KR101758862B1 - Method for Diagnosis of Amyloid Isotypes using Quantitative Analysis based Mass Spectrometry - Google Patents
Method for Diagnosis of Amyloid Isotypes using Quantitative Analysis based Mass Spectrometry Download PDFInfo
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Abstract
The present invention provides a standard peptide for quantitative analysis of amyloid subtypes and a synthetic standard peptide for amyloid subtype diagnosis. An amyloid subtype diagnostic kit comprising the synthetic standard peptide for amyloid subtype diagnosis of the present invention and a method for diagnosing an amyloid subtype using the same. The present invention enables rapid analysis and diagnosis of four representative types of amyloid subtypes. The present invention can be very useful in establishing a prediction and treatment strategy of amyloidosis.
Description
The present invention relates to a standard peptide for quantitative analysis of amyloid protein subtypes, a synthetic standard peptide for the detection of subtypes of amyloid proteins, and a method for diagnosing the subtypes of amyloid proteins using the same.
Amyloidosis is a disorder in which a protein mass called insoluble amyloid is formed in the process of protein formation and the formed amyloid component is deposited in the body organs and tissues. As a result, it is known as a disease which deteriorates the function of the organ and eventually loses its function.
Amyloidosis is classified according to the cause and the type of protein deposited. It is largely divided into primary amyloidosis, secondary amyloidosis and hereditary amyloidosis. Primary amyloidosis (AL) is the most common form of systemic amyloidosis. It is caused by immunoglobulin light chain (AL), which is abnormally made in the bone marrow. Abnormal antibody proteins made in plasma cells are found in various organs (heart, And autonomic nervous system, liver, soft tissue, etc.) and causing the organ dysfunction is the main etiology. This can lead to a variety of symptoms, often accompanied by multiple myeloma and, in severe cases, death.
The incidence of secondary amyloidosis (AA type) is less than 5% and is secondary to chronic infectious diseases. In the case of chronic inflammation such as tuberculosis, chronic osteomyelitis or rheumatoid arthritis, the protein called SAA (Serum Amyloid A) is increased in our body and this protein forms amyloid fiber and settle in organs. It mainly involves the kidneys, but it also deposits in the gastrointestinal tract, liver and heart as the disease progresses. Because the major organ involvement is kidney involvement, proteinuria, edema, weight loss, and fatigue are the main symptoms and are not associated with multiple myeloma. Typically, a high incidence of the disease is particularly high in rheumatoid arthritis, psoriatic arthritis, chronic juvenile arthritis, Ankylosing spondylitis, inflammatory bowel disease, and pyelonephritis. . Hereditary amyloidosis is very rare, and gene abnormalities in TTR (Transthyretin) proteins are known to be the most common cause. In the liver, a mutated TTR protein is synthesized, deposited in the heart, and inherited as autosomal dominant. If the parent is a patient, the child is inherited at a 50% probability, so gene screening of the patient's family is necessary. 20 to 40 generations occur, but the progression of the disease is slow, arrhythmia is common, and orthostatic hypotension develops in the nervous system. Other genes, such as ApoA protein, fibrinogen, and lysozyme, may also be caused by abnormal genes. As other subtypes, senile amyloidosis is a type of TTR protein, which is a type of protein such as heredity / familial amyloidosis, which is deposited in the tissue, but unlike genetic amyloidosis, a normal TTR protein is formed in the heart, not a mutant TTR protein. Arrhythmias are commonly associated, but the prognosis of the disease is relatively good.
Thus, the precise diagnosis of amyloid subtype is very important because the prognosis and treatment direction of the disease is determined according to the subtype of amyloid protein. Up to this point, the diagnosis of amyloid deposits through histological examination using a family history, muscle, bone, and adipose tissue, and immunohistochemistry of the patient to diagnose the subtype. However, immunohistochemical analysis suggests low specificity and low sensitivity due to the loss of the epitope reacting with the antibody in the preparation of the amyloid tissue in the subtype diagnosis. Due to these problems, a method for diagnosing amyloid protein subtypes based on mass spectrometry has recently been introduced. The mass spectrometry based amyloid subtype detection method is a method of isolating amyloid protein deposits deposited in organs using Laser Captured Microdissection (LCM) and identifying the proteins by mass spectrometry. Although the specificity of the diagnosis is high, it may cause diagnosis errors due to blood proteins in the analysis process, and it requires a high-performance equipment for clinical application and requires a well-educated expert. Accordingly, the present invention provides a method for determining the concentration of the representative four subtypes of proteins using multiple reaction monitoring-mass spectrometry based on mass spectrometry, which is relatively easy to apply in clinical applications with high sensitivity and measurement limit, The aim of this study was to develop a method for detecting subtypes of amyloid by relative ratio analysis.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
The present inventors have made extensive efforts to develop an accurate diagnostic method for amyloid subtypes that have important effects on the prognosis and treatment direction of amyloidosis. As a result, using the standard peptide for quantitative analysis of amyloid protein subtypes of SEQ ID NOS: 5 to 12 obtained by hydrolyzing amyloid protein, it was confirmed that four representative types of amyloid subtypes can be diagnosed quickly and accurately Thereby completing the present invention.
It is therefore an object of the present invention to provide a standard peptide for the quantitative analysis of subtypes of amyloid proteins.
Another object of the present invention is to provide a synthetic standard peptide for the subtype diagnosis of amyloid protein.
It is yet another object of the present invention to provide a method for diagnosing an amyloid subtype.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.
According to one aspect of the present invention, the present invention provides a standard peptide for quantitative analysis of amyloid subtypes obtained by hydrolyzing amyloid protein having any one of the amino acid sequences of SEQ ID NOS: 1 to 4 do.
The present inventors have intensively studied to develop an accurate diagnostic method for amyloid subtypes that have important effects on the prognosis and treatment direction of amyloidopathy. As a result, the present inventors have found that the amyloid- 12 subtype of amyloid protein subtype, the present inventors have found that four types of amyloid subtypes can be rapidly and accurately diagnosed using the standard peptide for quantitative analysis of the subtype of amyloid protein.
The standard peptide for quantitative analysis of the amyloid subtypes of the present invention has the amino acid sequence set forth in SEQ ID No. 5 to SEQ ID No. 12. The standard peptides are selected from peptides having a high ionization efficiency and high extraction efficiency from peptides obtained by hydrolyzing amyloid subtype proteins having amino acid sequences of
According to another aspect of the present invention, there is provided a synthetic standard peptide for amyloid subtype diagnosis comprising an amino acid sequence selected from the group consisting of SEQ ID No. 5 to SEQ ID No. 12.
The synthetic standard peptide for amyloid subtype diagnosis of the present invention is produced by hydrolysis of four typical amyloid subtypes and is labeled with a fluorescent substance, a pigment, a radioactive isotope or a mixture thereof.
According to one embodiment of the present invention, the synthetic standard peptide for amyloid subtype diagnosis of the present invention is labeled with a radioactive isotope.
According to another embodiment of the present invention, the synthetic standard peptide for amyloid subtype diagnosis of the present invention is labeled with a radioisotope 13 C or 15 N.
The labeling can be performed by adding an amino acid having a stable isotope in the course of peptide synthesis, or by labeling a functional group having a stable isotope at a specific amino acid after synthesis. In the present invention, since the radioactive isotope is used to distinguish the wild type peptide from the wild type peptide by binding to the wild type peptide by binding to the standard peptide, the radioactive isotope does not need to be included in the standard peptide and is included in a form bound to the OH end of the standard peptide It is possible.
As used herein, the term " wild-type peptide " refers to an amyloid peptide already present in a hydrolyzed sample of a subject. In the present invention, the concept is used in contrast with a standard peptide labeled or substituted with a radioisotope that is further added to the hydrolyzed sample.
As used herein, the term " diagnosis " means to determine whether an amyloid protein is present in a sample or to classify the type of amyloid protein subtypes present in the sample.
According to the present invention, four kinds of amyloid proteins are hydrolyzed with trypsin to obtain peptides for the four amyloid proteins, and then the MRM transition composition for a tryptic peptide consisting of more than 5 and less than 25 amino acids The peptide MRM transitions with high sensitivity and sensitivity to noise and signal ratio (S / N) of 8 or more were selected from all the peptide peaks and then the S / N ratio And the number of MRM transitions satisfying all of the ratio 8 or more and RT +/- 0.2 was more than 3 per the same peptide was selected as a peptide to be quantitated by MRM. The selected peptides were present specifically in the amyloid subtype and showed the highest signal peaks in the MRM scan (see Table 2).
The synthetic standard peptide for amyloid subtype diagnosis can be used as a peptide for SID-MRM-MS capable of rapidly analyzing four representative amyloid proteins.
According to another aspect of the present invention, there is provided an amyloid subtype diagnostic kit comprising the synthetic standard peptide for amyloid subtype diagnosis.
According to another aspect of the present invention, the present invention provides a method of diagnosing an amyloid subtype comprising the steps of:
(a) hydrolyzing a sample separated from a subject;
(b) adding the synthetic standard peptide to the hydrolyzed sample;
(c) quantitatively analyzing the peptide of wild-type amyloid subtype protein and the synthetic standard peptide from the sample of step (b); And
(d) determining the amyloid subtype by analyzing the quantitative analysis result.
According to the present invention, in order to measure the classification and concentration of amyloid subtypes, a sample is hydrolyzed, and a labeled synthetic standard peptide is added, followed by desalting and mass analysis.
In the present invention, the "sample" is a tissue, cell, blood or the like capable of detecting an amyloid peptide, and preferably includes tissues, blood, plasma, serum, secretions, and the like. When the amyloid tissue is used as the sample, the tissue is separated using Laser Captured Microdissection (LCM), and congo red is stained to separate the amyloid-deposited portion, and proteolytic enzyme (for example, Trypsin).
The hydrolysis may be performed using any protease capable of recognizing a cleavage site in a protein contained in a sample and degrading the peptide to a peptide level. Preferably, trypsin, chymotrypsin, pepsin, thermolysin, And according to one embodiment of the present invention, trypsin is used.
The quantitative analysis of step (c) can be performed by, but not limited to, quantitative analysis using SID-MRM-MS (stable isotope dilution-multiple reaction monitoring-mass spectrometry) and LC / MS mass spectrometry .
The term " stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) " of the present invention means that the expression level of the correct protein or modified protein is determined from all biological components including blood and tissues Means a mass spectrometry based quantitative method for direct quantification. The SID-MRM-MS is based on two principles, one is a stable radioisotope dilution theory and the other is the use of a synthetic peptide containing a stable radioisotope to mimic a natural counterpart after proteolysis. The peptides are synthesized by modification methods such as phosphorylation, methylation and acetylation, which allow direct quantitative analysis of the mutated proteins after decoding.
According to one embodiment of the present invention, in order to quantify the amyloid subtype protein in the sample, the mass (precursor ion m / z) of the peptide obtained by hydrolyzing the amyloid protein in the sample with trypsin is measured by MS1 spectrum, (Product ion m / z) is measured by MS2 spectrum. The transition of the MS1 precursor ion m / z and MS2 product ion m / z is specified. The transition for each protein is input to the mass spectrometer, and the concentration of the corresponding protein is set to absolute Quantitative analysis. At this time, standard synthetic peptide labeled with radioactive isotope is added to the sample as an internal control, so that absolute quantitative analysis is possible and sensitivity analysis of ng / ml concentration is possible.
Quantitative analysis is performed to compare the amounts of wild type amyloid peptide and synthetic standard peptide in the sample to determine the absolute value of the amount of wild type amyloid peptide, and the value is compared and analyzed to diagnose the amyloid subtype.
As a result of the quantitative analysis by the above method, the concentration difference of the amyloid subtype peptide was measured in the individual having the amyloid protein by the analysis using the standard peptide. Thus, the method of the present invention can be useful for the diagnosis of the amyloid subtype Respectively.
The features and advantages of the present invention are summarized as follows:
(I) The present invention provides a standard peptide for quantitative analysis of amyloid subtypes and a synthetic standard peptide for amyloid subtype diagnosis.
(Ii) an amyloid subtype diagnostic kit comprising the synthetic standard peptide for amyloid subtype diagnosis of the present invention and a method for diagnosing an amyloid subtype using the same.
(Iii) Mass analysis based on the present invention - Subtype classification by quantification of amyloid protein is advantageous in that it makes it easy to make a final diagnosis of a sample difficult to diagnose in IHC. In the present invention, four representative types of amyloid subtypes Rapid analysis and diagnosis.
(Iv) The present invention can be very useful in establishing a prediction and treatment strategy of amyloidosis.
FIG. 1 is a photograph showing tissue of amyloidosis obtained through laser microdissection (LCM).
Congo red, a stained tissue photograph showing the area of the amyloid protein (+) as a stained region. It also indicates the site of extraction through laser microdeposition.
Figure 2 shows MS / MS scan results of peptides derived from amyloid proteins.
a. MS / MS scan results of peptides SFFSFLGEAFDGAR and GPGGVWAAEAISDAR derived from amyloid protein SAA, b. MS / MS scan results of peptides TVAAPSVFIFPPSDEQLK and DSTYSLSSTLTLSK derived from amyloid protein IGK, c. MS / MS scan results of peptides GSPAINVAVHVFR and AADDTWEPFASGK derived from amyloid protein TTR, d. MS / MS scan results of peptides AAPSVTLFPPSSEELQANK and YAASSYLSLTPEQWK derived from amyloid protein IGL.
Fig. 3 is a spectrum showing the peak intensity and retention time of the peptide.
FIG. 3A shows extracted ion chromatography (EIC) obtained from six MRM transitions of representative peptides derived from the amyloid protein IGK. Figure 3b shows the EIC obtained from six MRM transitions of a representative peptide derived from the amyloid protein IGL. Figure 3c shows the EIC obtained from six MRM transitions of representative peptides derived from the amyloid protein SAA. Figure 3d shows the EIC from six MRM transitions of representative peptides derived from amyloid protein TTR.
4 is a schematic view showing the amyloid quantitative analysis method of the present invention.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
Example
Example 1: Selection of Peptide and MRM (Multiple Reaction Monitoring) Methods of Amyloid Proteins in Amyloid Deposits
The target peptide for SID-MRM-MS analysis was selected by virtually (In-silico) trypsin treatment of four amino acids of amyloid protein using MRM Pilot (ABSciex, Framingham, USA). Each 1 μg of the recombinant protein corresponding to the above four proteins (Table 1) was treated with trypsin and fragmented into peptides.
Among the peptide fragments, the best ionized and highly efficient peptides in LC-MS / MS systems were selected. For this purpose, up to five MRM transition compositions for tryptic peptides composed of more than 5 and less than 25 amino acids were predicted by in-silico method. At this time, the MRM transition for the final MRM quantitative analysis was selected as follows. First, both the noise and signal ratio (S / N) of all the peptide peaks detected in the MRM mode were determined, and the peptide MRM transitions with an S / N ratio of 8 or higher were selected. Second, the retention time (RT) of the selected peptide peaks was extracted and excluded if the retention time deviates from the range of ± 0.2 minutes for the MRM transition of the same peptide. Third, when the number of MRM transitions satisfying all of the S / N ratio 8 or more and RT ± 0.2 was more than 3 per the same peptide, the peptide was selected as the peptide to be analyzed by MRM. In order to determine the top three selected transitions as final MRM transitions, the optimal Q1 / Q3 value was determined by MS / MS scanning of peptides formed from recombinant proteins of four amyloid proteins and optimized for SID-MRM-MS system Parameters were determined (Figures 2, 3, and Table 2). FIG. 2A is a graph showing MS / MS scan results of peptides SFFSFLGEAFDGAR and GPGGVWAAEAISDAR derived from amyloid protein SAA, FIG. 2B is a graph showing MS / MS scan results of peptides TVAAPSVFIFPPSDEQLK and DSTYSLSSTLTLSK derived from amyloid protein IGK, FIG. 2D is a graph showing MS / MS scanning results of peptides AAPSVTLFPPSSEELQANK and YAASSYLSLTPEQWK derived from amyloid protein IGL. FIG. 2C is a graph showing MS / MS scanning results of peptide peptides GSPAINVAVHVFR and AADDTWEPFASGK derived from amyloid protein TTR. The final IS peptides and MRM conditions obtained by MS / MS scans of the 8 species of peptides are summarized in Table 2 below. Fig. 3 is a spectrum showing the peak intensity and retention time of the dual representative peptide. As a result, these selected peptide fragments were specifically present in the amyloid subtype and showed the highest signal peaks in the MRM scan (Table 2).
Example 2: Isolation of blood trypsin for SID-MRM-MS analysis
89 μl of 50 mM ABC (Ammonium bicarbonate) buffer was added to 1 μl of a serum (or plasma) sample of amyloidosis patients (corresponding to about 50-80 μg of protein), and the mixture was mixed with 50 mM DTT dithiothreitol) solution was added thereto, followed by reaction at 56 ° C for 30 minutes and cooling to room temperature. Then, 7.5 μl of 2 M IAA (iodoacetamide) was added and reacted for 20 minutes under dark conditions. 10 μl of trypsin solution (0.1 μg / μl in 50 mM ABC buffer) was added to the reaction solution, followed by reaction at 37 ° C for 12 hours. After completion of the reaction, the reaction product was applied to a vacuum concentrator (SpeedDry Vacuum Concentrator) and dried.
Example 3: Trypsin degradation process of amyloid tissue for SID-MRM-MS analysis
As shown in FIG. 1, RapiGest (waters) was added to 50 mM ABC buffer in a final 0.1% concentration in amyloid tissue (1.5 x 10 5 μm 2 ) obtained through laser microdissection (LCM) Deg.] C and reacted at 99 [deg.] C for 5 minutes. DTT solution was added to the reaction mixture to a final concentration of 5 mM, reacted at 56 ° C for 30 minutes, and then cooled at room temperature. Then, IAA was added to 200 mM and reacted for 30 minutes under dark conditions. Finally, 2.5 μg of trypsin solution was added to the reaction solution, followed by reaction at 37 ° C for 12 hours. After completion of the reaction, the reaction product was applied to a vacuum concentrator (SpeedDry Vacuum Concentrator) and dried.
Example 4: Addition and decalcification of peptide internal standards
(Table 3) corresponding to the 26 peptides shown in Table 2 were dissolved in distilled water (0.1% trifluoroacetic acid) at a concentration of 100 fmol / μl or 200 fmol / μl, respectively, 8 [mu] L of trypsin lysate (tissue or blood trypsin lysate) was added and redissolved. In this case, the internal standard (IS) is a compound known to be added to the sample or sample extract immediately before analyzing the sample. It has similar chromatographic characteristics, that is, retention time, And relative response, and refers to a substance used to check the amount of analyte present in each sample. In addition, the internal standard material is a synthetic peptide having the same sequence as the selected peptide in Table 2, but some amino acids are substituted with isotopes.
For the synthesis of the peptide used as the internal standard for the absolute quantification of the selected peptides, the position of the label portion (*) and the labeled radioisotope (15N, 13C) were determined as shown in Table 3 below.
The redissolved reagents were desalted by applying to a decontamination apparatus (OMIX Tips for peptide and protein desalting / concentrating 5-100 [mu] L size, C18, Agilent, Santa Clara, USA). Specifically, the OMIX tip was soaked twice with 100% ACN (0.1% TFA) buffer, washed with 75% ACN (0.1% TFA) buffer and then 10 times with distilled water (0.1% TFA). The re-dissolved reactant was added to the water-treated OMIX tip, the reaction was loaded upside down for 20 times, and washed three times with distilled water (0.1% TFA). Next, 50 μl of 75% ACN (0.1% TFA) buffer was added to the OMIX tip on which the sample was loaded, and the peptide fraction contained in the reaction product was eluted by inverting the
This step is carried out to remove the synthetic peptides substituted with the target peptide and the corresponding isotope, ie, the internal standard material and the salt components added to the sample pretreatment (disturbing mass spectrometry).
Example 5: SID-MRM-MS (stable isotope dilution-multiple reaction monitoring-mass spectrometry) method of four kinds of amyloid proteins
In Example 4, the peptide extract obtained through decontamination using an Eksigent 1D-plus nanoflow liquid chromatography system was dissolved in 8 μl of distilled water (0.1% formic acid), and 3 μl of the extract was transferred to a trapping column (200 μm x (0.1% formic acid) until it was eluted with an analytical column (75 μm × 150 mm, chromogenic C18-
The peptide eluate was analyzed by MR-mode of ABSciex Q-TRAP 5500. The MRM analysis conditions are as follows; ion spray voltages, 2,200-2,500 V; curtain gas, 20; spray gas, 20; collision gas, high; declustering potential (DP), 100 V; Mass resolution, unit; quadrupole resolution, low.
For the diagnosis of amyloid subtype, MRM quantification information of four representative peptides corresponding to each amyloid subtype protein was used. For the quantitative analysis of MRM of each peptide, one of the three types of MRM transitions was used to acquire the quantitative value of the target protein with excellent sensitivity and RT reproducibility. The other two transitions were used for peptide verification Respectively.
The peak area of the transition corresponding to each peptide was determined using MultiQuant (ABSciex, Framingham, USA). At this time, the measured parameter values were as follows. smoothing width, 2 points; Min.Peak Width, 3 points; Noise Percentage, 40%; baseline Sub.Window, 2 min; Peak Splitting Factor, 2 points.
To obtain quantitative information on each peptide, an internal standard (IS) corresponding to each peptide was injected together. At this time, the concentration of the internal reference material was 100 fmol / μL, and the peak area of the corresponding 100 fmol / μL IS was extracted by applying the above parameter values. The concentration of each amyloid subtype was calculated by extracting the peak area of the selected final peptide transition under the same conditions, dividing by the peak area of IS, and multiplying by the IS concentration value of 100 fmol / μL.
Example 6 Determination of Amyloid Subtype Diagnosis Method
For clinical verification of the invention described above, a clinical sample was injected into an LC-MS / MS system via the trypsin degradation process of the amyloid tissue of Example 3 and the addition and decontamination of the peptide internal standard material of Example 4, The concentration of the four representative amyloid subtypes in the clinical sample was calculated by SID-MRM-MS (stable isotope dilution-multiple reaction monitoring-mass spectrometry) At this time, verification of clinical specimens was performed using non-standard mass analysis based on mass analysis and Immunohistochemistry which are used in existing subtype analysis method.
Specifically, the concentrations of SAA subtypes were calculated by quantitative analysis of SFFSFLGEAFDGAR (SEQ ID NO: 5) and GPGGVWAAEAISDAR (SEQ ID NO: 6), TVAAPSVFIFPPSDEQLK (SEQ ID NO: 7), DSTYSLSSTLTLSK (SEQ ID NO: , And the concentration of TTR subtypes was calculated by quantifying GSPAINVAVHVFR (SEQ ID No. 9) and AADDTWEPFASGK (SEQ ID No. 10). And, AAPSVTLFPPSSEELQANK (SEQ ID No. 11) and YAASSYLSLTPEQWK (SEQ ID No. 12) were used for the diagnosis of IGL subtype. More specifically, ions having high detection specificity were determined based on MS / MS scans (FIG. 2) of 8 kinds of peptides (FIG. 3). The determined ions were applied to the SID-MRM-MS system to determine the concentration of the selected protein. Based on the concentration obtained from the target quantification of the above four kinds of amyloid proteins, the protein having the highest expression amount was determined. At this time, the concentration of the determined protein subtype should be 4 times or more as much as that of the remaining protein subtype.
Example 7 Clinical Validation of Four Amyloid Proteins
Non - amyloid lesions (control group) and amyloid lesions were studied for the differential diagnosis of four representative amyloid subtypes in clinical specimens. The determined detection concentration of the peptide was determined and the subtype of amyloid was determined based on the amyloid subtype that was measured specifically. Specifically, for the diagnosis of amyloid subtypes in amyloid lesion tissues, the concentration of each protein amount was calculated for a clinical sample (amyloid lesion and non-amyloid tissue) having four kinds of amyloid subtypes using SID-MRM-MS system . Table 5 shows the concentrations of four amyloid subtype proteins in non-amyloid lesion tissues and Table 6 shows the concentrations of four amyloid subtype proteins in various amyloid lesions. As shown in Table 5, a very small amount of 0.1 - 0.5 fmol / μL was detected in each non - amyloid lesion. This is thought to be due to contamination of blood during the tissue extraction process. On the other hand, in the case of amyloid lesions, the concentrations of four subtypes of protein were calculated and compared with each other, one type of subtype protein showed a significantly higher detection density than the other three types of amyloid. The difference in the detection concentration showed a difference in detection amount from several tens of times to several hundreds of times. Based on these differences in detection levels, we could differentiate amyloid subtypes. In order to verify the accuracy of the diagnostic results, IHC verification results usually used for diagnosis were presented. As shown in Table 6, in most cases the results were consistent with the SID-MRM-MS results.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
<110> Samsung Medical Center <120> Method for Diagnosis of Amyloid Isotypes using Quantitative Analysis based Mass Spectrometry <130> PN140025 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 122 <212> PRT <213> Human Serum Amyloid A Protein (SAA) <400> 1 Met Lys Leu Leu Thr Gly Leu Val Phe Cys Ser Leu Val Leu Gly Val 1 5 10 15 Ser Ser Arg Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala 20 25 30 Arg Asp Met Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile 35 40 45 Gly Ser Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys 50 55 60 Arg Gly Pro Gly Gly Val Trp Ala Ala Glu Ala Ile Ser Asp Ala Arg 65 70 75 80 Glu Asn Ile Gln Arg Phe Phe Gly His Gly Ala Glu Asp Ser Leu Ala 85 90 95 Asp Gln Ala Ala Asn Glu Trp Gly Arg Ser Gly Lys Asp Pro Asn His 100 105 110 Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr 115 120 <210> 2 <211> 236 <212> PRT <213> Human IGK protein <400> 2 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 20 25 30 Phe Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr 35 40 45 Val Phe Ser Ser His Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala 50 55 60 Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Ala Thr Gly Ile Pro 65 70 75 80 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Thr Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr 100 105 110 Gly Thr Ser Pro Ser Leu Thr Phe Gly Gly Gly Thr Arg Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Ser Ser Ser Ser Thr Ser Ser Ser Ser Thr Leu 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 3 <211> 147 <212> PRT <213> Human Prealbumin Protein (TTR) <400> 3 Met Ala Ser His Arg Leu Leu Leu Leu Cys Leu Ala Gly Leu Val Phe 1 5 10 15 Val Ser Glu Ala Gly Pro Thr Gly Thr Gly Glu Ser Lys Cys Pro Leu 20 25 30 Met Val Lys Val Leu Asp Ala Val Arg Gly Ser Pro Ala Ile Asn Val 35 40 45 Ala Val His Val Phe Arg Lys Ala Ala Asp Asp Thr Trp Glu Pro Phe 50 55 60 Ala Ser Gly Lys Thr Ser Glu Ser Gly Glu Leu His Gly Leu Thr Thr 65 70 75 80 Glu Glu Glu Phe Val Glu Gly Ile Tyr Lys Val Glu Ile Asp Thr Lys 85 90 95 Ser Tyr Trp Lys Ala Leu Gly Ile Ser Pro Phe His Glu His Ala Glu 100 105 110 Val Val Phe Thr Ala Asn Asp Ser Gly Pro Arg Arg Tyr Thr Ile Ala 115 120 125 Ala Leu Leu Ser Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Thr Asn 130 135 140 Pro Lys Glu 145 <210> 4 <211> 235 <212> PRT <213> Human Lambda Light chain protein <400> 4 Met Ala Ser Phe Pro Leu Leu Leu Thr Leu Leu Thr His Cys Ala Gly 1 5 10 15 Ser Trp Ala Gln Ser Val Leu Thr Gln Pro Ser Ala Ser Gly Thr 20 25 30 Pro Gly Gln Arg Val Pro Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile 35 40 45 Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp 65 70 75 80 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 85 90 95 Gly Leu Gln Ser Glu Asp Asp Ala Val Tyr His Cys Ala Thr Trp Asp 100 105 110 Asp Asn Leu Asn Ser Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val 115 120 125 Leu Ser Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Ser Ser 130 135 140 Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser 145 150 155 160 Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser 165 170 175 Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn 180 185 190 Asn Lys Tyr Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp 195 200 205 Lys Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr 210 215 220 Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 235 <210> 5 <211> 14 <212> PRT <213> SAA Peptide 1 <400> 5 Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala Arg 1 5 10 <210> 6 <211> 15 <212> PRT <213> SAA Peptide 2 <400> 6 Gly Pro Gly Gly Val Trp Ala Ala Glu Ala Ile Ser Asp Ala Arg 1 5 10 15 <210> 7 <211> 18 <212> PRT <213> IGK Peptide 1 <400> 7 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys <210> 8 <211> 14 <212> PRT <213> IGK Peptide 2 <400> 8 Asp Ser Thr Ser Ser Ser Ser Thr Leu Thr Leu Ser Ser 1 5 10 <210> 9 <211> 13 <212> PRT <213> TTR Peptide 1 <400> 9 Gly Ser Pro Ala Ile Asn Val Ala Val His Val Phe Arg 1 5 10 <210> 10 <211> 13 <212> PRT <213> TTR Peptide 2 <400> 10 Ala Ala Asp Asp Thr Trp Glu Pro Phe Ala Ser Gly Lys 1 5 10 <210> 11 <211> 19 <212> PRT <213> IGL Peptide 1 <400> 11 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 1 5 10 15 Ala Asn Lys <210> 12 <211> 15 <212> PRT <213> IGL Peptide 2 <400> 12 Tyr Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 1 5 10 15
Claims (11)
(a) hydrolyzing a sample separated from a subject;
(b) adding to the hydrolyzed sample eight kinds of synthetic standard peptides for amyloid subtype diagnosis, each of which is composed of a sequence list of any one of Sequence Listing 5 or Sequence Listing 12;
(c) calculating the SAA subtype concentration by quantifying the fifth and sixth sequences of the sequence listing, calculating the IGK subtype concentration by quantifying the seventh and eighth sequences of the sequence listing, Quantifying the TTR subtype concentration, and quantitating the sequence listing eleventh and twelfth sequences to calculate an IGL subtype concentration;
(d) determining the amyloid subtype by analyzing the result of the quantitative analysis step.
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| US20090104602A1 (en) * | 2005-05-23 | 2009-04-23 | Delmiro Fernandez-Reyes | Diagnosis of Tuberculosis |
| US20120149022A1 (en) * | 2009-02-20 | 2012-06-14 | Eva I-Wei Aw | Compositions and methods for diagnosis and prognosis of colorectal cancer |
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| US20090104602A1 (en) * | 2005-05-23 | 2009-04-23 | Delmiro Fernandez-Reyes | Diagnosis of Tuberculosis |
| US20120149022A1 (en) * | 2009-02-20 | 2012-06-14 | Eva I-Wei Aw | Compositions and methods for diagnosis and prognosis of colorectal cancer |
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