Method for improving sensitivity of mass spectrometer using reference protein

Abstract

The present invention relates to a method for optimizing mass spectrometer parameters for efficient detection of proteins. The present invention allows for the setting of optimal parameters tailored to a target protein by using a reference protein with a molecular weight similar to that of the target protein to pre-determine the pulse ion extraction (PIE) value, laser power, and / or detector gain that maximize the sensitivity and accuracy of the detection signal. Thus, the present invention enables the detection of trace amounts of target proteins with high sensitivity, accuracy, and reproducibility even using low-resolution mass spectrometry equipment such as MALDI-TOF, thereby providing high-sensitivity analytical results unaffected by environmental changes during detection. In addition, the present invention establishes a linear proportional relationship between the molecular weight of the protein to be detected and the optimal PIE value, deriving a linear functional formula therebetween, which enables the simple calculation of the optimal PIE value that maximizes the performance of the mass spectrometer by using only mass information on the protein to be analyzed even without a reference protein.

Description

Method for improving the sensitivity of a mass spectrometer using a reference protein

[0001] The present invention relates to a method for significantly improving analysis reliability and sensitivity by optimizing the main parameters of a mass spectrometer, specifically a MALDI-TOF (MALDI-TOF) instrument, in advance using a reference protein having a molecular weight similar to that of the protein to be analyzed.

[0002]

[0003] Mass spectrometry, compared to genetic sequencing methods such as PCR, offers a low-cost, high-efficiency identification system and can be a crucial tool for the rapid identification of biological material in samples. Protein analysis using a mass spectrometer is particularly suitable for the rapid and accurate detection and identification of microorganisms in samples. Mass spectrometers are fundamentally composed of three parts: the ionization region, the analysis region, and the detection region. Mass spectrometers are categorized based on the structure and principles of the ionization and analysis regions used.

[0004]

[0005] A Matrix Desorption / Ionization Time of Flight (MALDI-TOF) mass spectrometer measures the molecular weight of a target substance by drying a mixture of a sample and a matrix to create a crystal structure, irradiating it with a laser to desorb and ionize it, and measuring the time-of-flight (Time-of-Flight) required for the sample to reach a detector. Microbial identification using MALDI-TOF mass spectrometry requires no complex preprocessing, directly analyzing the cells. The time required for identification is only about 5 minutes per strain. Furthermore, since fragmentation does not occur for the target substance, mass analysis of macromolecules such as proteins can be performed quickly. This has led to its recent popularity as a method for microbial identification in clinical testing. However, MALDI-TOF has limitations in its sensitivity for detecting low-concentration proteins in such samples, and even with repeated testing on the same sample, results can vary significantly depending on the analytical environment, such as temperature and humidity. Therefore, in order to enhance sensitivity in the detection of trace proteins, improve repeatability, and enhance the reliability of data interpretation, it is important to set the optimal parameters of the MALDI-TOF mass spectrometer to maximize the signal intensity of trace target proteins.

[0006]

[0007] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the invention.

[0008]

[0009] The present inventors have diligently researched and strived to develop a stable mass spectrometry method capable of detecting and identifying a target protein with high sensitivity and reliability even when trace amounts of the target protein are present in the sample to be analyzed. As a result, by pre-injecting a reference protein with a molecular weight similar to that of the target protein into the mass spectrometer, the optimal phase ion extraction (PIE) value was searched for, which maximized signal intensity while reducing the full width at half maximum and increasing the peak-to-valley ratio. By applying this value as the optimal parameter tailored to the target protein, the sensitivity, accuracy, and repeatability of the analysis could be significantly improved, thereby completing the present invention.

[0010] Therefore, the purpose of the present invention is to provide a parameter optimization method for mass spectrometry of a target protein using a reference protein.

[0011] Another object of the present invention is to provide a method for deriving a phase ion extraction (PIE) value based on the mass value of a protein to be analyzed.

[0012]

[0013] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0014]

[0015] According to one aspect of the present invention, the present invention provides a parameter optimization method for protein mass spectrometry comprising the following steps:

[0016] (a) a step of injecting a reference protein having a mass of 80 to 120% of the mass of the protein to be analyzed or a reference protein having a mass value difference of ±5,000 Da or less from the protein to be analyzed into a mass spectrometer; and

[0017] (b) a step of selecting a phase-induced ion extraction (PIE) value that induces at least one selected from the group consisting of an increase in signal intensity, a decrease in full width at half maximum, and an increase in peak to valley ratio.

[0018] The present inventors have conducted extensive research to develop a stable mass spectrometry method capable of detecting and identifying target proteins with high sensitivity and reliability even when trace amounts are present in the sample being analyzed. As a result, by pre-injecting a reference protein with a molecular weight similar to that of the target protein into the mass spectrometer, we explored the optimal phase ion extraction (PIE) value that maximized signal intensity while reducing the full width at half maximum and increasing the peak-to-valley ratio. Applying this value as the optimal parameter tailored to the target protein revealed that the sensitivity, accuracy, and repeatability of the analysis could be significantly improved.

[0019] The term "protein" as used herein refers to a linear molecule formed by amino acid residues linked to each other by peptide bonds. In the present invention, the target protein to be detected through mass spectrometry may be a protein that serves as a biological marker, such as for determining the presence or absence of a pathogenic strain in a sample (i.e., for diagnosing pathogen infection), identifying its type and phenotype, or a blood protein such as hemoglobin protein, antibody protein, or M protein.

[0020] The term “pathogenic strain” as used herein includes any bacteria that cause infection or disease, including, but not limited to, Staphylococcus aureus, Streptococcus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas otitidis, Micrococcus luteus, Citrobacter coccinei, Protus mirabilis and Mycobacterium ulcerans.

[0021] The target protein to be analyzed in the present invention includes, for example, a marker protein that can predict the presence or absence of a pathogenic strain as well as the phenotype of the strain, such as antibiotic resistance.

[0022] As used herein, “resistant to an antibiotic” means that a specific pathogenic microorganism can grow in an environment where the antibiotic is present at a high concentration or in an effective amount. Antibiotic resistance can be determined by detecting the presence of a degrading enzyme, a protein secreted by the pathogenic microorganism that degrades the antibiotic and eliminates or reduces 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 thus do not inhibit pathogens that express them. Therefore, the term “resistance” is used synonymously with “resistance” or “low therapeutic response.”

[0023] As used herein, the term “mass spectrometry” refers to a series of analytical techniques for determining the mass and structural information of any particle, such as a neutral atom, molecule, cluster (an assembly of atoms or molecules), or polymer. Typically, the molecule of interest is bombarded with a high-energy beam to form ions, which are then accelerated using a magnetic, electrostatic, or electrodynamic field to separate the ions based on their mass-to-charge ratio (m / z), and an ion detector within the mass analyzer measures the relative abundance of each ion to produce a spectrum of signal intensity versus m / z.

[0024] In this specification, the term “reference protein” refers to a standard protein of known mass, which has a mass value similar to that of the target protein to be detected, and thus exhibits detection characteristics such as signal intensity, full width at half maximum, and peak-to-valley ratio similar to those of the target protein when subjected to mass analysis under the same conditions. By appropriately selecting any standard protein having a mass range similar to that of the target protein, parameter tuning can be indirectly performed in advance to maximize sensitivity and accuracy in the actual detection of the target protein.

[0025] As used herein, the term "signal intensity" refers to the intensity (i.e., height) of a peak corresponding to the mass value of a target protein. Signal intensity is proportional to the amount of target protein in a sample and provides quantitative information about the target protein. However, in the case of trace proteins in a sample, the peak may be weak, making detection difficult. Therefore, increasing signal intensity is very important for the accurate detection and identification of trace proteins.

[0026] In this specification, the term "full width at half maximum (FWHM)" refers to the width of a specific function, meaning the width of a line at half the maximum amplitude. Specifically, it is defined as the difference between the values ​​of two independent variables that are half the maximum values ​​of the function. A lower FWHM value results in a narrower and more distinct peak, making the signal indicating the mass value clearer.

[0027] The term “peak-to-valley ratio” as used herein refers to an index indicating the difference between the highest peak and the lowest valley in a mass spectrum, and is a quantitative value calculated by dividing the difference between the baseline and peak values ​​by the difference between the baseline and valley values. The peak-to-valley ratio is a measure of how well a specific peak indicating the mass value (m / z) of the target protein can be distinguished from peaks indicating other substances.

[0028] In this specification, the term “pulsed ion extraction (PIE)” means delaying ion extraction to compensate for the initial velocity diffusion of ions generated in a mass analyzer and improve mass resolution, thereby ensuring that all ions with the same m / z value are concentrated in time and space along the flight (z) axis within the detector plane. Therefore, the term “pulsed ion extraction (PIE) value” refers to the time delaying ion extraction from the ion source to the flight tube. The PIE value is typically in nanoseconds (ns), and the optimal PIE value for each target protein is a very important parameter for improving the accuracy and resolution of mass values.

[0029] In this specification, the term “laser power” refers to the intensity of laser energy irradiated to ionize a target protein in mass spectrometry.

[0030] In this specification, the term “detector gain” refers to the parameter used to measure and amplify the ion signal generated by the detector in mass spectrometry.

[0031] According to the present invention, the efficiency of mass spectrometry can be maximized by searching for an optimal PIE tailored to a target protein by pre-injecting a reference protein having a mass range similar to that of the target protein into a mass spectrometer and changing the PIE value, which is a key parameter that determines the accuracy of analysis, while increasing the signal intensity and peak-to-valley ratio and decreasing the full width at half maximum.

[0032] According to a specific embodiment of the present invention, the method of the present invention further comprises, after the step (b), a step (c) of selecting a primary laser power value, a primary detector gain value, or a combination thereof that induces at least one selected from the group consisting of an increase in signal intensity, a decrease in full width at half maximum, and an increase in peak-to-valley ratio under the selected PIE value.

[0033] According to the present invention, the present invention further improves the sensitivity and accuracy of analysis for the target protein by additionally performing a step of optimizing the laser energy range and / or the detector acquisition value range for ionization of the target protein under the same criteria (increase in signal intensity, decrease in full width at half maximum, increase in peak-to-valley ratio) after fixing the optimal PIE value set through the above-described step.

[0034] In this specification, the terms “primary laser intensity” and “primary detector gain” mean the optimal laser intensity value and detector gain value that are initially selected based on whether the signal intensity, full width at half maximum, and peak-to-valley ratio increase or decrease after fixing the optimal PIE value set by the method of the present invention. The primary laser intensity and the primary detector gain value may be the final parameter values ​​for directly analyzing a target protein in themselves, or may be preliminary values ​​for setting cut-off values ​​of the signal intensity and full width at half maximum value that serve as references for resetting the optimal laser intensity and detector gain value for analyzing the same target protein in a subsequent independent follow-up experiment (i.e., setting the “secondary laser intensity” and “secondary detector gain”).

[0035] According to a more specific embodiment of the present invention, the method of the present invention additionally includes a step (d) of selecting the signal intensity and full width at half maximum at the selected first laser power value, first detector gain value, or a combination thereof as a cut-off value for analysis of the target protein after the step (c).

[0036] According to a more specific embodiment of the present invention, the method of the present invention additionally includes a step (e) of selecting a secondary laser power value, a secondary detector gain value, or a combination thereof that satisfies the selected cut-off value after the step (d).

[0037] According to the present invention, after setting the first laser intensity value, in a future independent follow-up experiment on the same target protein, the signal intensity and the full width at half maximum at the preset first laser intensity / detector gain value are set as cut-off values, so that the laser intensity / detector gain value that satisfies this cut-off value can be set as an actual parameter value (i.e., “second laser intensity / second detector gain value”) to be applied in the follow-up experiment. In this way, the method of the present invention can efficiently overcome the disadvantage of mass spectrometry that variability in results occurs due to the analysis environment such as temperature and humidity during repeated inspections by resetting a new laser intensity value / detector gain value that passes the cut-off value for each experiment on the same target protein.

[0038] 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), LDI-TOF (Laser Desorption / Ionization Time of Flight), SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight) and ESI-TOF (Electrospray ionization time-of-flight), and more specifically, MALDI-TOF.

[0039] MALDI-TOF mass spectrometry is a method that analyzes the molecular weight of ions by irradiating a sample supported on a matrix with a laser to desorb and ionize the ions, and measuring the time it takes for the generated ions to reach the detector (time-of-flight). Since fragmentation of the target substance does not occur, it can quickly and accurately measure the mass of large biomolecules such as proteins. When the ionized molecule is accelerated by an electric field and the time-of-flight is measured, the mass-to-charge ratio (m / z) is generated, and the molecular weight of the target substance can be measured using this m / z value.

[0040] According to a specific embodiment of the present invention, the reference protein has a mass of 90 to 105% of the mass of the protein to be analyzed, more specifically, 95 to 105% of the mass, and most specifically, 98 to 102% of the mass.

[0041] According to a specific embodiment of the present invention, the reference protein has a mass value difference of ±4,000 Da or less from the target protein, more specifically, a mass value difference of ±3,000 Da or less, and most specifically, a mass value difference of ±2,000 Da or less.

[0042] According to another aspect of the present invention, the present invention provides a method for deriving a phase ion extraction (PIE) value for protein mass spectrometry, comprising the step of substituting the mass value of the target protein into Equation 1 below:

[0043] PIE(ns) = AX + B [Formula 1]

[0044] In the above equation 1, A is a coefficient of 0.010 to 0.011, B is a constant of 652 to 662, and X is the mass value (Da) of the protein to be analyzed.

[0045]

[0046] *The inventors investigated the correlation between the molecular weight of the target protein and the optimal PIE value and found a linear proportional relationship (R) between the two variables. 2 =0.9996) exists (Fig. 10). In addition to the method of the present invention described above for selecting a PIE value using a reference protein having a mass similar to that of the target protein, the optimal PIE value that maximizes the sensitivity, accuracy, and repeatability of mass spectrometry can be simply calculated using only the mass information on the target protein without a reference protein.

[0047] According to a specific embodiment of the present invention, A is a coefficient of 0.0104 to 0.0108 and B is a constant of 657 to 658, more specifically, A is a coefficient of 0.0106 and B is a constant of 657.75.

[0048]

[0049] According to another aspect of the present invention, the present invention provides a composition for performance tuning of a mass spectrometer, comprising as an active ingredient a reference protein having a mass of 80 to 120% of the mass of a protein to be analyzed or a reference protein having a mass value difference of ±5,000 Da or less from the protein to be analyzed.

[0050] Since the reference protein and mass spectrometer used in the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0051] In the present invention, the term “performance of a mass spectrometer” means the degree to which a mass spectrometer accurately separates a target protein based on the mass-to-charge ratio (m / z). More specifically, improved performance of a mass spectrometer means that at least one selected from the group consisting of an increase in signal intensity of a mass spectrum generated by ionizing a target protein, a decrease in full width at half maximum, and an increase in peak-to-valley ratio is achieved. By improving the performance of a mass spectrometer, an accurate mass value and structural information of the target protein can be obtained, thereby enabling a high degree of reliability in determining the presence of the target protein in a biological sample. Therefore, the term “performance tuning of a mass spectrometer” is used interchangeably with “improving the performance of a mass spectrometer” or “optimizing the performance of a mass spectrometer.”

[0052] In the present invention, the term “biological sample” is used to collectively refer to any material that may contain the target protein to be analyzed or cells expressing the same, microorganisms, culture medium thereof, etc., such as samples separated from a living organism (e.g., blood, plasma, urine, saliva, tissue, organ), materials collected from the environment (e.g., water, air, soil, etc.), or artificially mixed samples.

[0053]

[0054] The features and advantages of the present invention are summarized as follows:

[0055] (a) The present invention provides a method for optimizing mass spectrometer parameters for efficient detection of proteins.

[0056] (b) The present invention can set optimal parameters tailored to the target protein by searching in advance for the phase ion extraction (PIE) value, laser power value, and / or detector gain value that maximize the sensitivity and accuracy of the detection signal using a reference protein having a molecular weight similar to that of the target protein.

[0057] (c) The present invention can provide highly sensitive analysis results that are not affected by environmental changes at the time of detection by detecting trace amounts of target proteins with high sensitivity, accuracy, and repeatability even using low-resolution mass spectrometry equipment such as MALDI-TOF.

[0058] (d) In addition, the present invention clarifies that there is a linear proportional relationship between the molecular weight of the target protein and the optimal PIE value, and derives a first-order functional equation between them, thereby enabling the simple calculation of the optimal PIE value that maximizes the performance of a mass spectrometer using only mass information on the target protein without a reference protein.

[0059]

[0060] Figure 1 is a diagram showing the results of analyzing signal intensity and full width at half maximum according to phase ion extraction (PIE) values ​​during MALDI-TOF MS analysis of KPC-2 protein.

[0061] Figure 2 is a diagram showing the results of analyzing the signal intensity and full width at half maximum according to laser power after fixing the PIE value at 1,000 ns during MALDI-TOF MS analysis of the KPC-2 protein.

[0062] Figure 3 is a diagram showing the results of analyzing signal intensity and full width at half maximum according to PIE value during MALDI-TOF MS analysis of antibody heavy chain proteins.

[0063] Figure 4 is a diagram showing the results of analyzing the signal intensity and full width at half maximum according to laser intensity after fixing the PIE value at 1,200 ns during MALDI-TOF MS analysis of antibody heavy chain proteins.

[0064] Figure 5 is a diagram showing the results of analyzing signal intensity and full width at half maximum according to PIE value during MALDI-TOF MS analysis of full-length antibody proteins.

[0065] Figure 6 is a diagram showing the results of analyzing the signal intensity and full width at half maximum according to laser intensity after fixing the PIE value at 2,200 ns during MALDI-TOF MS analysis of a full-length antibody protein.

[0066] Figure 7 is a diagram showing the results of analyzing signal intensity and full width at half maximum according to PIE value during MALDI-TOF MS analysis of a sample in which CTX-M-15 and CTX-M-1 proteins were mixed at a ratio of 1:1.

[0067] Figure 8 is a diagram showing the results of analyzing the signal intensity and full width at half maximum according to laser intensity after fixing the PIE value at 900 ns during MALDI-TOF MS analysis of CTX-M-15 and CTX-M-1 protein mixture samples.

[0068] Figure 9 is a drawing showing the results of MALDI-TOF MS analysis of a mixed protein sample of CTX-M-15 and CTX-M-1 by fixing the laser intensity value to 82 and adjusting the detector gain value.

[0069] Figure 10 is a diagram showing the results of deriving a linear correlation between the molecular weight of the target protein and the PIE value applied to mass spectrometry.

[0070]

[0071] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0072]

[0073] Example

[0074] Example 1: Preparation of PT (performance tuning) samples

[0075] Expression and purification of proteins from E. coli

[0076] Escherichia coli transformed with plasmids containing KPC-2, CTX-M-1, and CTX-M-15 genes were inoculated into LB (Luria-bertani) liquid medium containing 50 μg / ml of ampicillin antibiotic and cultured at 37°C for more than 16 hours. For sample preparation, the culture broth of strains expressing KPC-2, CTX-M-1, and CTX-M-15 proteins was centrifuged at 4,000 RPM for 15 minutes, and the supernatant was removed to harvest the cells. The harvested cells were resuspended by adding hypertonic solution (500 mM NaCl, 20 mM Tris-HCl, pH 8.0). The suspension was reacted at room temperature for 10 minutes, centrifuged at 14,000 g at 4°C for 10 minutes, and the supernatant was removed. The remaining cells were resuspended in triple-distilled water and incubated at room temperature for 10 minutes. The cells were then centrifuged again at 14,000 g for 10 minutes at 4°C and the supernatant was retained.

[0077] The stored supernatant was purified through anion exchange chromatography, and the unreacted flowthrough was collected, concentrated through a 10 kDa filter, and washed with water. The purified sample was applied as a PT sample for analyzing proteins with a molecular weight of around 28 kDa.

[0078]

[0079] Antibody protein purification

[0080] 10 μg of antibody was treated with 20 units / μl of PNGaseF and deglycosylated at 37°C for 4 hours. The sample that was only deglycosylated was used as a PT sample used to analyze proteins with a molecular weight of around 150 kDa. The deglycosylated antibody was reduced by treating it with 10 mM DTT at 56°C for 30 minutes, and then alkylated by reacting it with 10 mM IAA in the dark for 30 minutes. The heavy chain of the antibody in the sample was used as a PT sample used to analyze proteins with a molecular weight of around 50 kDa.

[0081]

[0082] Example 2. Optimization of MALDI-TOF parameters using PT samples

[0083] Parameters were optimized using a Bruker Biotyper Smart LT MALDI-TOF MS instrument, and KPC-2, deglycosylated antibody [IgG1k (Immunoglobulin G, Subclass 1, k light chain) Merck, NIST8671], and the heavy chain of the deglycosylated antibody were used as PT samples, respectively. In addition, to determine the degree of peak separation, the combination of CTX-M-15 / CTX-M-1 (Δm= 102.08 m / z) proteins was used as a PT sample to perform MALDITOF parameter optimization. 1 μl of each sample was spotted on a MALDI plate, dried, and then 1 μl of a matrix containing 20 mg / mL sinapin acid dissolved in 0.1% TFA / 50% ACN was spotted, dried, and then MALDITOF analysis was performed.

[0084]

[0085] Optimization of 28 kDa protein analysis

[0086] One microliter of KPC-2 protein at a concentration of 60 ng / μl was used for analysis. KPC-2 protein was tested across the phase-induced ion extraction (PIE) range from 450 ns to 1,200 ns (Fig. 1). Examining the peak pattern of KPC-2 protein, we can see that the signal intensity is highest at 1,000 ns, while the full width at half maximum is narrowest.

[0087] After fixing the PIE value to 1,000 ns, a test was conducted to find the optimal value by adjusting the laser power value from aL82 to aL89.5 (Fig. 2). Looking at the peak pattern of the KPC-2 protein, as the laser power value increases, the signal intensity increases and the full width at half maximum broadens. At aL86.5, the full width at half maximum was 61 and the signal intensity was 62,573, and from aL88, the full width at half maximum broadens rapidly and the signal intensity shows a tendency to increase rapidly. Therefore, when analyzing with the laser power at aL86.5, the cut-off values ​​were set to a full width at half maximum of 65 or less and a signal intensity of 62,000 or more for the KPC-2 peak.

[0088] In the case of detector gain, the signal intensity increases as the voltage value increases, but it does not significantly affect the full width at half maximum. Therefore, to prevent unnecessary consumption of the detector, it was adjusted within the range of 30 V from the actual detector voltage value, and when the signal intensity was increased while maintaining the full width at half maximum, adjustment was made. In this experiment, the detector voltage was 2,963 V, and the detector gain was 2,967 V.

[0089] Afterwards, when analyzing the molecular weight around 28 kDa, 1 μl of KPC-2 protein at a concentration of 60 ng / μl is used, and the laser intensity and detector gain are adjusted to pass the cut-off value described above, and then the analysis is performed.

[0090]

[0091] Optimization of 50 kDa protein analysis

[0092] A 1 μl sample of antibody heavy chain at a concentration of 76 μg / μl was used in the experiment. The pulsed ion extraction (PIE) range for the heavy chain sample was tested from 600 ns to 3,000 ns, and the peak pattern at 1,200 ns showed the highest signal intensity and the narrowest full width at half maximum (Fig. 3).

[0093] After fixing the PIE value to 1,200 ns, a test was conducted to find the optimal value by adjusting the laser intensity value from aL83.5 to aL89.5. As shown in Fig. 4, the peak pattern of the heavy chain protein showed that as the laser intensity value increased, the signal intensity increased and the full width at half maximum widened. At aL86.5, the full width at half maximum was 147 and the signal intensity was 106,553, and from aL88 onwards, the increase in signal intensity was not large. Therefore, when analyzing with aL86.5, the cut-off criteria were a full width of 150 or less and a signal intensity of 106,000 or more of the heavy chain peak.

[0094] In the case of detector gain, the signal intensity increased as the voltage value increased, but it did not significantly affect the full width at half maximum. Therefore, in order to prevent unnecessary consumption of the detector, the actual detector voltage was adjusted within a range of 30 V, and as a result of adjusting in the direction of increasing the signal intensity while maintaining the full width at half maximum, the detector voltage in this experiment was 2,963 V, and the detector gain was 2,967 V.

[0095] Afterwards, when analyzing a molecular weight around 50 kDa, 1 μl of an antibody heavy chain sample with a concentration of 76 μg / μl is used, and the laser intensity and detector gain are adjusted so as to pass the cut-off criteria described above, and then analyzed.

[0096]

[0097] Optimization of 150 kDa protein analysis

[0098] A 1 μl antibody sample with a concentration of 86 ng / μl was used in the experiment. The phase-induced ion extraction (PIE) range for the antibody sample was tested from 600 ns to 3,500 ns. The peak pattern of the antibody protein showed the highest signal intensity and narrowest full-width at half-maximum (FWHM) at 2,200 ns (Fig. 5). Although the full-width at half-maximum (FWHM) was not the lowest at 2,200 ns, the optimal PIE was selected at 2,200 ns considering the increased signal intensity.

[0099] After fixing the PIE value to 2,200 ns, a test was conducted to find the optimal value by adjusting the laser intensity value from aL82 to aL89.5. Looking at the peak pattern of the antibody protein, it was found that as the laser intensity value increased, the signal intensity increased and the full width at half maximum widened (Fig. 6). At aL86.5, the full width at half maximum was 887 and the signal intensity was 41,504, and a decrease in signal intensity was observed from aL88. Therefore, when analyzing with aL86.5, the cut-off criteria were a full width at half maximum of 890 or less and a signal intensity of 40,000 or more of the antibody peak.

[0100] In the case of detector gain, as the voltage value increases, the signal intensity increases, but it does not significantly affect the full width at half maximum. Therefore, to prevent unnecessary consumption of the detector, the actual detector voltage value was adjusted within the range of 30 V, and as a result of adjusting in the direction of increasing the signal intensity while maintaining the full width at half maximum, the detector voltage in this experiment was 2,963 V, and the detector gain was 2,967 V.

[0101] Afterwards, when analyzing a molecular weight around 150 kDa, 1 μl of an antibody sample with a concentration of 86 ng / μl is used, and the laser intensity and detector gain are adjusted so as to pass the cut-off criteria described above, and then analyzed.

[0102]

[0103] Optimization for peak separation of similar proteins

[0104] CTX-M-15 and CTX-M-1 proteins, which have a mass difference of 102 m / z, were mixed in a 1:1 ratio and made into a concentration of 40 ng / μl. 1 μl of the sample was then tested over the PIE range of 700 ns to 1050 ns. As a result, the signal intensities of CTX-M-15 and CTX-M-1 were highest at PIE 900 ns, and the full width at half maximum was narrow as well as the peak-to-valley ratio was high (Fig. 7), so the optimal PIE was selected as 900 ns.

[0105] After fixing the PIE value to 900 ns, a test was conducted to find the optimal value by adjusting the laser intensity value from aL76 to aL88. As a result, when looking at the peak patterns of CTX-M-15 and CTX-M-1 proteins, it was found that as the laser intensity value increased, the signal intensity increased and the full width at half maximum widened. At aL82, the signal intensity of CTX-M-15 was over 28,000, the signal intensity of CTX-M-1 was over 36,000, the full width at half maximum was less than 50 for both CTX-M-15 and CTX-M-1, and the peak-to-valley ratio was over 2 (Fig. 8). The laser intensity value was set to 82, and the detector gain value was adjusted. As a result, it was found that as the detector gain value increased, the signal intensity increased, but the full width at half maximum did not significantly affect it (Fig. 9). Therefore, in order to prevent unnecessary consumption of the detector, the detector voltage was adjusted within the range of 30 V from the actual detector voltage value, and the signal intensity was adjusted in the direction of increasing while maintaining the full width at half maximum. As a result, the detector voltage in this experiment was 2,963 V, and the detector gain was 2,955 V. The signal intensity of CTX-M-15 was 34,000 or more, the signal intensity of CTX-M-1 was 46,000 or more, the full width at half maximum was 50 or less for both CTX-M-15 and CTX-M-1, and the peak-to-valley ratio was 2 or more. This value was used as the cut-off standard for distinguishing peaks of similar proteins.

[0106] Afterwards, when analyzing proteins with similar molecular weights around 28 kDa, CTX-M-15 and CTX-M-1 proteins are mixed in a 1:1 ratio and made into a concentration of 40 ng / μl, and then 1 μl of the sample is used to adjust the laser intensity and detector yield so that it passes the cut-off criteria described above, and then analyzed.

[0107] As a result of analyzing the quantitative correlation between the molecular weight of the target protein and the PIE value applied to mass spectrometry, a significant correlation (R) was found between the molecular weight and PIE. 2 =0.9996) was confirmed (Fig. 10).

[0108]

[0109] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Parameter optimization method for protein mass spectrometry, including the following steps: (a) a step of injecting a reference protein having a mass of 80 to 120% of the mass of the target protein or a reference protein having a mass value difference of ±5,000 Da or less from the target protein into a mass spectrometer; and (b) a step of selecting a phase ion extraction (PIE) value that induces at least one selected from the group consisting of an increase in signal intensity, a decrease in full width at half maximum, and an increase in peak to valley ratio.

2. In the first paragraph, the method is characterized in that it additionally includes a step (c) of selecting a first laser power value, a first detector gain value, or a combination thereof that induces at least one selected from the group consisting of an increase in signal intensity, a decrease in the full width at half maximum, and an increase in the peak-to-valley ratio under the selected PIE value after the step (b).

3. In the second paragraph, the method is characterized in that it additionally includes a step (d) of selecting the signal intensity and full width at half maximum at the selected first laser power value, first detector gain value, or a combination thereof as a cut-off value for analysis of the target protein after the step (c).

4. In the third paragraph, the method is characterized in that it additionally includes a step (e) of selecting a second laser power value, a second detector gain value, or a combination thereof that satisfies the selected cut-off value after the step (d) and applying it to the analysis of the target protein.

5. A method according to claim 1, characterized in that the mass spectrometry is selected from the group consisting of MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization Time of Flight), LDI-TOF (Laser Desorption / Ionization Time of Flight), SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight), and ESI-TOF (Electrospray ionisation time-of-flight).

6. A method according to claim 5, characterized in that the mass analysis is MALDI-TOF.

7. A method according to claim 1, characterized in that the reference protein has a mass of 95 to 105% of the mass of the protein to be analyzed.

8. Method for deriving particle ion extraction (PIE) values ​​for protein mass analysis, including the step of substituting the mass value of the target protein into Equation 1 below: PIE(ns) = AX + B [Formula 1] In the above equation 1, A is a coefficient of 0.010 to 0.011, B is a constant of 652 to 662, and X is the mass value (Da) of the protein to be analyzed.

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

10. A composition for performance tuning of a mass spectrometer, comprising as an effective ingredient a reference protein having a mass of 80 to 120% of the mass of the target protein or a reference protein having a mass value difference of ±5,000 Da or less from the target protein.

11. A composition according to claim 10, wherein the mass spectrometry is selected from the group consisting of MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization Time of Flight), LDI-TOF (Laser Desorption / Ionization Time of Flight), SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight), and ESI-TOF (Electrospray ionisation time-of-flight).

12. A composition according to claim 10, characterized in that the reference protein has a mass of 95 to 105% of the mass of the target protein.

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