method of the present invention effectively overcomes the variability of mass spectrometry results caused by analytical environmental factors (e.g., temperature and humidity) during repeated analyses by resetting new laser intensity and detector gain values ​​that satisfy the cutoff criteria for each experiment involving the same target protein.

[0030] According to one specific embodiment, the mass spectrometer is selected from the group consisting of: matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, laser desorption / ionization time-of-flight (LDI-TOF) mass spectrometry, surface-enhanced laser desorption / ionization time-of-flight (molecular weight) mass spectrometry, and electrospray ionization time-of-flight (ESI-TOF) mass spectrometry. More specifically, the mass spectrometer is matrix desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry.

[0031] MALDI-TOF mass spectrometry is a method in which a matrix-supported sample is desorbed and ionized by laser irradiation, and the molecular weight of the resulting ions is analyzed by measuring the time it takes for the ions to reach the detector (time of flight). This method allows for the rapid and accurate measurement of the mass of large biomolecules (such as proteins) because no breakage of the target material occurs. 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 this m / z value can be used to determine the molecular weight of the target material.

[0032] According to one specific embodiment, the mass of the reference protein is 90% to 105% of the target protein mass, more specifically 95% to 105%, and most specifically 98% to 102%.

[0033] According to one specific embodiment, the reference protein has a mass difference of ±4000 Da or less relative to the target protein, more specifically, the mass difference is ±3000 Da or less, and most specifically, ±2000 Da or less.

[0034] In another aspect of the invention, a method for obtaining pulsed ion extraction (PIE) values ​​for protein mass spectrometry is provided, comprising substituting the mass of the target protein into the following equation 1: PIE(ns) = AX + B [Equation 1] Where A is a coefficient between 0.010 and 0.011, B is a constant between 652 and 662, and X is the mass of the target protein (Da).

[0035] The inventors investigated the correlation between the molecular weight of the target protein and the optimal PIE value to determine a linear proportional relationship (Ri) between these two variables. 2 =0.9996)( Figure 10 Therefore, in addition to the above-described method of the present invention for determining PIE values ​​using a reference protein with a mass similar to that of the analyte protein, even without using a reference protein, the optimal PIE value that maximizes the sensitivity, accuracy, and reproducibility of mass spectrometry can be easily calculated using only the mass information of the analyte protein.

[0036] According to one specific implementation, A is a coefficient from 0.0104 to 0.0108, and B is a constant from 657 to 658. More specifically, A is a coefficient of 0.0106, and B is a constant of 657.75.

[0037] In another aspect of the invention, a composition for mass spectrometer performance tuning is provided, comprising a reference protein as an active ingredient, the reference protein having a mass of 80% to 120% of the target protein mass, or a reference protein having a mass difference of ±5000 Da or less relative to the target protein.

[0038] Since the reference protein and mass spectrometer used in this invention have been described in detail above, their descriptions are omitted to avoid excessive redundancy.

[0039] In this invention, the term "mass spectrometer performance" refers to the degree to which a mass spectrometer accurately separates a target protein based on its mass-to-charge ratio (m / z). More specifically, improved mass spectrometer performance means meeting one or more of the following criteria: increased intensity of the mass spectrometric signal generated by ionizing the target protein, decreased FWHM, and increased peak-to-valley ratio. Through such improvements in mass spectrometer performance, accurate mass values ​​and structural information of the target protein can be obtained, thereby enabling the determination of the presence of the target protein in biological samples with high reliability. Therefore, the term "mass spectrometer performance tuning" is synonymous with "mass spectrometer performance enhancement" or "mass spectrometer performance optimization."

[0040] As used herein, the term “biological sample” means any material that may contain the target protein to be analyzed or cells, microorganisms or their culture media expressing the protein, including samples isolated from living organisms (e.g., blood, plasma, serum, saliva, tissues, organs, etc.), materials extracted from the environment (e.g., water, air, soil, etc.), or artificially mixed samples.

[0041] Beneficial effects The features and advantages of this invention are summarized as follows: (a) The present invention provides an optimized mass spectrometer parameter method for efficient protein detection.

[0042] (b) By pre-screening pulsed ion extraction (PIE) values, laser power, and / or detector gain values ​​to maximize the sensitivity and accuracy of the detection signal, the present invention can establish optimal parameters for the target protein using a reference protein with a molecular weight similar to that of the target protein.

[0043] (c) Even when using low-resolution mass spectrometry equipment (e.g., MALDI-TOF), the present invention can detect trace amounts of target proteins with high sensitivity, accuracy and reproducibility, thereby providing highly sensitive analytical results that are unaffected by changes in the environment during detection.

[0044] (d) Furthermore, by establishing a linear proportional relationship between the molecular weight of the target protein and the optimal PIE value, and deriving a linear equation therefrom, the present invention can easily calculate the optimal PIE value, which maximizes the performance of the mass spectrometer using only the mass information of the target protein, without the need for a reference protein.

[0045] Brief description of the attached figures Figure 1 The results show the functional relationship between the measured signal intensity and full width at half maximum (FWHM) and the pulsed ion extraction (PIE) value during MALDI-TOF MS analysis of KPC-2 protein.

[0046] Figure 2 The analysis shows the relationship between signal intensity and half-width at half-maximum (FWHM) as a function of laser power during MALDI-TOF MS analysis of KPC-2 protein with PIE value fixed at 1000 ns.

[0047] Figure 3 The analysis shows the functional relationship between signal intensity and half-width at half-maximum (HWHM) and PIE value during MALDI-TOF MS analysis of antibody heavy chains.

[0048] Figure 4 The analysis shows the functional relationship between signal intensity and half-width at half-maximum (FWHM) and laser power during MALDI-TOF MS analysis of antibody heavy chains with the PIE value fixed at 1200 ns.

[0049] Figure 5 The analysis shows the functional relationship between signal intensity and half-width at half-maximum (FWHM) and PIE value during MALDI-TOF MS analysis of full-length antibodies.

[0050] Figure 6 The analysis shows the relationship between signal intensity and half-width at half-maximum (HWHM) as a function of laser power during MALDI-TOF MS analysis of full-length antibodies with the PIE value fixed at 2200 ns.

[0051] Figure 7 The analysis shows the functional relationship between signal intensity and half-maximum width at half-maximum (HWHM) and PIE value for a sample consisting of a 1:1 mixture of CTX-M-15 and CTX-M-1 proteins during MALDI-TOF MS analysis.

[0052] Figure 8The analysis shows the relationship between signal intensity and half-width at half-maximum (FWHM) as a function of laser power for a mixed sample of CTX-M-15 and CTX-M-1 proteins with a PIE value fixed at 900 ns during MALDI-TOF MS analysis.

[0053] Figure 9 The results of MALDI-TOF MS analysis of a mixed sample of CTX-M-15 and CTX-M-1 proteins are shown, with detector gain variations while laser intensity was fixed at 82.

[0054] Figure 10 The linear correlation between the molecular weight of the target protein and the PIE value applied in the mass spectrometry was shown.

[0055] Inventive Method The invention will be described in more detail below by way of examples. These examples are only for illustrating the invention in more detail, and those skilled in the art will understand that the scope of the invention is not limited to these examples.

[0056] Example: Example 1: Preparation of PT (Performance Conditioning) Samples Expression and purification of Escherichia coli proteins E. coli transformed with plasmids containing the KPC-2, CTX-M-1, and CTX-M-15 genes were inoculated into Luria Bertani (LB) liquid medium containing 50 μg / ml ampicillin and cultured at 37°C for at least 16 hours. For sample pretreatment, the culture supernatant of the strains expressing KPC-2, CTX-M-1, and CTX-M-15 proteins was centrifuged at 4000 RPM for 15 minutes, and then the supernatant was removed to harvest cells. The harvested cells were resuspended in hypertonic lysis buffer (500 mM NaCl, 20 mM Tris-HCl, pH 8.0). The suspension was incubated at room temperature for 10 minutes, then centrifuged at 14000 g for 10 minutes at 4°C, after which the supernatant was removed. The remaining cells were resuspended in triple-distilled water and reacted at room temperature for 10 minutes. The mixture was then centrifuged again at 14000 g for 10 minutes at 4°C, and the supernatant was collected and stored.

[0057] The collected supernatant was purified by anion exchange chromatography. The flow-through (the fraction not bound to the column) was collected, concentrated using a 10 kDa filter, and washed with water. The purified sample was used as a PT sample for the analysis of proteins with a molecular weight of approximately 28 kDa.

[0058] Purification of antibody proteins 10 μg of antibody was treated with 20 units / μl of PNGase F for deglycosylation at 37°C for 4 hours. The deglycosylated sample was used as a PT sample for analyzing proteins with a molecular weight of approximately 150 kDa. Hydroxylation was then performed by treating with 10 mM DTT at 56°C for 30 minutes, followed by reaction with 10 mM IAA in the dark for 30 minutes to reduce the amount of deglycosylated antibody. The heavy chain of the antibody in the sample was used as a PT sample for analyzing proteins with a molecular weight of approximately 50 kDa.

[0059] Example 2: Optimization of MALDI-TOF parameters using PT samples Parameters were optimized using a Bruker Biotyper Smart LT MALDI-TOF MS instrument. KPC-2, deglycosylated antibody [IgG1k (immunoglobulin G, subclass 1, k light chain) Merck, NIST 8671], and the heavy chain of the deglycosylated antibody were all used as PT samples. Furthermore, to assess peak resolution, protein pairs CTX-M-15 / CTX-M-1 (… ΔM =102.08 M / z ) PT samples were used for MALDI-TOF parameter optimization. After spotting 1 μL of each sample onto a MALDI plate and allowing it to dry, 1 μL of a matrix consisting of 20 mg / mL octaned acid dissolved in 0.1% TFA / 50% ACN was spotted and allowed to dry before MALDI-TOF analysis.

[0060] Optimization of 28kDa protein analysis The KPC-2 protein was analyzed using 1 μL of a concentration of 60 ng / μL. The KPC-2 protein was tested within a pulsed ion extraction (PIE) range of 450 ns to 1200 ns. Figure 1 The peak diagram of the KPC-2 protein shows that the signal intensity is highest at 1,000 ns, and the full width at half maximum (FWHM) is narrowest.

[0061] After fixing the PIE value at 1000 ns, the optimal value was searched by adjusting the laser power from aL82 to aL89.5. Figure 2 The peak plot of the KPC-2 protein shows that the signal intensity increases and the full width at half maximum (FWHM) broadens with increasing laser power. At aL86.5, the FWHM is 61 and the signal intensity is 62,573; after aL88, the FWHM broadens sharply and the signal intensity increases rapidly. Therefore, when the laser intensity is set to 86.5 for analysis, the cutoff values ​​are established as follows: the full width at half maximum (FWHM) of the KPC-2 peak is 65 or less, and the signal intensity is 62,000 or greater.

[0062] With the detector gain in place, the signal strength increases with increasing voltage, but this does not significantly affect the Free-Wheel-M (FWHM). Therefore, to prevent unnecessary wear on the detector, the actual detector voltage is adjusted to within 30V, and this adjustment is made to increase the signal strength while maintaining the FWHM. In this experiment, the detector voltage was 2963V, and the detector gain was 2967V.

[0063] Subsequently, when analyzing molecules with a molecular weight of approximately 28 kDa, 1 μl of KPC-2 protein at a concentration of 60 ng / μl was used. The laser power and detector gain were adjusted to ensure the sample met the aforementioned cutoff values ​​before analysis.

[0064] Optimization of 50kDa protein analysis The experiment used 1 μl of antibody heavy chain sample with a concentration of 76 μg / μl. The pulsed ion extraction (PIE) range for testing the heavy chain sample was 600 ns to 3000 ns. The results showed that the signal intensity was highest at 1200 ns, while the half-peak width at half-width was narrowest. Figure 3 ).

[0065] After fixing the PIE value at 1200 ns, the optimal value was searched by adjusting the laser intensity from aL83.5 to aL89.5. Figure 4 As shown in the peak diagram of the heavy chain protein, the signal intensity increases and the full width at half maximum (FWHM) broadens with increasing laser intensity. At aL86.5, the FWHM is 147 and the signal intensity is 106553. After aL88, the increase in signal intensity is not significant. Therefore, when performing analysis at aL86.5, an FWHM of 150 or lower and a signal intensity of 106000 or higher are set as the cutoff criteria for the heavy chain peak.

[0066] With the detector gain set, although the signal strength increases with increasing voltage, it does not significantly affect the half-width at half-maximum (FWHM). Therefore, to prevent unnecessary detector wear, the detector voltage was adjusted within ±30V of the actual detector voltage. By adjusting the voltage to increase signal strength while maintaining FWHM, the detector voltage used in this experiment was set to 2963V, and the detector gain was set to 2967V.

[0067] Subsequently, when analyzing molecules with a molecular weight of approximately 50 kDa, 1 μL of an antibody heavy chain sample at a concentration of 76 μg / μL was used. Prior to analysis, the laser power and detector gain were adjusted to meet the aforementioned cutoff criteria.

[0068] Optimization of 150kDa protein analysis In the experiment, 1 μl of antibody sample with a concentration of 86 ng / μl was used. Pulsed ion extraction (PIE) of the antibody sample was tested in the range of 600 ns to 3500 ns. The results showed that the peak of the antibody protein exhibited the highest signal intensity and the narrowest FWHM at 2200 ns. Figure 5 Although the FWHM at 2200ns is not the lowest value, it was chosen as the optimal PIE value considering the increase in signal strength.

[0069] After fixing the PIE value at 2200 ns, the optimal value was searched by adjusting the laser intensity from aL82 to aL89.5. The peak diagram of the antibody protein showed that the signal intensity increased and the FWHM broadened with increasing laser intensity. Figure 6 At aL86.5, the FWHM is 887 and the signal intensity is 41504, with a decrease in signal intensity observed starting from aL88. Therefore, when performing analysis at aL86.5, an FWHM of 890 or lower and a signal intensity of 40000 or higher are set as cutoff criteria for the antibody peak.

[0070] Regarding detector gain, although signal strength increases with increasing voltage, it has no significant effect on the half-width at half-maximum (FWHM). Therefore, to prevent unnecessary detector wear, the actual detector voltage was adjusted to within the 30V range. By adjusting the voltage to increase signal strength while maintaining FWHM, the detector voltage used in this experiment was set to 2963V, and the detector gain was set to 2967V.

[0071] Subsequently, when analyzing molecules with a molecular weight of approximately 150 kDa, 1 μl of an antibody sample at a concentration of 86 ng / μl was used. Prior to analysis, the laser power and detector gain were adjusted to meet the aforementioned cutoff criteria.

[0072] Optimization of differentiating similar protein peaks The CTX-M-15 and CTX-M-1 proteins with a mass difference of 102 M / z were mixed at a 1:1 ratio to a concentration of 40 ng / μl. Then, 1 μl of the sample was taken and tested within the PIE range of 700 ns to 1050 ns. The results showed that the signal intensity of CTX-M-15 and CTX-M-1 was highest at 900 ns PIE, with a narrow FWHM and a high peak-to-valley ratio. Figure 7 Therefore, PIE 900 ns was chosen as the optimal value.

[0073] After fixing the PIE value at 900 ns, the optimal value was found by adjusting the laser intensity from aL76 to aL88. Therefore, the peak plots of the CTX-M-15 and CTX-M-1 proteins show that the signal intensity increases with increasing laser intensity, and the full width at half maximum (FWHM) broadens. At aL82, the signal intensity of CTX-M-15 was confirmed to be 28000 or higher, and the signal intensity of CTX-M-1 was 36000 or higher. The FWHM of both CTX-M-1 and CTX-M-12 was 50 or lower, and the peak-to-valley ratio was 2 or higher. Figure 8 After setting the laser power to 82, the detector gain was adjusted. Therefore, it was observed that although the signal strength increased with increasing detector gain, this did not significantly affect the FWHM (Frequency-Wide Motion Detection). Figure 9 Therefore, to prevent unnecessary wear on the detector, the detector voltage was adjusted within ±30V of the actual detector voltage. By adjusting the voltage to increase signal strength while maintaining the FWHM, the detector voltage in this experiment was set to 2963V. At a detector gain of 2955V, the signal strength of CTX-M-15 was 34000 or higher, and the signal strength of CTX-M-1 was 46000 or higher; the FWHM of both CTX-M-15 and CTX-M-1 was 50 or lower; and the peak-to-valley ratio was 2 or higher. These values ​​were used as cutoff criteria to distinguish similar protein peaks.

[0074] Subsequently, when analyzing similar proteins with a molecular weight of approximately 28 kDa, the CTX-M-15 and CTX-M-1 proteins were mixed at a 1:1 ratio to a concentration of 40 ng / μl. Then, using 1 μl of sample, the laser power and detector gain were adjusted to meet the aforementioned cutoff criteria before analysis.

[0075] The quantitative correlation (R) between the molecular weight of the target protein and the PIE value applied in mass spectrometry was confirmed. ² =0.9996)( Figure 10 ).

[0076] Having described in detail the specific embodiments of the present invention above, it should be understood that variations and modifications within the spirit and scope of the present invention may become apparent to those skilled in the art, and the scope of the present invention will be determined by the appended claims and their equivalents.

Claims

1. A method for optimizing protein spectral parameters, comprising: (a) Inject a reference protein with a mass of 80% to 120% of the target protein mass, or a reference protein with a mass difference of ±5000 Da or less relative to the target protein, into the mass spectrometer; and (b) Select a pulsed ion extraction (PIE) value that induces at least one of the following: an increase in signal intensity, a decrease in full width at half maximum (FWHM), and an increase in peak-to-valley ratio.

2. The method as described in claim 1, wherein, Following step (b), the method further includes step (c), selecting a primary laser power value, or a primary detector gain value, or a combination thereof, to induce at least one of the following: an increase in signal intensity at the selected PIE value, a decrease in full width at half maximum (FWHM), and an increase in peak-to-valley ratio.

3. The method as described in claim 2, wherein, Following step (c), the method further includes step (d), selecting the signal intensity and full width at half maximum (FWHM) obtained at the selected primary laser power value, or primary detector gain value, or a combination thereof, as the cutoff value for the target protein to be analyzed.

4. The method of claim 2, wherein, Following step (d), the method further includes step (e), selecting a secondary laser power value, a secondary detector gain value, or a combination thereof that satisfies the selected cutoff value, and applying it to the analysis of the target protein.

5. The method of claim 1, wherein, The mass spectrometers were selected from the following group: matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, laser desorption / ionization time-of-flight (LDI-TOF) mass spectrometry, surface-enhanced laser desorption / ionization time-of-flight (SELDI-TOF) mass spectrometry, and electrospray ionization time-of-flight (ESI-TOF) mass spectrometry.

6. The method of claim 5, wherein, The mass spectrometer used is MALDI-TOF.

7. The method of claim 1, wherein, The mass of the reference protein is 95% to 105% of the mass of the target protein.

8. A method for obtaining pulsed ion extraction (PIE) values ​​for protein profiling, comprising substituting the mass of the target protein into the following Equation 1: PIE(ns) = AX + B [Equation 1] Where A is a coefficient between 0.010 and 0.011, B is a constant between 652 and 662, and X is the mass of the target protein (Da).

9. The method of claim 8, wherein, A is a coefficient ranging from 0.0104 to 0.0108, and B is a constant ranging from 657 to 658.

10. A composition for performance tuning of a mass spectrometer, wherein the active ingredient comprises a reference protein having a mass of 80% to 120% of the mass of the target protein, or a reference protein having a mass difference of ±5000 Da or less relative to the target protein.

11. The composition of claim 10, wherein, The mass spectrometers were selected from the following group: matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, laser desorption / ionization time-of-flight (LDI-TOF) mass spectrometry, surface-enhanced laser desorption / ionization time-of-flight (SELDI-TOF) mass spectrometry, and electrospray ionization time-of-flight (ESI-TOF) mass spectrometry.

12. The composition of claim 10, wherein, The mass of the reference protein is 95% to 105% of the mass of the target protein.

Images