CN118483346A - A group of markers for individualized monitoring of M protein in multiple myeloma patients and their application - Google Patents

Abstract

The present invention provides a group of markers and applications for individualized monitoring of M protein in multiple myeloma patients. The present invention belongs to the field of biomedical technology. The present invention uses the complete light chain of M protein with a specific mass-to-nuclear ratio as a detection marker, firstly uses a separation reagent to capture and separate the complete light chain of M protein in a plasma sample, uses a detection reagent and a detection device to detect the captured target sample, determines the molecular weight of the captured sample, and accurately quantifies the complete light chain of M protein by adding an internal standard, thereby determining the molecular weight and content of different M proteins, and realizing accurate personalized qualitative and quantitative analysis of different multiple myeloma patients.

Description

A set of markers for individualized monitoring of M protein in multiple myeloma patients and their applications

Technical Field

The present invention relates to the field of biomedical technology, and more specifically to a group of markers for individualized monitoring of M protein in multiple myeloma patients and applications thereof.

Background Art

Multiple Myeloma (MM) is a common hematological malignancy, the main characteristics of which are: abnormal proliferation of monoclonal plasma cells in the bone marrow. An abnormal protein secreted by monoclonal plasma cells, M protein, will appear in the serum or urine of MM patients.

Accurate monitoring of the micro-residues of M protein in the body is crucial for the individualized diagnosis, disease monitoring, treatment guidance, and prognosis and prognosis evaluation of MM, mainly including the following aspects: 1. Disease monitoring: The detection of micro-residues of M protein can help doctors monitor the treatment effect and disease progression. As the treatment progresses, the level of M protein should gradually decrease. If the level continues to rise, it may indicate disease progression or inappropriate treatment. 2. Guide treatment: According to the changes in the level of M protein, doctors can adjust the treatment plan, including changing the type of drug, dosage or treatment strategy, to better control the disease. 3. Predict prognosis: Changes in the level of M protein can help doctors predict the progression rate and prognosis of the disease, and help to develop a more reasonable treatment plan and lifestyle management. 4. Early detection of recurrence: By regularly testing the micro-residues of M protein, signs of disease recurrence can be detected early, which helps to take timely measures. In general, the detection of micro-residues of M protein is very important for the treatment and management of patients with multiple myeloma. It can provide key information to guide the medical team to develop the best treatment plan and improve the quality of life and prognosis of patients.

At present, there are many methods for detecting M protein, such as serum protein electrophoresis (SPE), immunofixation electrophoresis (IFE) and serum free light chain (sFLC). However, these methods have certain limitations, such as low sensitivity, low specificity, and cumbersome operation. Especially for free light chain detection, there is no existing technology and corresponding markers for quantitative detection of complete light chains of M protein based on MALDI-TOF-MS technology.

Therefore, how to develop a group of markers for individualized monitoring of M protein in multiple myeloma patients and their application is a technical problem that technical personnel in this field urgently need to solve.

Summary of the invention

In view of this, the present invention provides a group of markers for individualized monitoring of M protein in multiple myeloma patients and their applications, selects a specific mass-to-nuclear ratio of the complete light chain of the M protein as a detection marker, uses a separation reagent to separate the complete light chain of the M protein, uses a specific detection reagent and detection equipment to determine the precise molecular weight and content of the complete light chain of the M protein, and then realizes accurate personalized qualitative and quantitative analysis of different multiple myeloma patients according to the molecular weight and content of different M proteins.

In order to achieve the above object, the present invention adopts the following technical solution:

A set of markers for individualized monitoring of M protein in multiple myeloma patients, including complete light chains of M protein with specific mass-to-nuclear ratios, wherein the molecular weight m/z of the complete light chains of M protein, [M+H]+: 23243.359±5Da, 23218.878±5Da and 23139.142±5Da.

Another object of the present invention is to provide: application of the above-mentioned marker for detecting M protein in multiple myeloma patients in the detection of M protein.

Another object of the present invention is to provide: a detection system for the complete light chain of the M protein, comprising a separation reagent, a detection reagent and a detection device for the complete light chain of the M protein.

Preferably, the separation reagent comprises a PBS buffer solution, a capture microsphere solution prepared by mixing Capture Select Kappa XL and CaptureSelect LC-Lambda in a volume ratio of 1:1, and a TECP elution solution.

Preferably, the TECP elution solution is prepared with 1% formic acid, and the concentration of the TECP solution is 40-50 mM.

Preferably, the detection reagent includes KTI standard working solution.

Preferably, the preparation process of the KTI standard working solution is as follows: Kunitz soybean trypsin inhibitor powder is dissolved in 1% formic acid solution to prepare a KTI standard working solution with a concentration of C1 mg/mL.

Preferably, the detection equipment comprises a MALDI-TOF-MS detector.

Preferably, the use process of the detection system is as follows:

(1) Take a certain volume of plasma sample to be tested, add PBS buffer and mix thoroughly to obtain a mixed solution;

(2) adding the capture microsphere solution to the mixed solution of step (1) and shaking to allow it to react fully;

(3) After the reaction is completed, the supernatant is removed by low-speed centrifugation, the microsphere precipitate is retained, washed, eluted by oscillation, and centrifuged at high speed to obtain the supernatant to obtain the M protein complete light chain eluate;

(4) mixing the eluate with a KTI standard working solution having a concentration of C1 mg/mL to prepare a spiked working solution, and performing MALDI-TOF-MS detection on the spiked working solution to determine the molecular weight of the target molecule;

(5) The KTI mass spectrometry peak intensity (S1) and the IMLC peak intensity (S2) were obtained by software, and the IMLC concentration was calculated as C2 = 40*(S2/S1)/C1 mg/mL.

Another object of the present invention is to provide: application of the system in the detection of the molecular weight of the complete light chain of the M protein and/or the quantitative detection of the complete light chain of the M protein.

It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

The present invention uses the complete light chain of the M protein with a specific mass-to-nuclear ratio as a detection marker. First, a separation reagent is used to capture and separate the complete light chain of the M protein in the plasma sample, and a detection reagent and a detection device are used to detect the captured target sample to determine the molecular weight of the captured sample. The complete light chain of the M protein is accurately quantified by adding an internal standard, and then the molecular weight and content of different M proteins are determined, thereby achieving accurate personalized qualitative and quantitative analysis of different multiple myeloma patients.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

Figure 1 shows the mass spectra of M proteins of three patients with multiple myeloma, where A, B and C represent different patients, and 1, 2 and 3 represent before treatment, one cycle of treatment and two cycles of treatment, respectively.

DETAILED DESCRIPTION

The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1

A detection system for the complete light chain of an M protein, comprising a separation reagent, a detection reagent and a detection device for the complete light chain of an M protein;

The separation reagent includes a PBS buffer solution, a capture microsphere solution prepared by mixing Capture Select KappaXL and Capture Select LC-Lambda in a ratio of 1:1, and a TECP elution solution prepared by 1% formic acid and having a concentration of 50 mM;

The detection reagent includes KTI standard working solution;

The preparation process of KTI standard working solution is as follows: Kunitz soybean trypsin inhibitor powder is dissolved in 1% formic acid solution to prepare a KTI standard working solution with a concentration of C1 mg/mL. The 1% formic acid solution is prepared by mixing formic acid (mass spectrometry grade) and pure water in a ratio of 1% formic acid to 99% pure water.

Kunitz type soybean trypsin inhibitor, containing 216 amino acids, with a molecular weight of about 20 kDa, and an amino acid sequence of: MKSTIFFLFLFCAFTTSYLPSAIADFVLDN EGNPLENGGT YYILSDITAFGGIRAAPTGNERCPLTVVQSRNELDKGIGTIISSPYRIRFIAEGHPLSLKFDSFAVIMLCVGIPTEWSVVEDLPEGPAVK IGENKDAMDGWFRLERVSDDEFNNYKLVFCPQQAEDDKCGDIGISIDHDDGTRRLVVSKNKPLVVQFQKLDKESLAKKNHGLSRSE, SEQ ID NO. NO1;

The detection equipment includes a MALDI-TOF-MS detector.

Embodiment 2:

Application Example 1 Detection System for Individualized Multiple Myeloma M Protein Complete Light Chain Detection

1. Method for separating the complete light chain of M protein in plasma

(1) 20 μL of plasma was collected from three multiple myeloma patients (before treatment, 1 cycle of treatment, and 2 cycles of treatment), placed in a 0.6 mL centrifuge tube, and 160 μL of 10 mM PBS was added, followed by vortex mixing to dilute the plasma;

(2) Add 20 μL of Capture Select KappaXL and Capture Select LC-Lambda capture microspheres mixed in a 1:1 ratio and shake for 30 min to capture the complete light chain of M protein in plasma;

(3) After the reaction is completed, centrifuge at low speed, discard the supernatant, add 200 μL of 10 mM PBS buffer for three washes, and then wash with 200 μL of pure water for three washes. After each wash, centrifuge at low speed to retain the microsphere precipitate and remove non-specifically bound impurities;

(4) Add 20 μL of 50 mM TECP solution (prepared with 1% formic acid) to the washed microsphere precipitate to elute the complete light chain of the M protein on the microsphere;

2. Mass spectrometry detection of the complete light chain of the personalized M protein

(1) Dissolve KTI (Kunitz soybean trypsin inhibitor) powder in 1% formic acid solution to prepare a KTI standard working solution with a concentration of C1 mg/mL, add 20 μL of C1 mg/mL KTI standard working solution to 20 μL of IMLC eluent, mix well, and obtain a spiked working solution;

(2) 1 μL of the spiked working solution was mixed with 1 μL of 20 mg/mL α-cyano-4-hydroxycinnamic acid solution, spotted on the MTP AnchorChipTM target, evaporated at room temperature, and then analyzed by mass spectrometry;

(3) Detection instrument: MALDI-TOF MS, equipped with matrix-assisted laser desorption ionization source, flexControl 3.0.0 data acquisition software and flexAnalysis 3.3 (Bruker Daltonics) data processing software; Mass spectrometry conditions: using SmartBeam-II laser system, positive ion mode.

3. Qualitative and quantitative analysis of the complete light chain of the individualized M protein

(1) Qualitative analysis: The mass spectrum was calibrated using the precise molecular weight of the KTI molecule (19965.0630 Da) to obtain the accurate molecular weight of the IMLC molecule. The results are shown in FIG1 .

(2) Quantitative analysis: The KTI mass spectrum peak intensity (S1) and IMLC peak intensity (S2) in the MALDI TOF MS results were read by flexAnalysis software, and the IMLC concentration C2 = 40*(S2/S1)/C1 mg/mL was calculated. In this experiment, the C1 concentration was 10 mg/mL, and the quantitative results are shown in Table 1.

Table 1 Mass spectrometry results of complete light chain of M protein in 3 patients

Result analysis: Through the test, the molecular weight m/z of the complete light chain of the M protein with a specific mass-to-nuclear ratio in patients A, B and C was determined, [M+H]+: 23243.359±5Da, 23218.878±5Da and 23139.142±5Da. It can be used for the detection of M protein in patients A, B and C.

In this embodiment, the amount of complete light chain of M protein of patient A shows a downward trend with the treatment cycle, indicating that the patient's condition is effectively alleviated to varying degrees during the treatment process. Patient B shows a trend of first decreasing and then increasing, indicating that the possibility of relapse of the patient increases during the treatment process. Patient C shows a trend of first decreasing and then increasing, and the initial amount of complete light chain of M protein of the patient is high. After the second cycle of treatment, the amount of complete light chain of M protein increases again, indicating that the patient's condition worsens.

The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. A set of markers for the personalized monitoring of M proteins in patients with multiple myeloma, comprising a specific mass to core ratio of M protein complete light chain having a molecular weight M/z, [ m+h ] +: 23243.359.+ -. 5Da, 23218.878.+ -. 5Da and 23139.142.+ -. 5Da.

2. The use of the marker for detecting M protein of a patient with multiple myeloma according to claim 1 in the detection of M protein.

3. A detection system for an M protein complete light chain, which is characterized by comprising a separation reagent, a detection reagent and a detection device for the M protein complete light chain.

4. The system for detecting the complete light chain of M protein according to claim 3, wherein the separation reagent comprises a capture microsphere solution and a TECP elution solution prepared by mixing PBS buffer solution, capture SelectKappaXL and Capture Select LC-Lambda according to a volume ratio of 1:1.

5. The system for detecting the complete light chain of M protein according to claim 4, wherein the TECP elution solution is prepared from 1% formic acid, and the concentration of the TECP solution is 40 mM to 50mM.

6. The system for detecting an intact light chain of M protein of claim 5, wherein said detection reagent comprises KTI standard working fluid.

7. The system for detecting the complete light chain of the M protein according to claim 6, wherein the KTI standard working solution is prepared by the following steps: the Kunitz soybean trypsin inhibitor powder is taken and dissolved in 1% formic acid solution to prepare KTI standard working solution with the concentration of C1 mg/mL.

8. The system for detecting an intact light chain of M-protein of claim 7, wherein the detection device comprises a MALDI-TOF-MS detector.

9. The system for detecting the complete light chain of M protein of claim 8, wherein said detection system is used as follows:

(1) Taking a certain volume of plasma sample to be detected, adding PBS buffer solution, and fully mixing to obtain mixed solution;

(2) Adding the captured microsphere solution into the mixed solution in the step (1), and oscillating to fully react;

(3) After the reaction is finished, removing supernatant by low-speed centrifugation, retaining microsphere sediment, washing, oscillating and eluting, centrifuging at high speed, and taking supernatant to obtain M protein complete light chain eluent;

(4) Mixing the eluent with KTI standard working solution with the concentration of C1mg/mL to prepare a labeling working solution, carrying out MALDI-TOF-MS detection on the labeling working solution, and determining the molecular weight of target molecules;

(5) KTI mass spectrum peak intensity (S1) and IMLC peak intensity (S2) were obtained by software and IMLC concentration c2=40 (S2/S1)/C1 mg/mL was calculated.

10. Use of the system according to any one of claims 3-9 for the molecular weight detection of M protein intact light chains and/or for the quantitative detection of M protein intact light chains.