Peptide for high-efficiency quality control for evaluating performance quality of liquid chromatography-mass spectrometry system, and use thereof

Abstract

The present invention relates to a peptide, a composition, a kit, an evaluation method, and a manufacturing method for quality evaluation and performance verification of a liquid chromatography-mass spectrometry (LC-MS) system. The peptide for quality evaluation, of the present invention, is composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 7, and exhibits a consistent retention time (RT) and a mass-to-charge ratio (m / z) in an LC-MS system on the basis of a simple structure and high specificity, thereby ensuring data reproducibility and reliability. In addition, the present invention enables sensitivity, quantitative accuracy and data stability of the LC-MS system to be efficiently evaluated through the peptide, and the composition and kit comprising same.

Description

High-efficiency quality control peptides for liquid chromatography-mass spectrometry system performance quality evaluation and their uses

[0001] The present invention relates to a peptide for high-efficiency quality control for evaluating the performance quality of a liquid chromatography-mass spectrometry system and a method for manufacturing the same.

[0002] Liquid Chromatography-Mass Spectrometry (LC-MS) is widely used as a core tool for protein and peptide analysis in modern life science research and pharmaceutical development. This technology enables the qualitative and quantitative analysis of complex biomolecules with high sensitivity and precision, establishing itself as an essential analytical technique in various fields such as proteomics, metabolomics, drug discovery, and biomarker research. The LC-MS system is considered a powerful tool capable of accurately characterizing proteins and peptides in vivo by separating complex components of a sample and measuring the mass-to-charge ratio (m / z).

[0003] However, in order to stably maintain the performance of such LC-MS systems and ensure the accuracy and reproducibility of the data, suitable samples for quality control (QC) are essential. Currently, QC samples based on widely used HeLa cell digests have established themselves as a major tool for evaluating the quality of LC-MS analysis. However, HeLa digests consist of complex peptide mixtures and are not sensitive to specific LC-MS conditions, which can reduce the reproducibility of analysis results. Furthermore, the sample preparation process requires significant time and cost, and the process of culturing HeLa cells and digesting proteins demands high technical proficiency. Additionally, because HeLa cells are human-derived cells, they may be subject to ethical controversies and legal regulations, which limits their use in industrial and research environments requiring large-scale quality control.

[0004] To address these issues, there is an increasing demand for the development of new QC samples. Suitable QC samples must be cost-effective, simple to prepare, and capable of providing high reproducibility and reliability under various LC-MS conditions. Furthermore, an alternative approach is needed to overcome the ethical issues and legal restrictions associated with existing human-derived cell-based samples. As LC-MS technology continues to advance and its application scope expands, the development of new QC samples capable of complementing the limitations of existing HeLa digests is essential for improving data reliability and increasing research efficiency.

[0005] Therefore, there is a need for research on the development of efficient and reliable QC samples for quality control and performance verification.

[0006] The present invention aims to solve the aforementioned problem and other related problems.

[0007] One exemplary objective of the present invention is to provide a peptide for liquid chromatography-mass spectrometry (LC-MS) system quality evaluation selected from the group consisting of SEQ ID NOs 1 to 7.

[0008] Another exemplary objective of the present invention is to provide a composition for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system comprising a peptide for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system and a preparation for measuring the level thereof.

[0009] Another exemplary objective of the present invention is to provide a kit for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system comprising a composition for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system.

[0010] Another exemplary objective of the present invention is to provide a method for evaluating a peptide in a sample, comprising: (a) adding to a sample a composition for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system, the composition comprising one or more peptides selected from the group consisting of SEQ ID NOs 1 to 7; (b) analyzing the sample using liquid chromatography-mass spectrometry (LC-MS); (c) identifying liquid chromatography-mass spectrometry (LC-MS) data corresponding to one or more peptides selected from the group consisting of amino acid sequences of SEQ ID NOs 1 to 7; and (d) quantifying the amount of the peptide in the sample using the identified liquid chromatography-mass spectrometry (LC-MS) data.

[0011] Another exemplary objective of the present invention is to provide a monitoring method for monitoring the performance of a liquid chromatography-mass spectrometry (LC-MS) system, comprising: (a) adding to a sample a composition for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system comprising one or more peptides selected from the group consisting of SEQ ID NOs 1 to 7; (b) analyzing the sample using liquid chromatography-mass spectrometry (LC-MS); (c) identifying liquid chromatography-mass spectrometry (LC-MS) data corresponding to one or more peptides selected from the group consisting of amino acid sequences of SEQ ID NOs 1 to 7; and (d) evaluating the performance of the liquid chromatography-mass spectrometry (LC-MS) system using the identified liquid chromatography-mass spectrometry (LC-MS) data.

[0012] Another exemplary objective of the present invention is to provide a method for producing a peptide for liquid chromatography-mass spectrometry (LC-MS) system quality evaluation, comprising the steps of: extracting a cell pellet from a culture medium of Synechocystis (Synechocystissis p. PCC6803); adding acetone to the cell pellet and centrifuging to obtain a protein; ultrasonically grinding the obtained protein; and performing in-solution digestion.

[0013] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited by the specific descriptions provided below.

[0014]

[0015] As an embodiment for achieving the above objective, the present invention provides a peptide for evaluating the quality of a liquid chromatography-mass spectrometry (hereinafter, LC-MS) system selected from the group consisting of SEQ ID NOs 1 to 7.

[0016] The "peptides composed of SEQ ID NOs. 1 to 7" of the present invention are designed with specific amino acid sequences and are configured in an optimized manner to enable reliable quality evaluation in LC-MS. The peptides are suitable for evaluating the detection sensitivity, reproducibility, and reliability of an LC-MS system through unique ionization during analysis, and demonstrate consistent performance across various LC-MS platforms.

[0017] Furthermore, based on the simplicity and specificity of its sequence, the peptide of the present invention provides high reproducibility and reliability compared to conventionally used complex Quality Control (QC) samples (e.g., HeLa digest-based QC samples). Additionally, the peptide sequence can respond sensitively to changes in LC-MS conditions, offering a technical advantage that enables precise verification of system performance fluctuations. Moreover, the production process is simple, and since it is free from the ethical issues and legal restrictions associated with complex biological samples (e.g., human-derived cell-based samples), it is suitable for large-scale QC sample production and industrial application.

[0018] The above peptide has the ability to evaluate the signal strength and detection sensitivity of an LC-MS system, and can ensure consistency of analysis results through its unique ionic properties and RT (retention time) distribution.

[0019] Specifically, the present invention showed the lowest and most stable CV values ​​at 200 ng to 1000 ng, preferably 400 ng to 800 ng, more preferably 500 ng to 700 ng, and may have excellent reproducibility of PSM, number of peptides, number of proteins, and TIC intensity, but is not limited thereto.

[0020] The "LC-MS system" of the present invention is a key tool used to separate, detect, and analyze components within complex samples, and can be used as an essential technology in various fields, particularly in life sciences, pharmaceuticals, and chemical analysis. The LC-MS system is a combination of liquid chromatography (LC) and mass spectrometer (MS), and can perform qualitative and quantitative analysis of substances with high sensitivity and high precision.

[0021] The above liquid chromatography (LC) serves to separate various compounds within a sample. The sample passes through a column containing a mobile phase and a stationary phase, and each component can be separated by moving at different speeds according to its unique chemical properties (e.g., polarity, hydrophobicity). The components separated in this process are sequentially transferred to a mass spectrometer for detection and analysis.

[0022] The above-mentioned mass spectrometer (MS) can ionize components separated by LC and measure the mass-to-charge ratio (m / z) of each component to provide qualitative and quantitative information. The MS may be composed of an ionization device, a mass spectrometer, and a detector, and can be utilized to detect components within a sample with high sensitivity and to analyze the molecular structure and characteristics of the material.

[0023] The “quality evaluation” of the present invention is intended to continuously check and maintain the performance of the LC-MS system and may evaluate whether the LC-MS system can provide reliable analysis results based on high sensitivity and high precision, but is not limited thereto. Specifically, the quality evaluation can measure key performance indicators of the LC-MS system, detect variations or anomalies that may occur during the analysis process at an early stage, and contribute to maintaining the accuracy and reproducibility of the data.

[0024] The above quality evaluation includes, but is not limited to, the steps of injecting a QC sample into an LC-MS system and analyzing it; analyzing the data obtained through the LC-MS system; and comparing the collected data with preset reference values. Specific details are described in detail in the method for monitoring the performance of the liquid chromatography-mass spectrometry (LC-MS) system described below.

[0025]

[0026] In another embodiment for achieving the above objective, the present invention provides a composition for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system, comprising a peptide for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system and a formulation for measuring the level thereof. The "peptide," "liquid chromatography-mass spectrometry (LC-MS) system," "active ingredient," and "quality evaluation" of the present invention are as described above.

[0027] The “composition” of the present invention is a composition designed for performance evaluation and quality control of an LC-MS system and includes a standardized substance that exhibits high reproducibility and reliability in LC-MS analysis. Specifically, it includes a peptide composed of SEQ ID NOs 1 to 7, and may include an internal standard substance or a quantitative aid substance as needed.

[0028] The above internal standard material plays an essential role in evaluating the sensitivity and quantitative accuracy of the LC-MS system and can ensure the consistency and reliability of the quality evaluation results.

[0029] The above quantitative aid is a component capable of precisely adjusting or measuring the level of peptides, and is used to accurately measure or adjust the concentration of peptides, thereby minimizing data variability during the analysis process.

[0030]

[0031] In another embodiment for achieving the above objective, the present invention provides a kit for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system comprising a composition for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system. The "peptide," "liquid chromatography-mass spectrometry (LC-MS) system," "active ingredient," "quality evaluation," and "composition" of the present invention are as described above.

[0032] The “kit” of the present invention may consist of a peptide composition designed for performance evaluation and quality control of an LC-MS system and auxiliary components that support its use, but is not limited thereto.

[0033] Specifically, it may include internal standards capable of adjusting or accurately measuring peptide concentrations, solutions for diluting or preparing QC samples (e.g., buffer solutions, solvents), and standardized protocols and manuals guiding the analytical procedure. Internal standards play an essential role in evaluating the sensitivity and quantitative performance of the LC-MS system and minimize data variability during the analysis process. Additionally, dilution and preparation solutions maintain the stability of QC samples and allow the samples to be adjusted to suit the analytical conditions.

[0034] The above quality evaluation kit may be provided as an integrated unit containing all components necessary to evaluate the performance of an LC-MS system, and may be used to evaluate the system's sensitivity, signal-to-noise ratio (S / N), retention time stability, and quantitative reproducibility. The kit of the present invention is designed to be usable on various LC-MS platforms and analysis environments and provides simple preparation and usage procedures that enhance user convenience.

[0035]

[0036] In another embodiment for achieving the above objective, the present invention provides a method for evaluating a peptide in a sample, comprising: (a) adding to a sample a composition for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system, the composition comprising one or more peptides selected from the group consisting of SEQ ID NOs 1 to 7; (b) analyzing the sample using liquid chromatography-mass spectrometry (LC-MS); (c) identifying liquid chromatography-mass spectrometry (LC-MS) data corresponding to one or more peptides selected from the group consisting of amino acid sequences of SEQ ID NOs 1 to 7; and (d) quantifying the amount of the peptide in the sample using the identified liquid chromatography-mass spectrometry (LC-MS) data.

[0037] Step (a) above is a step of adding to a sample or directly injecting into a system a composition for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system comprising one or more peptides selected from the group consisting of SEQ ID NOs 1 to 7, wherein a peptide optimized to be designed with a specific amino acid sequence to exhibit a unique mass-to-charge ratio (m / z), retention time (RT), and signal intensity in LC-MS analysis is added.

[0038] The addition of peptides is adjusted according to the concentration of the sample and analysis conditions, and the concentration of the peptides can be set to ensure optimal detection sensitivity and quantitative accuracy.

[0039] Step (b) above is a step of analyzing a sample using LC-MS, which can provide high-sensitivity data by separating, ionizing, and detecting peptides and other components within the sample. In this process, the sample is separated via liquid chromatography, and peptides can be separated based on their inherent RT depending on the composition of the mobile phase and column. The separated peptides are transferred to a mass spectrometer, ionized, and then data is acquired by measuring the mass-to-charge ratio (m / z). The LC-MS system maximizes the detection efficiency of peptides by optimizing various setting conditions (e.g., mobile phase composition, flow rate, column type, ionization method) and has the effect of ensuring the reliability of the analysis data.

[0040] Step (c) above involves identifying data corresponding to peptides composed of SEQ ID NOs 1 to 7 based on data collected through LC-MS analysis. Key data include the RT, m / z, and signal intensity of the peptides, and the data may exhibit a specific pattern depending on the analysis conditions of the system. The analyzed peptides are designed to be clearly distinguishable from other components in the sample. During the data verification process, the detection and analysis of the peptides are reviewed by comparing them with preset reference values ​​to determine if they were properly performed. This can play a key role in evaluating the performance of the LC-MS system and quantifying the peptides in the sample.

[0041] Step (d) above involves quantifying the amount of peptide in a sample based on LC-MS data, and quantitative analysis can be performed to evaluate the reliability of the LC-MS system and accurately measure the peptide concentration in the sample. The peptides of the present invention can serve as internal standards, and they contribute to minimizing sample variability and increasing data accuracy. Furthermore, they can be used to evaluate the sensitivity, quantitative accuracy, and reproducibility of the LC-MS system by calculating the concentration by comparing the signal intensity of the peptide with a reference value during the quantification process.

[0042]

[0043] In another embodiment for achieving the above objective, the present invention provides a monitoring method for monitoring the performance of a liquid chromatography-mass spectrometry (LC-MS) system, comprising: (a) adding to a sample a composition for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system comprising one or more peptides selected from the group consisting of SEQ ID NOs 1 to 7; (b) analyzing the sample using liquid chromatography-mass spectrometry (LC-MS); (c) identifying liquid chromatography-mass spectrometry (LC-MS) data corresponding to one or more peptides selected from the group consisting of amino acid sequences of SEQ ID NOs 1 to 7; and (d) evaluating the performance of the liquid chromatography-mass spectrometry (LC-MS) system using the identified liquid chromatography-mass spectrometry (LC-MS) data.

[0044] The "method for monitoring the performance of a liquid chromatography-mass spectrometry (LC-MS) system" of the present invention includes efficient steps for maintaining the analytical accuracy and reproducibility of the LC-MS system and for detecting performance anomalies early, and is designed to evaluate key performance indicators of the LC-MS system, ensure the reliability of the analysis results, and maintain the sensitivity and quantitative accuracy of the system.

[0045] In step (a) above, a composition for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system comprising one or more peptides selected from the group consisting of SEQ ID NOs 1 to 7 may be prepared and added to a sample or directly injected into the system. The peptides are designed to exhibit a specific retention time (RT) and mass-to-charge ratio (m / z) in the LC-MS system, which can ensure the reproducibility of the analysis results. The concentration of the added peptides is optimized according to the analysis conditions and can provide a reference value for evaluating the detection sensitivity and quantitative accuracy of the system.

[0046] In step (b) above, the sample is analyzed using an LC-MS system, which is a process of measuring the RT and m / z of peptides in the sample for quality evaluation and collecting data. The LC-MS system can separate peptides using liquid chromatography and measure the characteristics of ionized peptides using a mass spectrometer. The analysis conditions are performed by optimizing various parameters such as mobile phase composition, flow rate, column selection, and ionization mode, which can increase the reliability of peptide detection and minimize data variability.

[0047] In step (c) above, LC-MS data is analyzed to identify key performance indicators of the peptide for quality evaluation. The analysis results evaluate the peptide's RT, m / z, signal intensity, signal-to-noise ratio (S / N), and quantitative accuracy, and these indicators can be used as key data to diagnose the performance status of the system. Each indicator is compared with a pre-set reference value, and the abnormality of the system can be determined through the deviation from the reference value.

[0048] In step (d) above, the performance of the LC-MS system is evaluated based on the verified data. The verified data is used to evaluate the sensitivity, quantitative accuracy, and analytical reproducibility of the system, and maintenance or calibration may be performed if a performance anomaly is detected. The present invention can maintain the consistency and reliability of the system by monitoring the status of the LC-MS system in real time and verifying whether the analysis conditions are optimized.

[0049]

[0050] In another embodiment for achieving the above objective, the present invention provides a method for producing a peptide for liquid chromatography-mass spectrometry (LC-MS) system quality evaluation, comprising the steps of: extracting a cell pellet from a culture medium of Synechocystis (Synechocystissis p. PCC6803); adding acetone to the cell pellet and centrifuging to obtain a protein; ultrasonically grinding the obtained protein; and performing in-solution digestion.

[0051] The "Synechocystis sp. PCC6803" of the present invention is a microalgae capable of performing photosynthesis and is a type of cyanobacteria containing chlorophyll a. The microorganism exists in a single-celled form and can fix carbon dioxide and grow using sunlight as energy in a natural environment. It has a relatively simple genome structure, making it suitable for genomic research and metabolic pathway analysis.

[0052] In the present invention, Synechocystis sp. PCC6803 is used as a protein source for producing peptides for quality evaluation of an LC-MS system. The microorganism exhibits a high growth rate under rapid and simple culture conditions and is capable of mass culture, making it highly suitable for producing quality evaluation samples. Furthermore, unlike human-derived samples, Synechocystis sp. PCC6803 is free from ethical issues and legal restrictions, making it safer and easier to comply with regulations compared to existing quality evaluation samples.

[0053] The *Synechocystis sp. PCC6803* of the present invention can be obtained and used from sources such as the American Type Culture Collection (ATCC) and the Korea Culture Collection Center (KCTC) of the Korea Research Institute of Biotechnology and Bioengineering.

[0054] In one embodiment of the present invention, the "in-solution digestion step" may include, but is not limited to, a step of adding urea; a step of adding a reducing agent to reduce disulfide bonds in the protein; and an alkylation reaction step of adding an alkylating agent to stabilize disulfide bonds in the protein.

[0055] In one embodiment of the present invention, the "method for preparing a peptide for liquid chromatography-mass spectrometry (LC-MS) system quality evaluation" may further include a desalting step, but is not limited thereto.

[0056] In step (a) of the present invention, cells are harvested from a Synechocytis culture medium and a cell pellet is extracted through centrifugation. The Synechocytis can be mass-produced through a simple culture process and is characterized by a rapid growth rate and high protein content, making it suitable for mass production of peptides for LC-MS system quality evaluation and contributing to increased cost efficiency.

[0057] In step (b) of the present invention, protein precipitation can be induced by adding acetone stored at -20°C to the obtained cell pellet, and then centrifuging at 14,000 g for 10 minutes to obtain the protein. Acetone is an excellent solvent for effectively removing impurities within cells and improving the purity of proteins, and it provides particularly superior purification capabilities compared to other solvents. Acetone increases the purity of proteins by dissolving or removing substances other than proteins among the cell components, and the obtained protein can be used as a key raw material for the production of peptides for quality evaluation.

[0058] In step (c) of the present invention, the obtained protein is homogenized using an ultrasonic grinding device such as a Covaris S2 Sonicator and the intracellular protein is eluted. This process is carried out at a temperature of 3°C to 8°C, preferably 4°C to 6°C, to prevent protein denaturation caused by heat, and the ultrasonic energy can induce complete elutation of the protein, thereby maximizing the protein yield and increasing the efficiency of the subsequent peptide generation step.

[0059] In step (d) of the present invention, the protein is digested into a peptide using an in-solution digestion method. This process involves reducing disulfide bonds by adding TCEP (Tris(2-carboxyethyl)phosphine) to the protein solution, performing an alkylation reaction using IAA (Iodoacetamide), adding Trypsin, and carrying out the digestion reaction at 30°C to 40°C for 15 to 20 hours. This ensures efficient production of the peptide and high reproducibility, and the generated peptide can be used for reliable quality evaluation in LC-MS analysis.

[0060] The present invention relates to a peptide for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system selected from the group consisting of SEQ ID NOs 1 to 7. The peptides consisting of SEQ ID NOs 1 to 7 provided in the present invention exhibit a consistent retention time (RT) and mass-to-charge ratio (m / z) in an LC-MS system based on a simple structure and high specificity, and have the effect of ensuring the reproducibility and reliability of the analysis results.

[0061] In particular, the peptide of the present invention utilizes Synechocystis sp. PCC6803 to enable the mass production and cost-effective manufacture of peptides for quality assessment while avoiding ethical issues and legal restrictions, which increases the data reliability of LC-MS systems, improves analytical efficiency, and can be utilized in various research and industrial applications.

[0062] The structure and effects of the present invention will be explained in more detail below through examples. These examples are intended solely to illustrate the present invention, and the scope of the present invention is not limited by them.

[0063]

[0064] <Experimental Example 1> Comparison of digestion methods for quality control sample analysis in Liquid Chromatography-Mass Spectrometry (hereinafter, LC-MS) (In-solution digestion method and FASP digestion method)

[0065] 1. Experimental Method

[0066] 1-1. Preparation of cell pellet from microalgae Synechocystis sp. PCC6803

[0067] In order to obtain a cell pellet from the microalgae Synechocystis sp. PCC6803, a sample containing Synechocystis sp. PCC6803 was received in a culture system, and stirring was performed to homogenize the sample. Subsequently, a cell pellet was collected through a stepwise centrifugation process.

[0068] Specifically, in the first step of centrifugation, 2,000 μL of the stirred sample was placed in a centrifuge tube and centrifuged at 4,000 rpm for 10 minutes at 4°C, and the supernatant was removed after centrifugation. This process was carried out using a total of two centrifuge tubes.

[0069] In the second stage of centrifugation, an additional 1,000 μL of sample was added to the same centrifugation tube, and centrifugation was performed again at 4,000 rpm, 10 minutes, and 4°C, and the supernatant was removed after centrifugation.

[0070] In the third stage of centrifugation, 1,000 μL of the sample was finally added to the same centrifugation tube and centrifuged at 4,000 rpm, 10 minutes, and 4°C, after which the supernatant was removed and the cell pellet was finally collected.

[0071]

[0072] 1-2. Protein extraction and impurity removal step via acetone precipitation

[0073] The present invention performed an acetone precipitation method to remove impurities from a cell pellet obtained from the microalgae Synechocystis sp. PCC6803 and to effectively extract proteins.

[0074] First, 1 mL of acetone stored at -20°C was added to the obtained cell pellet, and the sample was stirred to mix homogeneously. The mixed sample was reacted for 1 hour under vibration conditions of 50 rpm to induce protein precipitation.

[0075] The sample in which the precipitation reaction was completed was centrifuged at 14,000 g for 10 minutes to separate the protein precipitate, the supernatant was removed after centrifugation, and the precipitated protein was collected.

[0076]

[0077] 1-3. Protein elution and concentration quantification step via tube merging and cell disruption

[0078] The present invention performed a method comprising tube merging and cell disruption for efficient protein extraction and quantification from the microalgae Synechocystis sp. PCC6803.

[0079] First, the protein precipitates from two tubes obtained through centrifugation and precipitation processes were combined into a single sample, and 8M urea solution and a protease inhibitor were added to the combined sample to prevent protein degradation and homogenization was performed.

[0080] Subsequently, cells were lysed and protein elution was induced using a Covaris S2 Sonicator. The Focused Ultrasonicator (Covaris) was operated, the temperature of a bath filled with distilled water was set to 5°C, degassing was activated, the sample was placed in the bath, a glass tube was placed in the bath, and the lysis program (lysis_hp20min) was executed to lyse the cells.

[0081] After cell lysis was completed, the initial protein concentration was quantified using the Thermo Fisher BCA (Bicinchoninic Acid) Kit, and the protein concentration was calculated using a standard curve following the protocol provided by the manufacturer.

[0082]

[0083] 1-4. Peptide generation step through protein digestion (in-solution digestion and FASP)

[0084] The present invention is an effective and reproducible method for generating peptides from protein samples, and the efficiency of protein digestion and peptide recovery was confirmed through in-solution digestion (hereinafter, In-solution digestion) and FASP (Filter-Aided Sample Preparation) methods.

[0085] Specifically, for the in-solution digestion method for generating peptides from protein samples, the protein concentration was first quantified using a BCA kit. Then, 100 μg of protein from each sample was adjusted to a total volume of 100 μL using 8M urea solution, and a total of 5 repeated experiments were performed under the same conditions to ensure reproducibility. The protein samples were adjusted to a final concentration of 5 mM by adding 500 mM TCEP (Tris(2-carboxyethyl)phosphine), and the disulfide bonds were reduced at 37°C for 30 minutes. Subsequently, 500 mM IAA (Iodoacetamide) was added to adjust the final concentration to 15 mM, and the alkylation reaction was carried out at room temperature (25°C) under dark conditions for 30 minutes. The sample after the alkylation reaction was completed was diluted with a 50 mM ABC (Ammonium Bicarbonate in water, pH 8.0) solution to a final urea concentration of 2 M or less, Trypsin was added to adjust the enzyme-to-protein ratio to 1:50 (w / w), and the digestion reaction was performed at 37°C for 18 hours.

[0086] Experiments were conducted in a similar manner using the FASP (Filter-Aided Sample Preparation) method. Protein concentration was quantified using a BCA kit, and 100 μg of protein was adjusted to a total volume of 100 μL using 8M urea solution. A total of five replicate experiments were performed under identical conditions. For the reduction reaction, 500 mM TCEP was added to adjust the final concentration to 5 mM, and the reaction was carried out at 33°C for 30 minutes. Subsequently, the reduced sample was transferred to a filter and centrifuged at 14,000 xg at 20°C for 15 minutes; then, 100 μL of 50 mM IAA was added, and the alkylation reaction was carried out in the dark at 25°C for 30 minutes. After the reaction was complete, three repeated washes were performed using 8M urea buffer and 50 mM ABC solution, respectively. The washed filter was transferred to a new collection tube, 200 μL of a solution containing Trypsin (enzyme-to-protein ratio = 1:50, w / w) was added to 50 mM ABC, mixed at 600 rpm for 1 minute in a Thermo-mixer, and then the digestion reaction was performed at 37°C for 18 hours.

[0087] After digestion was complete, the filter was centrifuged at 14,000 xg at 20°C for 10 minutes to recover the peptides. Then, 40 μL of 50 mM ABC was added, and the remaining peptides in the filter were completely recovered by repeating the centrifugation twice under the same conditions.

[0088]

[0089] 1-5. In-solution digestion using a C-18 Spin Column (hereinafter, in-solution digestion) and salt removal step of FASP-digested peptides

[0090] The present invention improved the purity of peptides and removed impurities by utilizing a C-18 Macro Spin column during the salt removal process of peptides digested by in-solution digestion and FASP (Filter-Aided Sample Preparation).

[0091] Specifically, a C-18 Macro Spin column (HARVARD Apparatus, 74-4101) was used to purify in-solution digested peptides. First, the pH of the sample was adjusted to 2.5 to 3 using trifluoroacetic acid (TFA). Subsequently, the C-18 Macro Spin column was prepared, 150 μL of sol B (80% ACN and 0.1% TFA in HPLC-grade water) was added, and the column was washed by centrifuging at 200 xg for 1 minute. Next, 150 μL of sol A (0.1% TFA in HPLC-grade water) was added, and the column was centrifuged at 200 xg for 1 minute. This process was repeated twice at 600 xg for 1.5 minutes to prepare the column.

[0092] After loading the sample onto the prepared column, it was centrifuged at 600 xg for 1.5 minutes, and the flow-through was loaded back onto the column and processed twice under the same conditions. After loading the sample, 150 μL of sol A was added, and the centrifugation process at 600 xg for 1.5 minutes was repeated three times to remove salts and impurities from the sample.

[0093] Finally, the column was transferred to a new collection tube, 150 μL of sol B was added, and the mixture was centrifuged at 300 xg for 1.5 minutes. The glycolysis process was then centrifuged at 200 xg for 1 minute with the addition of 150 μL of ACN (Acetonitrile), followed by a final centrifugation at 700 xg for 1 minute. The eluted peptide solution was completely dried using a Speed ​​Vacuum.

[0094] In addition, a C-18 Micro Spin column (HARVARD Apparatus, 74-4601) was used to purify peptides digested by FASP. Samples were prepared by adding 75 μL of sol A (0.1% TFA in HPLC-grade water) to dried samples. A C-18 Micro Spin column was prepared, 75 μL of sol B was added, and the column was washed by centrifuging at 200 xg for 1 to 2 minutes. Subsequently, the column was prepared by adding 75 μL of sol A, centrifuging at 200 xg for 1 to 2 minutes, and repeating the centrifugation process at 400 xg for 1 to 2 minutes twice. After loading the peptide samples onto the prepared column, the column was centrifuged at 200 xg for 1 to 2 minutes, and the flow-through was loaded back into the column and processed twice under the same conditions. After loading, 75 μL of sol A was added, and the process of centrifuging at 200 xg for 1 to 2 minutes was repeated 3 times to remove salt and impurities from the sample.

[0095] Finally, the column was transferred to a new collection tube, 75 μL of sol B was added, and the mixture was centrifuged at 100 xg for 1 to 2 minutes. This process was repeated by adding 75 μL of ACN and centrifuging at 200 xg for 1 minute, followed by centrifugation at 400 xg for 1 minute. The eluted peptide solution was completely dried using a Speed ​​Vacuum.

[0096]

[0097] 1-6. Peptide Characterization Step via LC-MS Analysis

[0098] The present invention precisely analyzed the protein components of Synechocystis sp. PCC6803 using a high-efficiency LC-MS analysis method.

[0099] The LC system used in this experiment was the Thermo Fisher Ultimate 3000, and the MS system was the Q Exactive. Two types of C18 columns were used for sample separation and analysis; the trapping column consisted of a particle size of 3 μm, a pore size of 100 Å, and a size of 75 μm x 2 cm, while the analysis column consisted of a PepMap™ RSLC C18 with a particle size of 2 μm, a pore size of 100 Å, and a size of 75 μm x 25 cm. The mobile phase consisted of two solutions: Sol A was a solution of HPLC-grade water with 0.1% formic acid added, and Sol B was a solution of HPLC-grade water with 80% acetonitrile and 0.1% formic acid added.

[0100] For the LC conditions, the column temperature was maintained at an appropriate level, the sample injection volume was set to 1 μg / 1 μL, and the flow rate was controlled to 300 nL / min. An optimized protocol for the gradient was applied based on protein characteristics and column conditions. For MS analysis, Nanospray was adopted as the ionization method, and the MS scan range was set to 400–2000 m / z to cover a wide mass band.

[0101] For data analysis, Thermo Fisher’s Proteome Discoverer software was used, and protein information of Synechocystis sp. PCC6803 based on Uniprot KB was used as the database.

[0102] This process successfully performed protein profiling of Synechocystis sp. PCC6803 using an LC-MS analysis technique with high precision and repeatability, thereby providing meaningful data that can be utilized in various fields such as biotechnology and new drug development.

[0103]

[0104] 2. Experimental Results

[0105] The present invention compared and analyzed in-solution digestion and FASP digestion methods for the purpose of developing samples for quality control (QC) of an LC-MS system, and evaluated the analytical stability and reliability of the system.

[0106] Specifically, the in-solution digestion method recorded a high average digestion yield of 43.0%, demonstrating significantly higher efficiency compared to the FASP method's 1.2%. Furthermore, the coefficient of variation (CV) of the digestion yield was 3%, a lower value compared to the FASP method's 39%, proving the consistency of the digestion process (Table 1). In terms of protein identification results, the in-solution digestion method recorded a protein identification coefficient of variation (Protein ID CV) of 1.19%, providing more stable and reliable results than the FASP method's 3.24% (Table 2).

[0107] In addition, when comparing the Total Ion Chromatogram (TIC), which is an indicator of total ion signal intensity in LC-MS analysis and evaluates the stability and reproducibility of the analysis process, the intensity values ​​obtained from samples treated with in-solution digestion ranged from 6.12E9 to 6.59E9, and their coefficient of variation (CV) was calculated to be 2.7%. On the other hand, the intensity values ​​obtained from samples treated by the FASP method ranged from 3.14E9 to 5.53E9, and their coefficient of variation was 24.7%, showing relatively high variability. (Table 3)

[0108] As a result, reproducibility and stability are the most important factors in LC-MS quality control sample analysis, and it was confirmed that the in-solution digestion method satisfies these requirements and is the optimal choice for quality control sample production.

[0109]

[0110] DigestionSample IDConcentrationUnitExtinction Coeftotalconcentration(μg / μL)totalvolume(μL)totalquantity(μg)Yield(%)In-soultuondigestionIn-sol 010.954mg / mL310.98744.043.443.4%0.9870.997In-sol 020.9570.98344.043.343.3%0.9890.983In-sol 031.0351.01344.044.644.6%1.0131.009In-sol 040.9970.99744.043.943.9%1.120.972In-sol 050.9870.98744.043.443.4%0.9970.983FASPdigestionFASP 010.023mg / mL310.02344.01.01.0%0.0220.024FASP 020.0360.03144.01.41.4%0.0310.029FASP 030.010.01044.00.40.4%0.010.012FASP 040.0160.01544.00.70.7%0.0150.015FASP 080.0250.02544.01.11.1%0.0240.027

[0111] DigestionNo.PSMPeptideProteinRatioCVIn-soultuondigestion110311591389411.5 : 6.6 : 11.19%210590603788811.9:6.8:1310491591687811.9:6.7:1410707607186912.3 : 7.0 : 1510676613289212.0 : 6.9 : 1FASPdigestion1112726523108110.4 : 6.0 : 13.24 %2127937538115511.1 : 6.5 : 13129077534116211.1 : 6.5 : 14124217116110611.2 : 6.4 : 15111526390109610.2:5.8 : 1

[0112] DigestionNo.IntensityCVIn-soultuondigestion16.25 E92.7%26.27 E936.59 E946.12 E956.31 E9FASPdigestion13.50 E924.7 %25.53 E935.46 E944.75 E953.14 E9

[0113] <Experimental Example 2> Selection of Peptides for Quality Control of Liquid Chromatography-Mass Spectrometry (hereinafter, LC-MS) 1. Experimental Method

[0114] 2-1. Preparation of cell pellet from microalgae Synechocystis sp. PCC6803

[0115] In the present invention, after receiving a cultured Synechocystis sp. PCC6803 sample, stirring was performed to homogenize the sample, and a cell pellet was collected through a stepwise centrifugation process.

[0116] Specifically, 500 μL of the stirred sample was placed in a centrifuge tube, and centrifugation was performed at 4,000 rpm for 10 minutes at 4°C. After centrifugation, the supernatant was removed, and an additional 500 μL of the sample was added to the same tube, followed by repeated centrifugation under the same conditions to remove the supernatant. Finally, 1,000 μL of the sample was added to the same tube and centrifuged at 4,000 rpm for 10 minutes at 4°C, after which the final supernatant was removed and the cell pellet was collected.

[0117]

[0118] 2-2. Protein extraction and impurity removal step via acetone precipitation

[0119] The present invention performed an acetone precipitation method to remove impurities from a cell pellet obtained from the microalgae Synechocystis sp. PCC6803 and to effectively extract protein.

[0120] Specifically, 500 μL of acetone stored at -20℃ was added to the prepared cell pellet. The added acetone and cell pellet were stirred to homogeneously mix the sample. Subsequently, the mixed sample was vibrated at 50 rpm for 1 hour to induce protein precipitation.

[0121] The sample in which the precipitation reaction was completed was centrifuged at 14,000 g for 10 minutes to separate the protein precipitate, and after centrifugation, the supernatant was removed to collect the precipitated protein.

[0122]

[0123] 2-3. Cell lysis and protein concentration quantification step

[0124] The present invention performed a cell lysis method and a method for quantifying protein concentration using a BCA (Bicinchoninic Acid) Kit to efficiently elute protein from the microalgae Synechocystis sp. PCC6803.

[0125] Specifically, homogenization was performed by adding an 8M urea solution and a protease inhibitor to the sample after acetone precipitation was completed to prevent protein degradation. The homogenized sample was subjected to cell lysis and protein elution induction using a Covaris S2 Sonicator. The above process was prepared by operating a focused ultrasonicator, setting the temperature of a water bath filled with distilled water to 5°C, and activating degassing. The prepared sample was placed in a glass tube, placed in the water bath, and the cells were lysed by running the Covaris S2 Sonicator's lysis program (lysis_hp20min).

[0126] After cell lysis was completed, protein concentration was quantified using the Thermo Fisher BCA Kit. The quantification process was carried out according to the manufacturer's protocol, and the initial protein concentration was calculated based on a standard curve.

[0127]

[0128] 2-4. Steps of protein digestion through in-solution digestion

[0129] The present invention performed an in-solution digestion step for efficient and highly reproducible digestion of a protein sample.

[0130] Specifically, after quantifying the protein concentration using a BCA Kit, 100 μg of protein was prepared from each sample, and the prepared protein was adjusted to a final volume of 100 μL using 8M urea. This process was repeated a total of 5 times with samples of equal amount and volume.

[0131] 500 mM TCEP (Tris(2-carboxyethyl)phosphine) was added to the prepared protein solution to adjust the final concentration to 5 mM, and the disulfide bonds were reduced at 37°C for 30 minutes. After the reduction reaction was completed, 500 mM IAA (Iodoacetamide) was added to the sample to adjust the final concentration to 15 mM, and the alkylation reaction was carried out at room temperature (25°C) for 30 minutes under dark conditions.

[0132] Subsequently, the sample was diluted with a 50 mM ABC (ammonium bicarbonate in water, pH 8.0) solution to ensure the final urea concentration was 2 M or less, and Trypsin stock was added to the diluted sample to adjust the enzyme-to-protein ratio to 1:50 (w / w), and the digestion reaction was performed at 37°C for 18 hours.

[0133]

[0134] 2-5. Step for removing salts from peptides by in-solution digestion using a C-18 Macro Spin Column

[0135] The present invention performed an effective and highly reproducible salt removal step to remove salts and impurities from peptide samples digested by in-solution digestion. This step was designed to improve the purity of the peptides using a C-18 Macro Spin Column (HARVARD Apparatus, 74-4101) and to prepare a sample optimized for subsequent LC-MS analysis.

[0136] Specifically, the pH of the prepared peptide sample was adjusted to 2.5 to 3 pH using trifluoroacetic acid (TFA). Subsequently, a C-18 macro spin column was inserted into a tube, and 150 μL of sol B (80% ACN and 0.1% TFA in HPLC-grade water) was added to the column and washed by centrifugation at 200 xg for 1 minute. Next, 150 μL of sol A (0.1% TFA in HPLC-grade water) was added and centrifuged under the same conditions, after which an additional 150 μL of sol A was added and centrifuged at 600 xg for 1.5 minutes. This process was repeated twice to thoroughly wash the column.

[0137] After transferring the washed column to a new collection tube, the prepared peptide sample was loaded onto the column and centrifuged at 600 xg for 1.5 minutes. The flow-through effluent was loaded back into the column and centrifuged under the same conditions, and this step was repeated twice. Subsequently, 150 μL of sol A was added to the column, and the centrifugation process at 600 xg for 1.5 minutes was repeated three times to remove salts and impurities from the sample. All solutions collected during the washing process were gathered and dried for reuse if necessary.

[0138] After removing salts and impurities, the column was transferred to a new collection tube, 150 μL of sol B was added, and the column was centrifuged at 300 xg for 1.5 minutes. Subsequently, an additional 150 μL of sol B was added and the column was centrifuged at 200 xg for 1.5 minutes, followed by the addition of 150 μL of Acetonitrile (ACN) and centrifugation at 200 xg for 1 minute. Finally, 150 μL of sol B was added, and the column was centrifuged at 700 xg for 1 minute to elute the peptide. The eluted peptide solution was completely dried using Speed ​​Vacuum.

[0139]

[0140] 2-6. Confirmation of Peptide Characterization via LC-MS Analysis

[0141] The present invention performed a step of highly precise analysis of the characteristics of a peptide derived from Synechocystis sp. PCC6803 using LC-MS analysis.

[0142] Specifically, a Thermo Fisher Ultimate 3000 system was used for LC analysis, and MS analysis was performed on a Q Exactive system. A C18 column (3 μm, 100 Å, 75 μm x 2 cm) was used as the trapping column, and a PepMap RSLC C18 column (2 μm, 100 Å, 75 μm x 25 cm) was used as the analytical column. The mobile phase consisted of Sol A (0.1% formic acid in HPLC-grade water) and Sol B (80% acetonitrile, 0.1% formic acid in HPLC-grade water). For LC conditions, the sample injection volume was set to 1 μg / 1 μL and the flow rate to 300 nL / min, while the column temperature and gradient were set to optimized levels. Nanospray ionization was used for MS analysis, and the MS scan range was set to 400–2000 m / z. Data processing was performed using Thermo Fisher’s Proteome Discoverer software, and peptides were identified and quantified using the UniProt KB-based database for Synechocystis sp. PCC6803.

[0143]

[0144] 2. Experimental Results

[0145] 2-1. Selection of Peptide Candidates for Real-Time Check (RT-CHECK)

[0146] The present invention conducted experiments to select QC peptides and evaluate analytical stability in order to ensure quality control (hereinafter QC) and data reliability of the LC-MS system. Specifically, QC peptides were identified based on the variability of retention time (RT) and hydrophobicity / hydrophilicity indicators (GRAVY values) during LC-MS analysis, and peptides capable of stable detection under the system's analytical conditions were selected.

[0147] First, peptides were analyzed from samples prepared through in-solution digestion, and repeated experiments were performed twice. In the first experiment, peptides were identified and the mean RT values ​​and coefficients of variation (CV) were calculated from five files, while in the second experiment, 50 files were analyzed to calculate the mean RT values ​​and CVs. Through this, suitable candidates for QC peptides were selected.

[0148]

[0149] Sequence Number Peptide Primary Average RT Primary RT CV Secondary Average RT Secondary RT CVGRAVY1RIGQNPEPVTIK19.0542.4 %18.4631.69 %-0.8333333333333332YLSPGELDR25.6320.4 %25.5391.09 %-0.8888888888888893RPLVAIVGGSK21.6821.1 %21.3362.38 %0.627272727272734WYVEALK33.3980.3 %33.4750.88 %0.0285714285714295SGILYPVIVR41.6520.31 %41.5780.55 %1.266AYFDFVIGK47.5580.2 %47.470.38%0.777777777777787RYETLSYLPPLTDQQIAK41.9760.3 %42.030.48%-0.70555555555556

[0150] As a result, YLSPGELDR showed low variability in the first experiment with an average RT value of 25.632 and an RT CV of 0.4%, while maintaining stable results in the second experiment with an average RT value of 25.539 and an RT CV of 1.09%. WYVEALK showed low variability in the first experiment with an average RT value of 33.398 and an RT CV of 0.3%, and in the second experiment with an average RT value of 33.475 and an RT CV of 0.88%. SGILYPVIVR showed very stable results in the first experiment with an average RT value of 41.652 and an RT CV of 0.31%, and in the second experiment with an average RT value of 41.578 and an RT CV of 0.55% (Table 4). In addition, the balance of hydrophobicity and hydrophilicity was evaluated by analyzing the GRAVY values ​​of each peptide. The GRAVY value of YLSPGELDR was -0.888, indicating dominant hydrophilicity, while the GRAVY value of WYVEALK was 0.028, showing a value close to neutral. SGILYPVIVR exhibited strong hydrophobic characteristics with a GRAVY value of 1.26. This confirmed that system stability can be verified using peptides with various hydrophobic / hydrophilic characteristics when utilized as QC peptides in LC-MS analysis (Table 4).

[0151]

[0152] 2-2. Check Peptide Injection Amount

[0153] In order to perform experiments to determine the optimal injection amount for peptide analysis for quality control of an LC-MS system, the present invention injected peptides of various concentrations (200 ng, 400 ng, 600 ng, 800 ng, 1000 ng) into an LC-MS system and evaluated the coefficient of variation (CV) of the analysis results and the reproducibility of the analysis.

[0154] Specifically, peptide samples digested by the in-solution digestion method were prepared at various concentrations (200 ng, 400 ng, 600 ng, 800 ng, 1000 ng). Samples of each prepared concentration were analyzed using an LC-MS system (Thermo Fisher Ultimate 3000 and Q Exactive), and the same conditions were applied for the analysis.

[0155] LC-MS analysis was repeated 10 times for each concentration to measure the number of detected peptides (PSM), number of peptides, number of proteins, and total ion chromatogram (TIC) intensity, and the coefficient of variation (CV) was calculated based on these results. Consequently, the 600 ng concentration showed the lowest CV value and stable results, demonstrating excellent reproducibility for PSM, number of peptides, number of proteins, and TIC intensity. The sample at the 600 ng concentration exhibited a CV value of 1.45%, minimizing variation between analysis repetitions, while the CV value of TIC intensity was also stable at 8.87% compared to other concentrations. These results confirm that the 600 ng concentration is optimal as the injection volume for QC sample analysis.

[0156] In addition, the analysis results were compared for QC peptides (YLSPGELDR, WYVEALK, SGILYPVIVR) at various injection amounts (200 ng, 400 ng, 600 ng, 800 ng, 1000 ng), and the reproducibility and stability under each condition were evaluated.

[0157] Specifically, peptide samples digested by the in-solution digestion method were prepared and prepared at various concentrations. The prepared samples were analyzed using an LC-MS system (Thermo Fisher Ultimate 3000 and Q Exactive). In the LC-MS analysis, only the sample injection volume was varied while the column and mobile phase conditions were kept constant, and repeated experiments were performed for each injection volume to evaluate the consistency and reproducibility of the data.

[0158] The coefficient of variation (CV) was calculated based on the average Retention Time (RT), Retention Time Relative Standard Deviation (RT Relative Standard Deviation), average intensity, and intensity Relative Standard Deviation (Intensity Relative Standard Deviation) of the peptides according to each injection amount. As a result, a concentration of 600 ng was found to have the lowest RT and intensity standard deviation values ​​among all QC peptides, indicating the best analytical reproducibility. In the case of YLSPGELDR, the RT standard deviation was 0.17% and the intensity standard deviation was 5.37%, showing very low variability compared to other concentrations. WYVEALK was stable with an RT standard deviation of 0.12% and an intensity standard deviation of 6.69%. High reproducibility was also confirmed in SGILYPVIVR, with an RT standard deviation of 0.08% and an intensity standard deviation of 8.60%.

[0159] On the other hand, at concentrations of 200ng, 800ng, and 1000ng, the intensity standard deviations were 12.54%, 12.78%, and 10.71% or higher, respectively, indicating high variability; in particular, for 1000ng, the RT standard deviation was 0.50%, which is somewhat high, and the intensity standard deviation was 14.82%, showing the greatest variability.

[0160]

[0161] Sequence Number Peptide Concentration RT Intensity (Intensity) Average RT Intensity Average Intensity Median RT Standard Deviation Strength Standard Deviation 2YLSPGELDR 200ng 26 2.22E+0825.99 22.16E+082.21E+080.19%6.39% 26.06 2.27E+0826.05 1.96E+0825.93 1.92E+0826.01 2.21E+0826.03 2.06E+0826 2.22E+0825.97 2.26E+0825.92 2.19E+0825.95 2.34E+084 00ng 25.74 4.05E+0825.66 13.94E+084.02E+080.27%6.00% 25.71 4.19 E+0825.733.81E+0825.674.18E+0825.74.02E+0825.643.87E+0825.61 3.87E+0825.634.01E+0825.674.07E+0825.513.38E+08600ng25.524.4 8E+0825.4584.7E+084.62E+080.17%5.37%25.485.22E+0825.484.89E+ 0825.54.98E+0825.494.46E+0825.474.51E+0825.414.65E+0825.44.5 4E+0825.434.72E+0825.44.58E+08800ng25.325.2E+0825.285.28E+08 5.4E+080.24%8.53%25.245.5E+0825.245.32E+0825.225.53E+0825.21 5.75E+0825.275.71E+0825.275.47E+0825.285.28E+0825.44.29E+082 5.354.75E+081000ng25.24.46E+0825.3055.71E+085.82E+080.58%10. 71%25.275.26E+0825.235.6E+0825.136.36E+0825.276.31E+0825.675 .23E+0825.45.51E+0825.296.17E+0825.36.04E+0825.296.16E+084WY VEALK200ng33.981.43E+0833.9661.43E+081.45E+080.21%12.54%34.0 51.49E+0834.071.05E+0833.931.25E+08341.69E+0834.021.39E+0833.971.41E+0833.891.48E+0833.851.52E+0833.91.61E+08400ng33.642.79 E+0833.5832.76E+082.81E+080.21%8.62%33.682.96E+0833.662.67E+08 33.592.96E+0833.622.8E+0833.572.69E+0833.522.87E+0833.512.85E+ 0833.582.83E+0833.462.14E+08600ng33.413.14E+0833.3422.93E+083.0 4E+080.12%6.96%33.343.04E+0833.332.89E+0833.362.81E+0833.42.56E+0833.353.08E+0833.283.07E+0833.293.05E+0833.333.07E+0833.332. 63E+08800ng33.233.6E+0833.2235.09E+085.41E+080.27%12.78%33.115.4E+0833.175.32E+0833.155.54E+0833.155.52E+0833.25.57E+0833.255 .41E+0833.235.41E+0833.394.35E+0833.354.82E+081000ng33.134.82E +0833.2645.37E+085.54E+080.50%14.82%33.225.57E+0833.25.79E+0833 .044.17E+0833.236.26E+0833.675.41E+0833.355.51E+0833.246.19E+0 833.295.98E+0833.274.01E+085SGILYPVIVR200ng42.036754306441.9466 6015215670285120.10%11.09%41.986651396041.975567728841.925510326041.947216507241.986838837641.926187718041.936510136441.91680 5730241.8879725280400ng41.721.34E+0841.6871.32E+081.3E+080.08%11.81%41.71.6E+0841.691.29E+0841.711.44E+0841.711.29E+0841.681.27E+0841.671.28E+0841.721.36E+0841.661.32E+0841.6197648121600ng41.561 .36E+0841.5281.43E+081.39E+080.08%8.60%41.511.72E+0841.481.52E+0841.54 1.37E+0841.561.33E+0841.571.37E+0841.551.29E+0841.521.45E+0841.491.47E +0841.51.4E+08800ng41.412.13E+0841.3732.06E+082.14E+080.09%13.26%41.38 2.28E+0841.342.12E+0841.342.14E+0841.352.37E+0841.362.2E+0841.322.26E +0841.391.91E+0841.431.49E+0841.411.73E+081000ng41.311.73E+0841.3562.3 1E+082.37E+080.26%11.45%41.312.27E+0841.272.1E+0841.232.55E+0841.352.5 4E+0841.632.18E+0841.362.25E+0841.362.55E+0841.382.46E+0841.362.5E+08.

[0162] As described above, specific embodiments of the present invention have been described in detail; however, those skilled in the art who understand the spirit of the present invention will be able to easily propose other inventions that are inferior or other embodiments included within the scope of the spirit of the present invention by adding, changing, or deleting other components within the same spirit. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims set forth below rather than by the detailed description above, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included within the scope of the present invention.

Claims

1. Any one liquid chromatography-mass spectrometry (LC-MS) system quality evaluation peptide selected from the group consisting of SEQ ID NOs 1 to 7.

2. A composition for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system comprising the peptide for evaluating the quality of a liquid chromatography-mass spectrometry (LC-MS) system according to claim 1 and a preparation for measuring the level thereof.

3. A liquid chromatography-mass spectrometry (LC-MS) system quality evaluation kit comprising the composition for liquid chromatography-mass spectrometry (LC-MS) system quality evaluation of claim 2.

4. As a method for evaluating peptides in a sample, (a) a step of adding to a sample a composition for liquid chromatography-mass spectrometry (LC-MS) system quality evaluation comprising one or more peptides selected from the group consisting of SEQ ID NOs 1 to 7; (b) a step of analyzing the sample using liquid chromatography-mass spectrometry (LC-MS); (c) a step of confirming liquid chromatography-mass spectrometry (LC-MS) data corresponding to any one or more peptides selected from the group consisting of the amino acid sequences of SEQ ID NOs 1 to 7; and (d) An evaluation method comprising the step of quantifying the amount of peptide in a sample using the confirmed liquid chromatography-mass spectrometry (LC-MS) data.

5. In Paragraph 4, Evaluation method in which the above sample is a biological sample containing body fluids, secreted proteins, cell surface proteins, plant-derived materials, or microorganisms.

6. As a method for monitoring the performance of a liquid chromatography-mass spectrometry (LC-MS) system, (a) a step of adding to a sample a composition for liquid chromatography-mass spectrometry (LC-MS) system quality evaluation comprising one or more peptides selected from the group consisting of SEQ ID NOs 1 to 7; (b) a step of analyzing the sample using liquid chromatography-mass spectrometry (LC-MS); (c) a step of confirming liquid chromatography-mass spectrometry (LC-MS) data corresponding to any one or more peptides selected from the group consisting of the amino acid sequences of SEQ ID NOs 1 to 7; and (d) A monitoring method comprising the step of evaluating the performance of a liquid chromatography-mass spectrometry (LC-MS) system using the confirmed liquid chromatography-mass spectrometry (LC-MS) data.

7. In Paragraph 6, Evaluation method in which the above sample is a biological sample containing body fluids, secreted proteins, cell surface proteins, plant-derived materials, or microorganisms.

8. A method for preparing peptides for quality evaluation of a liquid chromatography-mass spectrometry (LC-MS) system, A step of extracting a cell pellet from a culture medium of Synechocystis (Synechocystissis p. PCC6803); A step of adding acetone to the cell pellet and centrifuging to obtain protein; The step of ultrasonically grinding the protein obtained above; and A method for preparing a peptide comprising the step of in-solution digestion.

9. In Paragraph 8, The in-solution digestion step is, Step of adding urea; A step of reducing disulfide bonds within the protein by adding a reducing agent; and A method for preparing a peptide comprising an alkylation reaction step of stabilizing disulfide bonds within a protein by adding an alkylating agent.

10. In Paragraph 8, A method for preparing a peptide that further includes a desalting step.