KR20110103902A - Development of diagonstic and prognostic methods for chronic myelodal leukemia by measurement of expression levels of bcr-abl and drug-resistant bcr-abl mutants with quantitiative mass spectrometry - Google Patents

Abstract

The present invention provides a method for quantitatively analyzing a Bcr / Abl hybrid protein, which is a causative protein of chronic myelogenous leukemia, in a biological sample (bone marrow / blood / plasma) using a mass spectrometer, using an inhibitor of a Bcr / Abl hybrid protein. The present invention relates to a method for quantitative analysis of drug resistance mutations of Bcr / Abl generated in the course of treatment, the diagnosis and treatment prognosis of chronic myeloid leukemia using the same, and peptides used in the method.

Description

Development of diagonstic and prognostic methods for chronic myelodal leukemia by measurement of expression levels of Bcr-Abl and drug-resistant Bcr-Abl mutants with quantitiative mass spectrometry }

The present invention is a quantitative analysis method of Bcr-Abl, the causative protein of chronic myelogenous leukemia using an isotope dilution method, the amount of mutant protein of Bcr-Abl having drug resistance that occurs during the treatment and phosphorylation that has an important effect on the prognosis of the disease. It relates to a method of quantitatively analyzing a patient's biological sample (plasma, bone marrow) and a peptide used in the method.

The present invention is derived from a study carried out as part of the Cancer Conquest Promotion Research and Development Project of the Ministry of Health and Welfare [Task serial number: 0520070-1, Task name: Diagnosis of chronic myelogenous leukemia using quantitative proteomic analysis technology]

Leukemia is a malignant blood disease that occurs in the hematopoietic system of the human body, including hematopoietic stem cells that produce blood cells and lymphocytes. Immature malignant white blood cells proliferate in the bone marrow caused by disorders in the hematopoietic process in which leukocytes mature. It is a type of blood cancer in which malignant leukocytes occupy more than 30% of the total bone marrow hematopoietic stem cells due to the disturbance of the production. Leukemia is classified into 4 types according to acute and chronic, myeloid and lymphocytic, and classified into 20 types of leukemia by subdividing them in histological aspect. Any type of leukemia can appear at any age, but acute lymphocytic leukemia is predominantly present in children (2-10 years old), and acute myelogenous leukemia or chronic myelogenous leukemia is commonly seen in middle or old age. In Korea, the incidence rate of leukemia is 0.96 per 100,000 people as of 1992, 3,000-4,000 people are diagnosed as new leukemia every year, and 1,306 people, which account for 0.54% of the total cause of death, died from leukemia in 2000. Ministry of Health and Welfare Cancer Patient Survey Report, 1996; Statistical Yearbook by Cause of Death, National Statistical Office, 2000).

As a method for diagnosing leukemia known to date, a method of diagnosing peripheral blood cells and bone marrow cells by using conventional optical microscopes and staining is known, but even tumors showing the same findings show very different clinical results depending on the patient, and each treatment is different. Since they may react differently, diagnosis and classification methods according to pathology and morphological specificity have limitations in determining an appropriate treatment method for treating leukemia. In recent years, molecular genetics has been developed, and abnormalities of leukemia-specific chromosomes and genes have been identified in more than 80-90% of malignant blood diseases. hybridization), polymerase chain reaction, etc. are used to improve the diagnostic success rate. Nevertheless, the diagnostic accuracy for determining the type of leukemia is only about 30 to 40%, and the disadvantage of requiring a large amount of time for diagnosis has not been improved. Accordingly, there is an urgent need to develop a new molecular genetic diagnostic method that can diagnose various leukemia-specific genes more accurately and quickly and determine the prognosis after disease treatment by measuring the presence of microscopic residual diseases. It is not reported.

A biological sample is a state in which a number of proteins are mixed. After separating proteins or peptides obtained by hydrolyzing proteins by a method such as one-dimensional SDS-PAGE or liquid chromatography, the tandem mass spectrum of the peptides is analyzed using a mass spectrometer. Get

Using the protein sequence database, the amino acid sequence of the peptide corresponding to each tandem mass spectrum can be found, and the protein can be identified by integrated analysis of them. This protein retrieval process includes SEQUEST® (Eng et al., J. Am. Soc. Mass Spectrom. 5:976-989, 1994; Thermo Electron Corp., USA), Mascot (Perkins et al., Electrophoresis, 20:3551). -3567, 1999; Matrix Science Ltd., USA, http://www.matrixscience.com/search_form_select.html), Sonar (Field, HI et al., Proteomics, 2:36-47,2002; http:// knxs.bms.umist.ac.uk/prowl/sonar/sonar_cntrl.html), X!Tandem (Craig et al., 　 Bioinformatics, 20:1466-1467, 2004; Proteom Software Inc., USA), Phenyx, PeptideProphet ( Keller A., et al., Anal.Chem. 2002, 74, 5383-5392), ProteinProphet (Nesvizhskii AI, et al., Anal.Chem. 2003, 75,4646-4658), DTASelect (Tabb DL, et al ., Proteome Res. 2002, 1, 21-26) or OMSSA (Syka JE, et al., Proc Natl Acad Sci USA. 2004. Jun 29, 101(26). 9528-33).

In general, studies that quantitatively understand the amount of expression of a specific protein or a specific post-transcriptional modification process are of great importance. In particular, since there is no process of amplifying the amount of protein in the sample, mass spectrometry based on the isotope dilution method was developed to measure a small amount of protein. This analysis method enables quantitative analysis of a small amount of protein present in cells of less than 1000 cells/cell, which is a very suitable method in that a sufficient amount of sample cannot be prepared in clinical studies. In this assay, once the amino acid sequence of the target protein is known, peptide synthesis, physicochemical property analysis, and analysis method development were completed within a few weeks.

The present invention solves the above problems and has been conceived by the necessity of the above, and an object of the present invention is to provide a method for quantifying a protein related to chronic myelogenous leukemia.

Another object of the present invention is to provide a method for quantifying a specific degree of phosphorylation that can predict the prognosis of chronic myelogenous leukemia.

Another object of the present invention is to provide a method for quantifying a protein related to chronic myelogenous leukemia drug resistance.

Another object of the present invention is to provide a standard peptide for quantification of proteins associated with chronic myelogenous leukemia.

Another object of the present invention is to provide a standard peptide for the quantification of proteins related to chronic myelogenous leukemia drug resistance.

In order to achieve the above object, the present invention is LGGGQHGEVYEGVWK (SEQ ID NO: 1), LGGGGQYGEVYEGVWK (SEQ ID NO: 2), LGGGQFGEVYEGVWK (SEQ ID NO: 3), LGGGQYGVVYEGVWK (SEQ ID NO: 4), LGGGQYGVYEGVWK (SEQ ID NO: 4), SEQ ID NO: 5), PPFYIITE (SEQ ID NO: 6), PPFYIIIE (SEQ ID NO: 7), KQSSKALQRPVASDF (SEQ ID NO: 8), EEALQR (SEQ ID NO: 9), and TGQIWPNDGEGAFHGDAEALQR (SEQ ID NO: 10). to provide.

In the above peptide, the sequences of SEQ ID NOs: 1 and 2 are preferably phosphorylated, and the greeted peptide is more preferably LGGGQpYGEVYEGVWK, LGGGQYGEVpYEGVWK, LGGGQpYGEVpYEGVWK, or LGGGQHGEVpYEGVWK (p represents phosphorylation), but is not limited thereto.

In addition, the present invention provides a peptide set for quantitative measurement of one or more Bcr-Abl hybrid proteins selected from the group consisting of KQSSKALQRPVASDF (SEQ ID NO: 8), and EEALQR (SEQ ID NO: 9), and TGQIWPNDGEGAFHGDAEALQR (SEQ ID NO: 10).

In addition, the present invention is LGGGQHGEVYEGVWK (SEQ ID NO: 1), LGGGQYGEVYEGVWK (SEQ ID NO: 2), LGGGQFGEVYEGVWK (SEQ ID NO: 3), LGGGQYGVVYEGVWK (SEQ ID NO: 4), LGGGQYGK (SEQ ID NO: 5), PPFYIITE (SEQ ID NO: 6) and PPFYIITE (SEQ ID NO: 6). A set of peptides for quantitative measurement of drug resistance-causing mutations occurring in at least one Abl or Bcr-Abl hybrid protein selected from the group consisting of number 7) is provided.

In addition, the present invention provides a peptide set for quantitative measurement of the degree of phosphorylation of one or more Abl or Bcr-Abl hybrid proteins selected from the group consisting of peptides in which the tyrosine residues of the peptide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 are phosphorylated. The greeted peptide is more preferably LGGGQpYGEVYEGVWK, LGGGQYGEVpYEGVWK, LGGGQpYGEVpYEGVWK, or LGGGQHGEVpYEGVWK (p indicates phosphorylation), but is not limited thereto.

In one embodiment of the present invention, valine (Val,V), asisoleucine (Ile, I), lysine (Lys, K), or leucine (Leu, L) of the peptide sequence is preferably substituted with an isotope. However, it is not limited thereto.

In another aspect, the present invention comprises the steps of: a) separating a target protein from a sample; b) treating the protein with a protease; And c) mass spectrometry of the treated peptides, and a method for quantifying a Bcr-Abl hybrid protein by adding the peptide set of the present invention thereto is provided.

In one embodiment of the quantification method of the present invention, the target protein is the Bcr-Abl hybrid protein, and the molecular weight is preferably about 200 kDa or more, more preferably p190, depending on the various exon combinations b3a2, b2a2, and e1a2 of the two proteins. p210, or p230, but is not limited thereto.

In addition, the present invention comprises the steps of: a) separating a target protein from a sample; b) treating the protein with a protease; And c) mass spectrometry of the treated peptides, and adding the peptide set of the present invention to the Abl mutant protein quantification method.

The target protein is referred to as p145 because the molecular weight of the Abl protein that normally exists in cells where hybridization has not occurred is about 145 kDa.

In one embodiment of the quantification method of the present invention, the proteolytic enzyme is preferably trypsin or Glu-C, but is not limited thereto.

The term "leukemia" as used herein includes, but is limited to, chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL), particularly Philadelphia-chromosome positive acute lymphocytic leukemia (Ph+ ALL) and imatinib-resistant leukemia. It doesn't work. Preferably, this

The modified form of leukemia to be treated by the methods disclosed herein is CML.

"Gleevec(imatinib)-resistant leukemia" as used herein, in particular, defines leukemia in which imatinib is no longer therapeutically effective or has a reduced therapeutic effect.

Among the peptide sequences used in the present invention, valine (Val, V), asisoleucine (Ile, I) lysine (Lys, K), or leucine (Leu, L) is well known in the art. , In Korean Patent Application Publication No. 2001-0108475, site-specific isotope-enriched amino acids such as leucine, isoleucine, lysine and valine biochemical precursors, as well as site-specific isotope-enriched amino acids The content that provides itself is known in detail.

Glu-C used in the present invention is a serine protease that selectively cleaves a peptide bond at the C-terminus of a glutamic acid residue, and is also referred to as Staphylococcus aureus Protease V8.

Hereinafter, the present invention will be described.

The present invention performed the quantification of the Bcr-Abl hybrid protein, which is the etiology of the most aggressive and fatal chronic myelogenous leukemia (CML) using the method of the present invention.

In CML, translocation of mutually balanced chromosomes in hematopoietic stem cells (HSC) produces the Bcr-Abl hybrid gene.

The Bcr-Abl gene is a gene found in patients with positive Philadelphia chromosomes that are derived from the reciprocal shift between chromosomes 9 and 22 in humans. Ninety-five percent of patients with chronic myelogenous leukemia test positive for the Philadelphia chromosome. This Philadelphia chromosome is composed of a translocation of the long arm of chromosome 22 and the long arm of chromosome 9.

The ABL gene, a proto-oncogene on chromosome 9, binds to the BCR on chromosome 22 to form a Bcr-Abl fusion gene, and leukemia is caused by the protein produced therein. This rearrangement of the Bcr-Abl gene is found in all erythrocytes, granulocyte-monocytic, and platelet-based immature cells.

The diagnosis of chronic myelogenous leukemia is made by detection of the Philadelphia chromosome (Philadelphia chromosome) or the Bcr-Abl gene, which is derived from a translocation between chromosomes 9 and 22. The Bcr-Abl gene produces different fusion proteins of various sizes due to the breakpoints present in the Bcr gene. It is the p210 or p190 proteins made from the BCR-ABL oncogene that play a specific role in the pathogenesis of chronic myelogenous leukemia, and they play a crucial role in cell proliferation and cancerous changes. In addition, the difference in prognosis between acute and chronic myelogenous leukemia is thought to be due to the difference in the size of these cancer proteins. Looking at the types of Bcr-Abl mRNA that have been specifically identified to date, in general, the Bcr gene has three breakpoints. Each is divided into major (M-bcr: b2a2, b3a2-P210), minor (m-bcr: e1a2-P190), and micro (μ-bcr: e19a2(or c3a2)-P230). More than 95% of patients with Philadelphia chromosome-positive chronic myelogenous leukemia have a breakpoint in the M-bcr area. Two important breakpoints are b2a2 (e13a2) and b3a2 (e14a2), and their fusion mRNAs produce the p210Bcr-Abl protein. The breakpoint e1a2 binding site in the m-bcr segment triggers the production of a smaller p190 Bcr-Abl protein, in two-thirds of patients with acute lymphocytic leukemia (ALL), and very rare chronic myelogenous leukemia and acute myeloid disease. It appears in people with leukemia. In some cases, a breakpoint between Bcr exon19 and 20 present in the μ-bcr region produces a larger p230 Bcr-Abl hybrid protein. In addition to these breakpoints, transcripts of the Bcr-Abl hybrid protein with other binding sites are known to have been reported in several rare cases. As a result, if there is a Bcr-Abl fusion, you will necessarily develop chronic myelogenous leukemia.

The Bcr-Abl hybrid gene encodes the oncogene Bcr-Abl fusion protein. Abl encodes a tightly regulated protein tyrosine kinase, which plays a fundamental role in regulating cell proliferation, adhesion and apoptosis, while the Bcr-Abl fusion gene encodes a constitutively activated kinase, which is deregulated. Clonal proliferation, transforming HSCs to produce a phenotype that exhibits a reduced ability to attach to bone marrow lipids, reducing the apoptosis response to mutagenic stimuli, which makes it possible to accumulate more malignant transformations in progress. do. The resulting granulocytes do not develop into mature lymphocytes and are released into the circulatory system, which leads to a deficiency of mature cells and an increased susceptibility to infection. Bcr, which prevents the kinase from activating the mitogenic and anti-apoptotic pathways (e.g., P-3 kinase and STAT5), causing the death of Bcr-Abl phenotype cells, thereby providing an effective therapy for CML. ATP-competitive inhibitors of -Abl are described.

In May 2001, N-{5-[4-(4-methyl-piperazino-methyl)-benzoylamido]-2-methylphenyl}-4-(3-pyridyl)-2-pyrimidine- The mesylate salt of amine (imatinib mesylate, STI571, hereinafter referred to as'Gleevec®') has been approved by the FDA for the treatment of CML in patients who have failed treatment from IFN-alpha therapy.

Imatinib (formerly STI571, Gleevec®), a selective tyrosine kinase inhibitor, is used for platelet-derived growth factor receptor (PDGF) alpha and beta and human stem cell factor (SCF) c-kit. It has been known to block phosphorylation of tyrosine residues by occupying the ATP binding site of the receptor as well as the Abl tyrosine kinase Bcr-Abl, c-Abl, v-Abl and Abl-related genes (ARG). Based on numerous studies of chronic myelogenous leukemia (CML), including studies of patients in early chronic stage (CP), advanced stage (AP), and myeloblastosis (BC), imatinib is a novel drug for chronic myelogenous leukemia. It is considered a reliable standard of care. Additionally, imatinib is a tumor entity that exhibits constitutive activation of c-kits, gastrointestinal stromal tumors (GIST).

In individuals suffering from myeloproliferative disease, and in patients whose PDGF-R-beta gene on chromosome 5q33 is rearranged, a sustained response is induced.

Despite these promising results, especially in CP, resistance to imatinib often occurs in AP and BC, and the remission generally lasts only for 6 to 12 months. Therefore, there is an urgent need to ensure a synergistic treatment strategy, especially in the case of late stage disease.

To further enhance therapeutic success, there is a need to establish new screening strategies for synergistic treatment approaches. Differential protein expression analysis of imatinib-treated cell lines, as compared to untreated Bcr-Abl positive cell lines, is regulated by Bcr-Abl expression and potentially serves as a novel target for synergistic therapeutic intervention. It is used for the purpose of detecting proteins.

Chronic myelogenous leukemia is a type of hematologic cancer, and the tyrosine kinase activity of the Bcr-Abl hybrid protein is always enhanced. It is reported that the level of expression of the Bcr-Abl hybrid protein in CML cells and its transition to the acute phase are closely related. Bcr-Abl hybrid proteins exist as 8 or more different hybrid proteins by translocation between Bcr exon b2, b3, b19 on chromosome 22 and Abl exon a2, a3 on chromosome 9.

With the introduction of Gleevec, one of the molecular target treatments for CML, the mortality rate was greatly reduced to less than 2%, but if this drug was administered for a long time. Mutations in the Bcr-Abl protein result in a significant decrease in sensitivity to the drug, which worsens the disease.

The most common Gleevec resistance in Korean CML patients is formed by the T315I and Y253H point mutations of the Bcr-Abl hybrid protein (FIG. 2), and the expression level of this mutant protein was quantitatively analyzed.

Bcr-Abl hybridization using conditions obtained by synthesizing a standard peptide corresponding to the Bcr-Abl mutant protein and studying the properties seen in the mass spectrometer of the standard peptides, and using CML cell lines K562 and BaF3 cells expressing the Bcr-Abl protein. Ex-vivo analysis conditions were established for quantitative analysis of proteins and mutant proteins.

By analyzing the correlation between the onset and prognosis of CML by quantitatively analyzing the expression level of Bcr-Abl protein in the bone marrow samples of patients diagnosed with CML using this analysis method, it enables early selection of the treatment appropriate for the patient's prognosis. I want to improve the survival rate.

The present invention will be described in detail.

In the present invention, by selecting a specific amino acid sequence of a Bcr/Abl hybrid protein, valine (Val, V), asisoleucine (Ile, I), or leucine (Leu, L) contained in the sequence is replaced with an isotope for quantification. Standard peptides are synthesized. The synthesized peptide was constructed based on the peptide amino acid sequence that can be generated when a target protein present in a biological sample is treated with a characteristic protease such as trypsin. The synthesized standard peptide has the same physical properties as seen in other mass spectrometers and HPLC, except for the peptide and molecular weight that can be produced from the target protein. Using the synthesized standard peptide, the analysis conditions of the peptide in HPLC and the analysis conditions in a mass spectrometer (ionization energy, ms/ms conditions, CID conditions (collison induced dissociation)) are optimized. Using the established optimal analysis conditions, a Selective Reaction Monitoring (SRM) analysis method is established.

The method of quantitatively analyzing the cancer-related proteins (hybrid proteins) and mutant types of the proteins presented through the present invention in the femtomole range using mass spectrometry is very original, and is a very excellent result that has not been reported at all. It is expected to be used extensively in tumor biology research.

In addition, it has the effect of performing clinical diagnosis and prognosis by opening the possibility of researching the temporal analysis of clinical patients (from the time of onset to during treatment, before and after the time of onset of resistance).

1 is a diagram illustrating a quantitative analysis process of Bcr-Abl, a protein that causes chronic myelogenous leukemia, using an isotope dilution method.
2 is a diagram showing a Bcr-Abl hybrid protein, which is a CML etiology, and a Bcr-Abl mutant protein produced by Gleevec resistance.
3 is a grip for establishing the conditions for mass spectrometry of the AQAU peptide (for example, T315I peptide). The spectrum on the left is the precursor ion, and the spectrum on the right is the fragment ion (@, Collision energy).
FIG. 4 shows 100ug (microgram), 50ug, 25ug, and 10ug of proteins of the BaF3 cell line, respectively, separated by SDS-PAGE. The hybrid protein has a molecular weight of 210 kDa and a normal protein (Wild type) of 145 kDa.
5 is a quantitative analysis of Bcr-Abl hybrid protein using the K562 cell line. The lower panel is the Bcr-Abl hybrid protein present in the K562 cell line, and the upper panel is the spectrum of the synthetic peptide when 1 pmol/ul of B3A2 synthesized in the K562 cell line is added.
6 is a quantitative analysis of a Bcr-Abl hybrid type mutant protein using a BaF (Y253H type) cell line. The lower panel is the Bcr-Abl hybrid protein present in the K562 cell line, and the upper panel is the spectrum of the synthesized peptide when 1 pmol/ul of the Y253H peptide synthesized in the BaF cell line was added.
7 shows the quantification curve of the T315I standard peptide.
8 is an analysis of the amount of T315I mutant protein and T315T (Wild type) protein present in the BaF3 cell line through mass spectrometry.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are for explaining the present invention, and the scope of the present invention should not be construed as being limited by the following examples.

Example 1: Standard for quantitative analysis Peptide Synthesis and Characterization Study

A standard peptide for quantitative analysis to quantify the phosphorylation of Y253H and T315I and Y253 and Y257 mutants of Abl resistant to the combination of b3a2,b2a2,e1a2 and Gleevec, a CML treatment, with various exon combinations of the hybrid protein Bcr-Abl that causes CML. Was devised (Table 1).

When designing most standard peptides for quantitative analysis, most of them are selected as peptides produced by trypsin enzymes, but trypsin is cut off the C-terminus of basic amino acids (Lysine, Arginine), which creates a cation per peptide fragment. Because side branches are included, they not only help ionization but also enable quantification. However, in the case of the standard peptide for T315I mutation analysis, when trypsin is used, the length of the peptide is lengthened and it is too hydrophobic, so the resolution and retention time in LC are unstable and the ionization efficiency is not good, so Glu-C enzyme It was produced using.

Table 1 shows the design and synthesis of standard peptides for quantitative measurement of Bcr-Abl hybrid and mutant proteins. [p, phosphorylation (mass shift of +79 Da) on tyrosine; *, isotope labeled 13C/15N]

Example 2 : Synthesized standard Peptide Establish mass spectrometry

Mass spectrometry conditions were established using the standard peptides shown in Table 1 above. Using the synthesized standard peptide direct injection method (syringe), m/z of the precursor ion of the standard peptide is selected, and fragmentation is performed with an appropriate energy for the selected ion, and the ms/ms condition of the fragment ion showing the strongest signal among them Was established (Fig. 3). Selective reaction monitoring (SRM), an analysis method for observing fragmentation of selective precursor ions, was determined using the obtained ms/ms condition.

After establishing the AQUA peptide analysis conditions, the'Limit Of Detection (LOD)'and'Limit Of Quantitation (LOQ)' for each peptide were analyzed by LC-MS (Table 2). Since each peptide has a different sequence and length, the degree of ionization is different, so the concentration that can be measured by mass is different.

Table 2 shows the minimum detectable concentration (Limit Of Dectection, LOD) and the minimum quantifiable concentration (Limit Of Qauntication, LOQ) according to the AQUA peptide type by mass spectrometry.

Example 3 : Separation of samples ( In - gel digestion )Wow LC - MS / MS analysis

Leukemia cell lines (K562, BaF3) 1X10 ⁷ or the corresponding patient sample proteins were isolated from SDS-PAGE Gel (4-12% Bis-Tris Gel, invitrogen) (Fig. 4), and the target protein (p210, p145) site Cut out. After removing the dyed Coomassie blue for mass analysis for protein concentration and visualization in the cut gel, dehydration the gel using 100% Acetonitrile (ACN). Trypsin or Glu-C enzyme is added to the dehydrated gel. At this time, the synthesized AQUA peptide is diluted to a concentration of 1 pmol/ul and added together. Incubate this sample overnight at 37°C. To extract the protein in the gel, add the harvest solution (50%ACN/5% formic acid) to the incubated gel. The sample thus obtained (protein cut by the enzyme to form a peptide form) is stored after drying the solvent completely. After dissolving the completely dried sample with 3ul of solvent A (Water/1% formic acid) for mass analysis, it was analyzed by nano-flow LC-MS. For sample analysis, Ultimate 3000 system (Dionex) HPLC and Thermo's LTQ linear ion trap mass spectrometry were used. Samples were separated with a 12 cm long homemade analytic column filled with C _{18 (5 um).}

Example 4 : Mass Quantitative analysis of Bcr - Abl hybrid protein and resistant protein through spectrometry

Using the synthesized standard peptide, mutations of the oncogenes Bcr-Abl and Abl were quantitatively analyzed (Ref. AQUA quantitative analysis).

Example 4 -One: Bcr - Abl Quantitative analysis of hybrid proteins of ( b3a2 type )

Using the determined LC-SRM-MS/MS method, the amount of Bcr-Abl hybrid protein was confirmed in the cell line (K562) (FIG. 5). In the case of the leukemia cell line K562, there is a protein form of b3a2 among Bcr-Abl hybrid proteins, and from the ratio of 1 pmol of b3a2 standard peptide area, it was found that 3.4 fmol (650 femtogram) of b3a2 hybrid protein out of a total of 25 ug proteins. Could.

Using this analysis method, a quantitative analysis of Bcr-Abl hybrid protein was performed using bone marrow samples from CML patients.In this case, the b2a2 hybrid protein was 18.5 fmol (3.7 picogram) of a total of 120 ug proteins through the AQUA assay. It was found that this exists (Table 3).

For reference, the AQUA ( Absolute Quantification of Protein ) method quantitative analysis will be briefly described.

AQUA ( Absolute Quantification of protein) Law Professor Steve Gygi group of Harvard Medical School in 2003, he developed a method to quantitatively measure the degree of the transfer After a formula that exists at a specific location of a desired protein and the expression levels of proteins present in the cell. This method selects a tryptic peptide having an amino acid sequence specifically present in the protein to be measured, and synthesizes an AQUA peptide containing an isotope substituted amino acid (C13/N15-Ile, Leu, Val. Pro). The synthesized peptide has the same characteristics except for the protein and mass to be analyzed present in the cell. After investigating the physicochemical properties of the synthesized AQUA peptide in an LC-Mass spectrometer, administer a predetermined amount (usually 500 femto mole) to a biological sample, This is a method of measuring the amount of protein present in the sample to be analyzed by measuring the relative amount of peak. This method can measure even hundreds of atto-moles of protein in the case of partially purified protein, and in the case of a protein mixture, it has a very good selectivity of 3 ppm (measures the amount of three proteins present in a mixture of 3,000,000 proteins) It is a protein quantitative analysis method. This analysis method is more excellent than the Western Analysis method, which is the most excellent protein analysis method, and it is an analysis method that has a sensitivity almost similar to that of the mRNA expression analysis method using real-time PCR. In addition, this assay is also applied to identify and quantify post-transcriptional modifications indicating protein activity.

Table 3 is an analysis of the amount of Bcr-Abl hybrid protein present in the patient sample and the BaF3 cell line.

Example 4-2: Abl or Bcr - Abl mutant ( Y253H , T315I ) protein showing Gleevec resistance

Example 4-2-1: Y253H Mutant protein

The analysis range of the quantitative assay was determined using BaF3_Y253H, a cell line expressing the Y253H mutant Bcr-Abl (FIG. 6). After the Y253H standard peptide was added to the cell sample, the Y253H mutant protein was quantified, and 9.92 fmol (1.8 ng) of the total 100 ug protein was present. Among CML patients, samples that showed Gleevec resistance due to the Y253H mutation in the Bcr-Abl hybrid protein were analyzed (Table 4). As a result of the measurement, it was confirmed that this assay showed quantitative properties within the range of attomole to picomole.

Table 4 is a quantitative analysis of the Bcr-Abl mutant protein using the bone marrow of a CML patient.

Example 4-2-2: T315I Mutant protein

As a result of measuring the assay sensitivity of the standard peptide of T315I produced in a new way, excellent quantitation was shown even at a concentration lower than 10 femtomoles (FIG. 7).

Compared to the standard peptide produced by trypsin, the peptide produced by Glu-C enzyme can overcome the hydrobphobic property, so that the resolution in LC and the ionization efficiency in ESI are increased. After establishing the SRM condition with the standard peptide, the T315I mutant protein present in the leukemia cell line BaF3-T315I was quantified. The newly established LC_SRM-MS/MS analysis method shows good retention time under the concentration gradient conditions suggested in the experimental method. 495 femtomoles of T315I mutation and 7 femtomoles of T315T (wild type) protein were quantified in 200 ug of total cell lysate of cells (FIG. 8).

<110> Konkuk University Industrial Cooperation Corp. <120> Development of diagonstic and prognostic methods for chronic myelodal leukemia by measurement of expression levels of Bcr/Abl and drug-resistant Bcr/Abl mutants with quantitiative mass spectrometry <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 1 Leu Gly Gly Gly Gln His Gly Glu Val Tyr Glu Gly Val Trp Lys 1 5 10 15 <210> 2 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 2 Leu Gly Gly Gly Gln Tyr Gly Glu Val Tyr Glu Gly Val Trp Lys 1 5 10 15 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 3 Leu Gly Gly Gly Gln Phe Gly Glu Val Tyr Glu Gly Val Trp Lys 1 5 10 15 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 4 Leu Gly Gly Gly Gln Tyr Gly Val Val Tyr Glu Gly Val Trp Lys 1 5 10 15 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 5 Leu Gly Gly Gly Gln Tyr Gly Lys 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 6 Pro Pro Phe Tyr Ile Ile Thr Glu 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 7 Pro Pro Phe Tyr Ile Ile Ile Glu 1 5 <210> 8 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 8 Lys Gln Ser Ser Lys Ala Leu Gln Arg Pro Val Ala Ser Asp Phe 1 5 10 15 <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 9 Glu Glu Ala Leu Gln Arg 1 5 <210> 10 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 10 Thr Gly Gln Ile Trp Pro Asn Asp Gly Glu Gly Ala Phe His Gly Asp 1 5 10 15 Ala Glu Ala Leu Gln Arg 20

Claims (9)

A composition for quantitative determination of Bcr-Abl hybrid protein comprising at least one peptide selected from the group consisting of KQSSKALQRPVASDF (SEQ ID NO: 8), EEALQR (SEQ ID NO: 9), and TGQIWPNDGEGAFHGDAEALQR (SEQ ID NO: 10). According to claim 1, Valine (Val, V), isoleucine (Ile, I), lysine (Lys, K) or leucine (Leu, L) in the peptide sequence is Bcr, characterized in that substituted with isotopes -Abl composition for quantitative determination of hybrid proteins. A composition for quantitative determination of Bcr-Abl hybrid protein consisting of KQSSKALQRPVASDF (SEQ ID NO: 8), EEALQR (SEQ ID NO: 9), and TGQIWPNDGEGAFHGDAEALQR (SEQ ID NO: 10) peptides. 4. The Bcr of claim 3, wherein valine (Val, V), isoleucine (Ile, I), lysine (Lys, K) or leucine (Leu, L) in the peptide sequence is substituted with an isotope. -Abl composition for quantitative determination of hybrid proteins. a) separating the target protein from the sample;
b) treating said protein with a protease; And
c) mass spectroscopy the treated peptides,
Bcr-Abl hybrid protein quantification method of adding the peptide composition of claim 1 in this process. The method of claim 5, wherein the target protein is p190, p210, or p230.  The method of claim 5, wherein the proteolytic enzyme is trypsin or Glu-C. A kit for quantitative determination of Bcr-Abl hybrid protein comprising at least one peptide selected from the group consisting of KQSSKALQRPVASDF (SEQ ID NO: 8), EEALQR (SEQ ID NO: 9), and TGQIWPNDGEGAFHGDAEALQR (SEQ ID NO: 10). According to claim 8, Valine (Val, V), isoleucine (Ile, I), lysine (Lys, K) or leucine (Leu, L) of the peptide sequence is Bcr, characterized in that substituted with isotopes Kit for quantitative determination of Abl hybrid protein.

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