CN118033014A - Single-cell proteomics pretreatment method, kit and application thereof in HSPE, TGS1 and MDH1 target discovery - Google Patents

Single-cell proteomics pretreatment method, kit and application thereof in HSPE, TGS1 and MDH1 target discovery Download PDF

Info

Publication number
CN118033014A
CN118033014A CN202410049094.5A CN202410049094A CN118033014A CN 118033014 A CN118033014 A CN 118033014A CN 202410049094 A CN202410049094 A CN 202410049094A CN 118033014 A CN118033014 A CN 118033014A
Authority
CN
China
Prior art keywords
cells
pretreatment
sample
single cell
pretreatment method
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202410049094.5A
Other languages
Chinese (zh)
Inventor
杨梦甦
戴钧
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Futian Research Institute Of City University Of Hong Kong
Original Assignee
Shenzhen Futian Research Institute Of City University Of Hong Kong
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Futian Research Institute Of City University Of Hong Kong filed Critical Shenzhen Futian Research Institute Of City University Of Hong Kong
Priority to CN202410049094.5A priority Critical patent/CN118033014A/en
Publication of CN118033014A publication Critical patent/CN118033014A/en
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

The invention discloses a single-cell proteomics pretreatment method, a kit and application thereof in HSPE, TGS1 and MDH1 target discovery, and the single-cell proteomics pretreatment method and the single-cell proteomics pretreatment kit provided by the invention can effectively break single cells, extract and purify proteins, and simultaneously maintain the stability and activity of the proteins. The pretreatment method and the kit provided by the invention are suitable for treating various types of single-cell samples, including cell line samples and clinical samples, can extract high-quality proteins, and provide a reliable basis for subsequent mass spectrometry. The pretreatment kit provided by the invention has the advantages of simple operation, low cost, good repeatability, wide market application range and good prospect.

Description

Single-cell proteomics pretreatment method, kit and application thereof in HSPE, TGS1 and MDH1 target discovery
Technical Field
The invention belongs to the technical field of biology, and relates to a single-cell proteomics pretreatment method, a kit and application thereof in HSPE, TGS1 and MDH1 target discovery.
Background
Single cell proteomics is an emerging field of research aimed at studying protein expression, modification and interaction at the single cell level. This research approach is of great importance for understanding cellular heterogeneity, for finding new biomarkers, and for developing new therapeutic strategies. However, implementation of single cell proteomics presents challenges, one of which is the difficulty and complexity of sample pretreatment. Sample pretreatment refers to the process of extracting, purifying, quantifying and enzymatically hydrolyzing proteins from cells, which directly affects the efficiency and quality of proteomic analysis. Since each cell contains only a very small amount of protein (about 200 pg), efficient and sensitive pretreatment methods are needed to extract, purify and analyze these proteins. Most of the kits on the market today are designed for processing multicellular samples, which often do not meet the demands of single-cell proteomics. For example, they may not be effective in disrupting individual cells, or may not be amenable to stable protein extraction and purification at low protein concentrations. Therefore, it is necessary to develop a kit specifically for single cell proteomics pretreatment.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides the following technical scheme:
the invention provides a pretreatment method of a single-cell proteomics analysis sample, which comprises the following steps:
1) Adding a triethylammonium bicarbonate buffer solution containing Rapigest to the single cells, and heating at 75 ℃;
2) Acetonitrile was added followed by trypsin, incubation at 37 ℃;
3) And 2) after the reaction is finished, freeze-drying and preserving the sample, or adding a formic acid solution to obtain a detectable sample, and directly carrying out mass spectrometry sample loading analysis.
Further, the volume ratio of the triethylammonium bicarbonate buffer solution, acetonitrile, trypsin and formic acid solution is in the range of 1:0.2-0.4:0.2:5-10 microliters/single cell.
In some embodiments, the volume ratio of the triethylammonium bicarbonate buffer, acetonitrile, trypsin, formic acid solution in the pretreatment method comprises 1:0.2:0.2:5、1:0.25:0.2:5、1:0.3:0.2:5、1:0.35:0.2:5、1:0.4:0.2:5、1:0.2:0.2:6、1:0.25:0.2:6、1:0.3:0.2:6、1:0.35:0.2:6、1:0.4:0.2:6、1:0.2:0.2:7、1:0.25:0.2:7、1:0.3:0.2:7、1:0.35:0.2:7、1:0.4:0.2:7、1:0.2:0.2:8、1:0.25:0.2:8、1:0.3:0.2:8、1:0.35:0.2:8、1:0.4:0.2:8、1:0.2:0.2:9、1:0.25:0.2:9、1:0.3:0.2:9、1:0.35:0.2:9、1:0.4:0.2:9、1:0.2:0.2:10、1:0.25:0.2:10、1:0.3:0.2:10、1:0.35:0.2:10、1:0.4:0.2:10.
Further, the volume ratio of the triethylammonium bicarbonate buffer solution to the acetonitrile to the trypsin to the formic acid solution is 1:0.4:0.2:5-10 microliters/single cell.
Further, the concentration of the triethylammonium bicarbonate buffer ranges from 50 to 100mM.
In some embodiments, the concentration of the triethylammonium bicarbonate buffer comprises 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM.
Further, the Rapigest concentration range is 0.1% -0.2%.
In some embodiments, the concentration of Rapigest comprises 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%.
Further, the Rapigest concentration is 0.2%.
Further, the heating time at 75 ℃ in the step 1) is 15-20min.
In some embodiments, the time range of heating at 75 ℃ in step 1) includes 15min, 16min, 17min, 18min, 19min, 20min.
Further, the concentration of trypsin was 0.1 ng/. Mu.L.
Further, the temperature keeping time at 37 ℃ in the step 2) is 8-12h.
In some embodiments, the incubation time range of 37 ℃ in step 2) includes 8h, 9h, 10h, 11h, 12h.
Further, the samples may be stored at-20 ℃ or-80 ℃ after lyophilization.
Further, the concentration of the formic acid solution was 0.1%.
The term "mass spectrometry" (MS) refers to a technique used for the identification and/or quantification of molecules in a sample. MS involves ionizing molecules in a sample, thereby forming charged molecules; separating the charged molecules according to their mass to charge ratio; and detecting the charged molecule. MS allows for qualitative and quantitative detection of molecules in a sample. The molecules may be ionized and detected by any suitable method known to those skilled in the art. Some examples of mass spectrometry are "tandem mass spectrometry" or "MS/MS", which are techniques in which multiple rounds of mass spectrometry occur using more than one mass analyzer simultaneously or using a single mass analyzer sequentially. The term "mass spectrometry" may refer to the application of mass spectrometry to protein analysis. In some embodiments, electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) may be used in this context. In some embodiments, intact protein molecules may be ionized by the techniques described above and then introduced into a mass spectrometer. Alternatively, the protein molecules may be broken down into smaller peptides, for example, by enzymatic digestion with proteases, such as trypsin. Subsequently, the peptide is introduced into a mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry.
Further, the single cells include T cells, B cells, tumor cells, myeloid cells, blood cells, normal cells, fetal cells, maternal cells, artificial cell lines.
In some embodiments, the artificial cell line comprises the SV40 transformed monkey kidney CV1 strain (COS 7, atcc CRL 1651); human embryonic kidney system; baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary cells/DHFR; mouse supporting cells (TM 4); monkey kidney cells (CV 1 ATCC CCL 70); african green monkey kidney cells (VERO 76, ATCC CRL 1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); brugo rat (buffalo rat) hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562,ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human liver tumor lines (Hep G2).
Further, the sample is freeze-dried and stored, and then formic acid solution can be added according to the volume of 5-10 microliter/single cell, so that a detectable sample is obtained and is used for mass spectrometry loading analysis.
The terms "cell", "host cell" are used interchangeably and encompass plant and animal cells, and include invertebrate, non-mammalian vertebrate and mammalian cells. All such designations include cell populations and offspring. Exemplary non-mammalian vertebrate cells include, for example, avian cells, reptilian cells, and amphibian cells. Exemplary invertebrate cells include, but are not limited to, insect cells such as army (spodoptera frugiperda) cells, mosquito (aedes aegypti) cells, drosophila (drosophila melanogaster) cells, schneider cells, and bombyx mori cells. See, for example, luckow et al, bio/Technology 6:47-55 (1988). The cells may be differentiated, partially differentiated or undifferentiated, such as stem cells, including embryonic stem cells and pluripotent stem cells. Additional tissue samples derived from organs or organ systems may be used in accordance with the present invention. Exemplary mammalian cells include, for example, rodent cells, including mouse, hamster, rat, and guinea pig cells, and any derivatives and offspring thereof, derived from humans, non-human primates, cats, dogs, sheep, goats, cows, horses, pigs, rabbits.
The term "cell line" refers to all stages of development of a cell type, from the earliest precursor cells to fully mature cells (i.e., specialized cells).
The present invention provides a composition comprising an agent for use in the pretreatment method described above.
Further, the composition included acetonitrile, triethylammonium bicarbonate buffer, 0.1 ng/. Mu.L trypsin, surfactant, 0.1% formic acid solution.
Further, the surfactant includes anionic surfactant, nonionic surfactant, and cationic surfactant.
Further, the surfactant includes Rapigest.
Further, the surfactant may be added to the triethylammonium bicarbonate buffer.
The term "composition" encompasses and discloses any physical entity comprising (consisting of or consisting essentially of) the respective recited substances, the physical form of the composition being not limited. For example, the term "composition" encompasses and discloses a powder wherein the listed materials are each present in powder form. In one embodiment, the term "composition" also encompasses and discloses a liquid solution in which the listed materials are each present in a soluble form.
The term "surfactant" refers to a compound that reduces the surface tension of water or reduces the interfacial tension between two normally immiscible liquids (e.g., oil and water) when dissolved in water or an aqueous solution. Any surfactant known in the art may be used, including any naturally occurring pulmonary surfactant. Examples of suitable surfactants include, but are not limited to: oleic acid; sorbitan trioleate; cetyl pyridinium chloride; soybean lecithin; polyoxyethylene (20) sorbitan monolaurate; polyoxyethylene (10) stearyl ether; polyoxyethylene (2) oleyl ether; polyoxypropylene-polyoxyethylene ethylenediamine block copolymers; polyoxyethylene (20) sorbitan monostearate; polyoxyethylene (20) sorbitan monooleate; polyoxypropylene-polyoxyethylene block copolymers; castor oil ethoxylates; and combinations thereof.
The term "buffer" or "buffer" refers to a material that when added to a solution causes the solution to resist a change in pH.
The present invention provides a pretreatment kit comprising the composition described above, and a reaction vessel.
Further, the reaction vessel comprises a low adsorption vessel.
Further, the low adsorption container comprises a low adsorption liquid chromatography sample injection bottle.
Further, the pretreatment kit also includes instructions describing the method of use of the kit.
The terms "pretreatment" and "sample pretreatment" refer to concentration of trace amounts of the components to be tested, so as to improve the sensitivity of the method and reduce the detection limit; removing the matrix and other interfering substances in the sample; the measured object is converted into a substance with higher detection sensitivity or a substance which can be separated from the interference component in the sample through derivatization by other reactions, so that the sensitivity and the selectivity of the method are improved; the mass and the volume of the sample are concentrated, so that the sample is convenient to transport and store, and the stability of the sample is improved, so that the sample is not influenced by air; sample processing means for protecting the analytical instrument and the test system from affecting the performance and lifetime of the device. Sample pretreatment techniques are largely classified into solid, liquid, and gas pretreatment techniques.
The term "kit" refers to any delivery system for delivering materials. In the case of immunotherapeutic agents, such delivery systems include systems that allow for storage, transport, or delivery of the immunogenic agent and/or support material (e.g., written instructions for using the material, etc.) from one location to another.
The invention provides a single cell proteomics detection method, which comprises the following steps: taking a detectable sample obtained by the pretreatment method, and detecting the detectable sample by adopting a trapped ion mobility mass spectrum.
The term "ion mobility mass spectrometry" refers to a novel two-dimensional mass spectrometry technique that combines ion mobility separation with mass spectrometry, the ion mobility separation principle being based on the difference in collision cross section of ions in a drift tube when they collide with a buffer gas, the ions being separable according to size and shape. The term "ion mobility", also known as ion mobility, refers to the velocity of movement of positive or negative ions in m/V at an electric field strength of 1V/m or an electric field force of 1N. Ion mobility separation is based primarily on the shape and size of the ions, and is of particular advantage for analysis of isomers or complexes, etc., which cannot be distinguished by conventional mass spectrometry. After the ions are pre-separated according to mobility, the mass number is obtained through the mass-to-charge ratio of each component, and then the two-dimensional spectrum or the three-dimensional spectrum of the ion mobility mass spectrum can be obtained.
The invention provides a pretreatment method as described above, or the use of a composition as described above for detecting single cell proteins.
The present invention provides the use of the previously described pretreatment methods, or the previously described compositions, in a system or device for automated pretreatment of a single cell proteome sample.
The term "automated" refers to an optional computer control system capable of sensing and/or adjusting any condition, process, flow, input, output (e.g., temperature, pH, gas content, flow, and/or other measurable characteristics) within the plan, receiving data, optionally using artificial intelligence or other adaptive control, optionally processing the data by a computer to determine whether adjustments to any operating parameters are likely to be needed, sending one or more signals to one or more systems that then complete one or more physical adjustments to the operating parameters of the plan.
The terms "one or more" and "single or multiple" are interchangeable and refer to a number greater than one. For example, the term "one or more" encompasses any of the following: a number of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, twenty or more, fifty or more, 100 or more, or even more.
The terms "system" and "apparatus" encompass not only the configuration of multiple computers, hardware or apparatus connected by a communication medium such as a network (including a connection supporting one-to-one communication), but also the configuration implemented by a single computer, hardware or apparatus. The terms "device" and "system" are used interchangeably. It is obvious that the term "system" includes not only manually arranged social constructions (social systems).
When processing is performed by each module or each time a plurality of processes are performed within a module, information to be processed is retrieved from the storage device, and the processing result is written back to the storage device after the processing. Thus, descriptions retrieved from storage before processing and written back to storage after processing may be shortened or omitted in some cases. Note that the storage device herein may include a hard disk, a Random Access Memory (RAM), an auxiliary or external storage medium, a storage device accessed via a communication link, a register in a Central Processing Unit (CPU), and the like.
The present invention provides a single cell pretreatment system or device comprising a processor for performing the steps of the pretreatment method described above.
Further, the system or apparatus also includes a memory.
Further, the memory stores a computer program.
Further, the computer program is for programming the steps of the pre-processing method described above.
Further, the computer program is operative by a processor.
The term "memory" includes any type of integrated circuit or other memory device suitable for storing digital data, including, but not limited to, random Access Memory (RAM), pseudo Static RAM (PSRAM) Dynamic RAM (DRAM), synchronous Dynamic RAM (SDRAM) including Double Data Rate (DDR) type memory and graphic DDR (GDDR) and variations thereof, ferroelectric RAM (FeRAM), magnetic RAM (MRAM), resistive RAM (ReRAM), read-only memory (ROM), programmable ROM (PROM), electrically erasable PROM (EEPROM or E2 PROM), DDR/2SDRAM, EDO/FPMS, reduced Latency DRAM (RLDRAM), static RAM (SRAM), a "flash" memory (e.g., NAND/NOR), phase Change Memory (PCM), 3-dimensional cross point memory (3D Xpoint), stacked memory such as HBM/HBM2, and Magnetoresistive RAM (MRAM), such as spin torque transfer RAM (STT RAM).
The term "storage device" refers to, but is not limited to, a computer hard disk drive (e.g., hard Disk Drive (HDD), solid state drive (SDD)), flash drive, DVR device, memory, RAID device or array, optical media (e.g., CD-ROM, laser disc, blu-ray, etc.), or any other device or medium capable of storing content or other information, including semiconductor devices capable of maintaining data in the absence of a power source.
The term "processor" is intended to include any integrated circuit or other electronic device and mechanical device that is capable of operating on at least one instruction word, such as executing instructions, code, computer programs, and scripts accessed from a storage medium. However, the term "processor" should not be construed as limited to only hardware capable of executing software, but refers to a processing device in a general manner.
In certain embodiments, the single cell pretreatment system or device is implemented in the context of a computer program controlled processor:
operation 1: adding a triethylammonium bicarbonate buffer containing Rapigest to the single cells;
Operation 2: heating at 75deg.C;
Operation 3: acetonitrile was added followed by trypsin;
operation 4: preserving heat at 37 ℃;
Operation 5: freeze-drying and preserving, or adding formic acid solution to obtain a detectable sample.
In some embodiments, the system or apparatus includes one or more processors, and memory for storing a single or multiple computer programs for execution by the processors, the single computer program being executable by the single or multiple processors, the multiple computer programs being executable by the single or multiple processors.
The invention has the advantages and beneficial effects that:
1. Improving the sensitivity and accuracy of protein detection: because the kit is specially designed for single-cell samples, fewer operation steps and sample transfer steps can reduce protein loss in the pretreatment process to the greatest extent, thereby improving the sensitivity and accuracy of protein detection.
2. The operation steps are simplified: the kit has simple operation steps, can greatly reduce the time and complexity of experiments, and ensures that the single-cell proteomics research is more efficient.
3. The cost of scientific research is reduced: the kit has the advantages of low use cost, no need of building a complete platform, low difficulty of the hand, and no need of having specific background and skill to master. Can reduce the cost and the difficulty of the upper hand of the scientific research of single-cell proteomics.
4. Promotes the development of single cell proteomics: the kit can solve a great difficulty in single-cell proteomics research, is helpful for promoting the development of the field, and provides more possibility for disease diagnosis and treatment.
Drawings
FIG. 1 is a graph of the results of single cell proteome mass spectrometry analysis of SK-Hep-1 cell lines;
FIG. 2 is a graph of single cell proteome mass spectrometry results for 11 circulating tumor cells of non-small cell lung cancer;
FIG. 3 is a graph showing the results of a thermographic analysis and a principal component analysis of different circulating tumor cells;
FIG. 4 is a graph of volcanic analysis results of circulating tumor cell co-expressed proteins.
Detailed Description
In order to make the person skilled in the art better understand the solution of the present invention, the technical solution of the embodiment of the present invention will be clearly and completely described below with reference to the embodiment of the present invention and the accompanying drawings. It should be apparent that the embodiments described herein are only some, but not all, of the embodiments in which the invention may be practiced. All other embodiments, which can be derived by a person skilled in the art from the embodiments according to the invention without any creative effort, are within the protection scope of the invention.
Aspects, features, and advantages of several embodiments herein will become better understood with regard to the following description in conjunction with the accompanying drawings. It should be apparent to those skilled in the art that the embodiments of the present invention provided herein are intended to be exemplary only and not limiting and are intended to be illustrative only. All features described herein may be replaced by alternative features serving the same or similar purpose unless explicitly stated otherwise. Thus, many other embodiments of the modifications thereof may be made while remaining within the scope of the invention as defined herein and equivalents thereof. Thus, use of unconditional terms such as "will", "will not", "should", "must" and "must" are not intended to limit the scope hereof, as the embodiments described herein are exemplary.
The term "about" or "approximately" means that the particular value measured by one of ordinary skill in the art is within acceptable tolerances, depending in part on how the value is measured or measured, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation per practice in the art.
Example 1 Single cell proteome pretreatment kit and Single cell proteome pretreatment analysis of cell line samples
1. Composition of single cell proteome pretreatment kit
Reaction vessel (low adsorption liquid chromatography vial), acetonitrile (ACN), triethylammonium bicarbonate (TEAB) buffer (50-100 mM), trypsin (0.1 ng/. Mu.l), surfactant (0.1% to 0.2% Rapigest), formic acid solution (0.1%).
2. Experimental materials
SK-Hep-1 cell line (three replicates), single cell proteome pretreatment kit, timsToF Pro mass spectrometer as mentioned in example 1.
3. Single cell proteome pretreatment method
1) Cell lysis: after placing single cells into a reaction vessel, 1. Mu.l of Rapigest triethylammonium bicarbonate buffer containing 0.1% to 0.2% was added and the cells were allowed to lyse well by heating at 75℃for 15-20 minutes.
2) Denaturation: then 0.2-0.4. Mu.l acetonitrile was added to the reaction vessel. The purpose of denaturation is to break down the spatial structure of the macromolecular proteins for subsequent enzymatic digestion in combination with trypsin. Acetonitrile has the function of increasing the solubility of polar proteins, in addition to helping protein denaturation.
3) Protein digestion: subsequently 0.2. Mu.l of 0.1 ng/. Mu.L trypsin was added to the reaction vessel. Preserving the temperature for 8-12 hours at 37 ℃.
4) Sample preservation: after the end of the protein digestion reaction, the sample needs to be lyophilized. The lyophilized sample can be stored at-20deg.C or-80deg.C for several months. A quantity (5 to 10 μl) of formic acid solution was then added for mass spectrometry analysis.
4. Experimental results
After mass spectrometry analysis of the samples treated by the single cell proteome pretreatment kit, the results are shown in fig. 1, and the results show that about 1200 proteins can be detected in each cell, and 1600 proteins are detected in total in three repeated samples. In addition, the results showed that the reproducibility of the three replicates was better. EXAMPLE 2 Single cell proteome pretreatment and library-free proteome analysis of clinical Single cell samples Using Single cell proteome pretreatment kit
1. Experimental materials
A blood sample from a patient with non-small cell lung cancer, a single cell proteome pretreatment kit as described in example 1, timsToF Pro mass spectrometer.
2. Experimental method
1) A non-small cell lung cancer patient was subjected to blood collection and 11 circulating tumor cells were isolated from 4mL of the blood sample.
2) The single circulating tumor cells were proteomic pretreated using the single cell proteomic pretreatment kit provided in example 1, specifically comprising:
A. Cell lysis: after placing single cells into a reaction vessel, 1. Mu.l of Rapigest triethylammonium bicarbonate buffer containing 0.1% to 0.2% was added and the cells were allowed to lyse well by heating at 75℃for 15-20 minutes.
B. Denaturation: then 0.2-0.4. Mu.l acetonitrile was added to the reaction vessel. The purpose of denaturation is to break down the spatial structure of the macromolecular proteins for subsequent enzymatic digestion in combination with trypsin. Acetonitrile has the function of increasing the solubility of polar proteins, in addition to helping protein denaturation.
C. Protein digestion: subsequently 0.2. Mu.l of 0.1 ng/. Mu.L trypsin was added to the reaction vessel. Preserving the temperature for 8-12 hours at 37 ℃.
D. Sample preservation: after the end of the protein digestion reaction, the sample needs to be lyophilized. The lyophilized sample can be stored at-20deg.C or-80deg.C for several months. A quantity (5 to 10 μl) of formic acid solution was then added for mass spectrometry analysis.
3) Mass spectrometry was performed using a no library search mode.
3. Experimental results
The results of single cell protein mass spectrometry of circulating tumor cells are shown in FIG. 2, which shows that 429 proteins were detected in total from 11 circulating tumor cells, and 25 cancer driving genes were identified. Moreover, due to cancer heterogeneity, there is a difference in protein expression between different circulating tumor cells, and the results of the thermographic analysis and the principal component analysis are shown in FIG. 3, which shows that these circulating tumor cells can be divided into three groups according to protein expression. There was a clear difference in protein expression in these three groups of circulating tumor cells.
Volcanic analysis was then performed on 288 proteins co-expressed by three sets of circulating tumor cells, as shown in FIG. 4, which shows that HSPE.sup.1 is the most differentially expressed protein, which may be associated with the metastatic mechanism of CTC. In contrast to G1, TGS1 and MDH are upregulated in both G2 and G3, which may be key proteins for tumor progression, and can serve as novel tumor markers or therapeutic targets. Previous studies have shown that this protein is a biomarker for tumor metastasis, which results are consistent with previous studies, demonstrating the reliability of this assay.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (10)

1. A method of pretreatment of a single cell proteomic analysis sample, the method comprising:
1) Adding a triethylammonium bicarbonate buffer solution containing Rapigest to the single cells, and heating at 75 ℃;
2) Acetonitrile was added followed by trypsin, incubation at 37 ℃;
3) And 2) after the reaction is finished, freeze-drying and preserving the sample, or adding a formic acid solution to obtain a detectable sample, and directly carrying out mass spectrometry sample loading analysis.
2. The pretreatment method according to claim 1, wherein the volume ratio of the triethylammonium bicarbonate buffer, acetonitrile, trypsin, formic acid solution is in the range of 1:0.2-0.4:0.2:5-10 microliters/single cell;
preferably, the volume ratio of the triethylammonium bicarbonate buffer solution to the acetonitrile, the trypsin and the formic acid solution is 1:0.4:0.2:5-10 microliters/single cell;
Preferably, the concentration of the triethylammonium bicarbonate buffer is in the range of 50-100mM;
Preferably, the Rapigest is present in a concentration range of 0.1% to 0.2%;
preferably, the Rapigest is at a concentration of 0.2%;
preferably, the heating time at 75 ℃ in the step 1) is 15-20min;
Preferably, the concentration of trypsin is 0.1 ng/. Mu.L;
preferably, the temperature keeping time at 37 ℃ in the step 2) is 8-12h;
preferably, the sample may be stored at-20 ℃ or-80 ℃ after lyophilization;
Preferably, the concentration of the formic acid solution is 0.1%.
3. The pretreatment method according to claim 1 or 2, wherein the single cells comprise T cells, B cells, tumor cells, myeloid cells, blood cells, normal cells, fetal cells, maternal cells, artificial cell lines.
4. The pretreatment method according to claim 1 or 2, wherein the sample is lyophilized and stored, and then a formic acid solution can be added in a volume of 5 to 10 microliters per single cell to obtain a detectable sample for mass spectrometry.
5. A composition comprising an agent for use in the pretreatment method of any one of claims 1 to 4;
Preferably, the composition comprises acetonitrile, triethylammonium bicarbonate buffer, 0.1 ng/. Mu.L trypsin, surfactant, 0.1% formic acid solution;
Preferably, the surfactant comprises an anionic surfactant, a nonionic surfactant, a cationic surfactant;
preferably, the surfactant comprises Rapigest;
Preferably, the surfactant may be added to the triethylammonium bicarbonate buffer.
6. A pretreatment kit comprising the composition of claim 5, and a reaction vessel;
Preferably, the reaction vessel comprises a low adsorption vessel;
Preferably, the low adsorption vessel comprises a low adsorption liquid chromatography sample injection bottle;
preferably, the pretreatment kit further comprises instructions describing the method of use of the kit.
7. A method of single cell proteomics, the method comprising: taking a detectable sample obtained by the pretreatment method of any of claims 1-4, and detecting the detectable sample by using trapped ion mobility mass spectrometry.
8. Use of the pretreatment method of any one of claims 1 to 4, or the composition of claim 5, for detecting single cell proteins.
9. Use of the pretreatment method of any one of claims 1 to 4, or the composition of claim 5, in a system or device for automated treatment of single cell proteome pretreatment samples.
10. A single cell pretreatment system or device, comprising a processor for performing the steps of the pretreatment method of any of claims 1-4;
preferably, the system or apparatus further comprises a memory;
Preferably, the memory stores a computer program;
preferably, the computer program is for programming the steps of the pretreatment method according to any of claims 1 to 4;
preferably, the computer program is operated by a processor.
CN202410049094.5A 2024-01-12 2024-01-12 Single-cell proteomics pretreatment method, kit and application thereof in HSPE, TGS1 and MDH1 target discovery Pending CN118033014A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202410049094.5A CN118033014A (en) 2024-01-12 2024-01-12 Single-cell proteomics pretreatment method, kit and application thereof in HSPE, TGS1 and MDH1 target discovery

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202410049094.5A CN118033014A (en) 2024-01-12 2024-01-12 Single-cell proteomics pretreatment method, kit and application thereof in HSPE, TGS1 and MDH1 target discovery

Publications (1)

Publication Number Publication Date
CN118033014A true CN118033014A (en) 2024-05-14

Family

ID=90997961

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202410049094.5A Pending CN118033014A (en) 2024-01-12 2024-01-12 Single-cell proteomics pretreatment method, kit and application thereof in HSPE, TGS1 and MDH1 target discovery

Country Status (1)

Country Link
CN (1) CN118033014A (en)

Similar Documents

Publication Publication Date Title
Mallick et al. Proteomics: a pragmatic perspective
Karpievitch et al. Liquid chromatography mass spectrometry-based proteomics: biological and technological aspects
Gundry et al. Preparation of proteins and peptides for mass spectrometry analysis in a bottom‐up proteomics workflow
Cho et al. Oncoproteomics: current trends and future perspectives
Coombs Quantitative proteomics of complex mixtures
Lovric Introducing proteomics: from concepts to sample separation, mass spectrometry and data analysis
Gutstein et al. Microproteomics: analysis of protein diversity in small samples
Kline et al. Protein quantitation using isotope-assisted mass spectrometry
CN105637098A (en) Methods and systems for aligning sequences
CN111272889B (en) Method for analyzing differential expression protein of aeromonas hydrophila infected macrobrachium nipponensis blood cells based on proteomic quantitative technology
Eichelbaum et al. Combining pulsed SILAC labeling and click-chemistry for quantitative secretome analysis
WO2023134169A1 (en) Pretreatment method, storage method, automatic treatment system, and detection method for urine sample
Greening et al. The peptidome comes of age: mass spectrometry-based characterization of the circulating cancer peptidome
Nakajima et al. Toward proteome‐wide exploration of proteins in dried blood spots using liquid chromatography‐coupled mass spectrometry
Bose et al. Proteomics: tools of the trade
Cheng et al. Peptide labeling using isobaric tagging reagents for quantitative phosphoproteomics
López Applications of proteomics in marine ecology
Espejo et al. Early cancer biomarker discovery using DIA-MS proteomic analysis of EVs from peripheral blood
Nelson et al. Global mass spectrometry-based analysis of protein ubiquitination using K-ε-GG remnant antibody enrichment
O'riordan et al. Technology Insight: renal proteomics—at the crossroads between promise and problems
Di Silvestre et al. Multidimensional protein identification technology for direct-tissue proteomics of heart
Winnik et al. Analysis of proteins using DIGE and MALDI mass spectrometry
CN118033014A (en) Single-cell proteomics pretreatment method, kit and application thereof in HSPE, TGS1 and MDH1 target discovery
Masuo et al. Brain proteomics: sample preparation techniques for the analysis of rat brain samples using mass spectrometry
Sigdel et al. Interpreting the proteome and peptidome in transplantation

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination