阅读说明：本技术 基于稳定同位素半胱氨酸代谢标记的棕榈酰化修饰蛋白质定量分析方法 (Palmitoylation modified protein quantitative analysis method based on stable isotope cysteine metabolic labeling ) 是由 陆豪杰 张晓勤 方彩云 于 2018-07-06 设计创作，主要内容包括：本发明属于生物分析技术领域,涉及一种基于稳定同位素半胱氨酸代谢标记的棕榈酰化修饰蛋白质定量分析方法。本发明采用含重标稳定同位素半胱氨酸的培养基培养细胞,使细胞蛋白质中的半胱氨酸被重标半胱氨酸完全替代,将不同细胞分别培养在含轻、重标半胱氨酸的培养基中,蛋白质提取混合,采用固相载体选择性富集,色谱-质谱分离分析后,可定量分析不同细胞中的棕榈酰化修饰蛋白质。与现有的定量分析方法相比,本方法可减少因繁冗的并行操作带来的误差,以及减少样本的复杂性,简化谱图；促进低丰度蛋白的准确定量；可用于定量分析包括C末端肽段在内的所有潜在的棕榈酰化修饰位点,本方法适用于所有的细胞样品。(The invention belongs to the technical field of biological analysis, and relates to a palmitoylation modified protein quantitative analysis method based on stable isotope cysteine metabolic labeling. The invention adopts a culture medium containing heavy-mark stable isotope cysteine to culture cells, so that the cysteine in cell protein is completely replaced by the heavy-mark cysteine, different cells are respectively cultured in the culture medium containing light-mark cysteine and heavy-mark cysteine, the protein is extracted and mixed, the palmitoylation modified protein in different cells can be quantitatively analyzed after the selective enrichment by a solid phase carrier and the chromatographic-mass spectrometry separation and analysis. Compared with the existing quantitative analysis method, the method can reduce errors caused by complicated parallel operation, reduce the complexity of samples and simplify spectrograms; accurate quantification of low-abundance proteins is facilitated; can be used for quantitative analysis of all potential palmitoylation modification sites including C-terminal peptide fragments, and the method is suitable for all cell samples.)

1. A method for quantitatively analyzing palmitoylation modified protein based on stable isotope cysteine metabolic labeling (SILAC-Cys), which is characterized in that the palmitoylation modification level of the protein is quantitatively analyzed by a mass spectrometry method by using cysteine as a mass label, and comprises the following steps:

(1) MHCC97L and/or HCCLM3 cells are cultured by adopting a culture medium containing light and heavy isotope cysteine, so that cell proteins are completely marked by the light or heavy isotope cysteine, and the marking efficiency is 100 percent;

(2) collecting cells with different passage times, and inspecting the marking efficiency of cysteine in the protein by using a mass spectrometry method;

(3) collecting HCCLM3 cells with light and heavy isotope cysteine labeling efficiency of 100%, and inspecting feasibility of quantitative analysis of palmitoylation modified protein by SILAC-Cys technology through protein extraction, selective enrichment of palmitoylation modified protein and mass spectrometry;

(4) collecting MHCC97L and HCCLM3 cells with light and heavy isotope cysteine labeling efficiency of 100%, extracting proteins, selectively enriching palmitoylation modified proteins and analyzing mass spectrum, and quantitatively comparing the difference of the palmitoylation modification levels of the proteins in the two cells with different transfer potentials by using an SILAC-Cys technology combined with mass spectrum analysis;

(5) the potential palmitoylation modified differential protein in the isotopic cysteine metabolism labeled (SILAC-Cys) palmitoylation modified protein is verified by using a Western Blot method.

2. The method of claim 1, wherein said MHCC97L and HCCLM3 cells are liver cancer low metastatic potential cells and liver cancer high metastatic potential cells, respectively.

3. The method of claim 1, wherein in step (1), when the cell is cultured in the medium containing the heavy isotope cysteine, the cysteine in the cell protein is completely labeled with the heavy isotope cysteine, so that the cell proteins cultured in the medium containing the light isotope cysteine and the heavy isotope cysteine, respectively, have the peptide fragments with the same amino acid composition, which are resolved by mass spectrometry due to the difference in the number of the cysteine masses.

4. The method of claim 1, wherein the isotope mass labels for mass spectrometric quantitative analysis are introduced at the beginning of the cell culture, i.e. the experiment, and the two groups of samples to be analyzed can be mixed as early as possible and subjected to subsequent sample processing, enrichment and mass spectrometric analysis operations simultaneously, reducing the operating errors.

5. The method of claim 1, wherein the cysteine residue is a modification site and comprises a mass tag for quantifying all potential palmitoylated modified peptides including the C-terminal peptide.

6. The method of claim 1, wherein said method is further used for quantitative analysis of the whole proteome or other types of modifications occurring at cysteine residues.

7. Use of the method of claim 1 for quantitatively detecting the level of protein palmitoylation modification in a biological sample.

Technical Field

The invention belongs to the technical field of biochemical analysis, and relates to a method for quantitatively analyzing palmitoylation modified protein/peptide fragment in cells, in particular to a method for quantitatively analyzing palmitoylation modified protein based on stable cysteine isotope metabolism labeled cells.

Background

Disclosure of Invention

The invention aims to provide a novel method for quantitatively analyzing the palmitoylation modification level of a protein aiming at the defects in the prior art, and particularly relates to a method for quantitatively analyzing the palmitoylation modification of the protein based on a stable isotope cysteine metabolic labeling culture cell combined mass spectrometry technology.

Specifically, the invention provides a palmitoylation modified protein quantitative analysis method based on stable isotope cysteine metabolic labeling, which comprises the steps of carrying out cell culture by using a culture medium containing stable isotope cysteine, introducing a quality label in the cell culture process, mixing different samples together at the initial stage of an experiment to carry out sample treatment, enrichment and analysis, reducing errors caused by complicated parallel operation, and being beneficial to obtaining more accurate quantitative results; meanwhile, cysteine is a modification site and also contains a quantitative label, so that all potential palmitoylation modification sites can be quantitatively analyzed.

According to the invention, the liver cancer cell lines with different transfer potentials are cultured by using a culture medium containing cysteine which is a light stable isotope and cysteine which is a heavy stable isotope, the palmitoylation modified peptide segments/proteins in the sample are selectively enriched after the extracted proteins are mixed according to the ratio of 1: 1, and the quantitative analysis of the palmitoylation modification level in the liver cancer cells with different transfer potentials can be realized by combining the characteristic of mass spectrum precision analysis, and the information of differential proteins can be obtained.

In the quantitative method, firstly, a culture medium containing cysteine which is a light stable isotope and a heavy stable isotope is used for carrying out cell culture, cell protein is collected, palmitoylation modified protein is enriched, and then mass spectrometry detection is carried out.

The quantitative analysis of all potential palmitoylation modified peptide fragments (containing C-terminal peptide fragments) in the protein by taking cysteine as a mass label is an advantageous supplement of the classical SILAC method (taking lysine K and/or arginine R as mass labels).

In the present invention, the established method can be used for qualitative and quantitative analysis of palmitoylated modified protein/peptide fragments in all cells including the liver cancer cells with different metastatic potential, and can also be used for quantitative detection of other types of post-translational modification (such as nitrosylation modification and the like) occurring on cysteine residues.

More specifically, the invention relates to a method for detecting the difference of palmitoylation modification level based on the stable isotope cysteine metabolism labeled cell culture (SILAC-Cys) technology, which comprises the following steps:

(1) the method comprises the following steps of (1) carrying out cell culture by utilizing a culture medium containing light and heavy isotope stable cysteine, wherein the light isotope cysteine in cells can be completely replaced by the heavy isotope cysteine in the heavy isotope cysteine culture medium, so as to obtain heavy isotope cysteine cells which are completely metabolically labeled;

in the invention, the cultured cells are selected from MHCC97L and/or HCCLM3 cells, MHCC97L and HCCLM3 are liver cancer low-metastasis potential cells and liver cancer high-metastasis potential cells respectively, and the marking efficiency is 100 percent;

(2) after the cells are cultured for more than 6 generations, collecting the cells marked by the light and heavy isotopes cysteine, extracting the protein in the cells, and detecting the marking condition of the isotopes cysteine by using mass spectrometry;

in the embodiment of the invention, HCCLM3 cells with the efficiency of marking heavy isotope cysteine of 100 percent are collected, and the feasibility of quantitative analysis of palmitoylation modified protein by an SILAC-Cys technology is inspected through protein extraction, selective enrichment of palmitoylation modified protein and mass spectrum analysis;

(3) respectively culturing MHCC97L and HCCLM3 in a culture medium containing light stable isotope cysteine and heavy stable isotope cysteine to obtain light standard MHCC97L and heavy standard HCCLM3 cells, respectively extracting proteins in the light standard MHCC97L and heavy standard HCCLM3 cells, mixing the light standard MHCC97L and heavy standard HCCLM3 cells according to a ratio of 1: 1, selectively enriching potential palmitoylation modified proteins/peptide segments in the light standard MHCC97 and heavy standard HCCLM3 cells by using a commercial material Thiopropyl Sepharose 6B, and quantitatively detecting the difference of palmitoylation modification levels in the two cells with different transfer potentials through mass spectrometry;

(4) potential palmitoylation modified differential proteins were verified using the Western Blot method.

The invention provides a selective enrichment method based on a cell culture stable isotope cysteine metabolic labeling (SILAC-Cys) technology and combined with palmitoylation modified protein, quantitatively analyzes palmitoylation modified protein in MHCC97L and HCCLM3 cells with different transfer potentials, and performs Western Blot verification on 1 potential modified protein. The invention provides an effective means for detecting the palmitoylation modification level of the protein in different cell samples.

The invention has the advantages and beneficial effects that:

the method utilizes stable isotope cysteine as a mass label to quantitatively analyze the palmitoylation modification level of protein, and combines a palmitoylation modification enrichment method to quantitatively analyze the difference of the palmitoylation modification level in liver cancer cells MHCC97L and HCCLM3 with different transfer potentials through chromatography-mass spectrometry detection; compared with the existing quantitative analysis method, the method introduces a quality label at the initial stage of the experiment, has high labeling efficiency, and can reduce errors caused by complicated parallel operation; by utilizing the characteristics that cysteine is highly active and low-frequency but exists in most proteins, the complexity of a sample can be greatly reduced, a spectrogram is simplified, and accurate quantification of low-abundance proteins is promoted by combining a high-specificity enrichment technology; cysteine is a modification site and contains a quantitative label, can be used for quantitatively analyzing all potential palmitoylation modification sites including a C-terminal peptide fragment, and is an effective supplement of the traditional SILAC quantitative method (marked by using lysine and/or arginine stable isotope); the characteristic that the palmitoylation modified peptide fragment forms a peak in a primary mass spectrogram can be used for realizing double verification on the identified modified peptide fragment.

The quantitative method is suitable for all cell samples, and can be used for the quantitative determination of cysteine modification groups such as palmitoylation and the like and the quantitative determination of complete proteome.

Description of the drawings:

FIG. 1 is a gel electrophoresis image of cellular proteins after different passages in a medium containing heavy isotope cysteine, stained with Coomassie Brilliant blue, with arrows indicating the molecular weight regions of β -actin, in which H-LM3-2(3, 4, 5, 6, 7, 8, 9) represents the 2 nd passage cellular protein of HCCLM3 labeled with heavy cysteine and L-LM3 represents the cellular protein of HCCLM3 labeled with light cysteine.

FIG. 2 shows an example of β -actin, which is performed by mass spectrometry to analyze labeling efficiency of heavy isotope cysteine on cellular protein, and is performed by culturing cells after passage 0 (a), 6 (B) and 9 (C) in a medium containing heavy isotope cysteine, extracting protein, performing polyacrylamide gel electrophoresis, separating, selecting target bands (as shown in FIG. 2), performing enzymolysis in gel, and performing mass spectrometry, wherein graphs A and B are mass spectrograms of peptide fragments LC (+57) YVALDFEQEMATAASSSSLEK (m/z 2553.1) and C (+57) PEALFQPSFLGMESCGIHETTFNSIMK (m/z 3254.4) after different passage times, as shown in the figure, the content of light isotope cysteine peptide fragments originally existing in cellular protein is gradually reduced and the content of heavy isotope cysteine peptide fragments is gradually increased with the increase of passage times, and only heavy isotope cysteine peptide fragments can be detected by passage 9, the peptide segment of the light mark cysteine can not be detected basically, which shows that the light mark cysteine contained in the cell after 9 passages is almost completely replaced by the heavy isotope cysteine;

in the figure, L sample represents light cysteine-labeled HCCLM3 cell protein, and H6sample and H9sample represent heavy cysteine-labeled HCCLM3 passage 6 and 9 cell proteins, respectively.

FIG. 3 is a view of the feasibility of the method for quantitative analysis by mass spectrometric detection, taking beta-actin as an example, wherein HCCLM3 cells cultured for 9 passages in a medium containing light and heavy isotopes cysteine are collected respectively, after protein extraction, mixing at a ratio of 1: 1, polyacrylamide gel electrophoresis separation, and the target bands between molecular weight regions of beta-actin are intercepted for in-gel enzymolysis, peptide fragment extraction and mass spectrometric analysis;

the signal intensity of the light standard peptide segment LCYVALDFEQEMATAASSSSLEK and the heavy standard peptide segment LCYVALDFEQEMATAASSSSLEK in a mass spectrogram is shown in the figure, and the result shows that the peak intensities of the light standard peptide segment and the heavy standard peptide segment are basically consistent and are consistent with a theoretical value, which indicates that heavy isotope cysteine can enter into protein through cell culture metabolic labeling and can be used in quantitative proteomics research;

light and Heavy are shown in the figure to represent the cell protein samples of Light and Heavy cysteine, respectively.

FIG. 4 shows the Western Blot to detect the difference of the palmitoylation modification level of the proteins in MHCC97L and HCCLM3 of two liver cancer cells with different metastatic potentials,

in the figure, (A) after incubation of the anti-palmitoylation modified pan-antiserum, the MHCC97L protein of the liver cancer cell with low metastatic potential presents a more obvious positive band than the HCCLM3 cell with high metastatic potential; (B) the MHCC97L and HCCLM3 cell proteins with the same amount are separated by polypropylene gel electrophoresis and stained by Coomassie brilliant blue, and the color development degrees are basically consistent; (C) the result is the Western Blot result of the reference protein beta-actin.

FIG. 5 shows the results of Western Blot analysis of potential palmitoylation modification differential protein Double-stranded RNA-specific adenosine deaminase (DSRAD) showing the sensitivity of thioester binding to hydroxylamine treatment, and Western Blot analysis of the level of palmitoylation modification of DSRAD in MHCC97L and HCCLM3 cells using protein antibodies against DSRAD after enrichment with Thiopropyl Sepharose 6B, shows that both DSRAD expression and palmitoylation modification are higher in MHCC97L than in HCCLM3 at the same amount. 97L and LM3 are shown in the figure as MHCC97L and HCCLM3 cell protein samples, respectively.

Detailed Description

The present invention is further described with reference to the drawings and the specific embodiments, so that the technical solutions of the present invention can be more clearly understood by those skilled in the art, and the present invention is not limited thereto.