A method for proteome scale absolute quantification based on peptide segment sensitization

Abstract

The present application relates to a kind of based on peptide segment sensitization's high-precision scale absolute quantification method of proteome.It is using chemical derivatization technique to be modified on peptide segment, to change the physicochemical property of peptide segment, improve its mass spectrum signal response intensity.The derivatization sensitizer used generally includes positive charge or proton affinity group, it is helpful to improve the ionization efficiency of peptide segment.Due to the difference of the physicochemical property of peptide segment itself, after being labeled by the same derivatization sensitizer, usually low-abundance peptide segment signal response greatly improves, and high response peptide segment changes less, to cause the difference of the mass spectrum response signal between peptide segment to reduce, so that intensity information can more accurately reflect the content of peptide segment.The advantage of this method is: high accuracy of quantification, can realize large-scale proteome absolute quantification analysis.

Description

Technical Field

[0001] This invention belongs to the field of absolute protein quantification analysis, and particularly relates to a highly accurate, large-scale absolute quantification method for proteomics based on peptide sensitization. Background Technology

[0002] Absolute quantification in proteomics is a method for determining the precise quantity of proteins in a sample, providing detailed information about protein expression levels, such as copy number or concentration, which is crucial for understanding biological processes. For liquid chromatography-mass spectrometry (LC-MS)-based absolute quantification of proteomics, various methods have been developed, including absolute quantification (AQUA), concatenator of quantification peptides (QconCAT), and protein standard absolute quantification (PSAQ). These methods achieve accurate quantification of target peptides by synthesizing standard peptides or constructing recombinant proteins and then analyzing them by mass spectrometry. Although these methods are considered the gold standard for absolute quantification, the limited number of internal standards and high synthesis costs restrict their application in large-scale analysis of absolute proteomic content. Commonly used large-scale protein absolute quantification methods are divided into two main categories: those based on the number of secondary spectra and those based on the intensity of precursor ions. Both are based on the principle that the number of spectra or signal intensity of all / partial peptides constituting a protein in a sample is linearly correlated with their abundance in the sample. The former method achieves absolute protein quantification by statistically analyzing the number of identified peptides and secondary spectra, including methods such as emPAI and APEX. Its quantification principle is simple, calculation is convenient, and it was widely used in the early stages. The latter method calculates the absolute protein quantity using precursor ion intensity or peak area, including commonly used methods such as iBAQ and Top3. Its quantification accuracy is superior to the former. However, it is affected by two factors: low precursor ion signal-to-noise ratio and the randomness of acquisition in the data-dependent acquisition (DDA) mode. Furthermore, absolute quantification methods based on fragment ion intensity in the data-independent acquisition (DIA) mode provide another option for large-scale quantification. However, the types and intensities of fragment ions are affected by various factors such as peptide sequence, physicochemical properties, and fragmentation, which increases interference factors in the linear relationship between the quantification results and the actual protein concentration, leading to low correlation and affecting quantification accuracy.

[0003] Chemical derivatization is an effective way to improve peptide ionization efficiency by forming fixed-charge peptide derivatives and increasing their intensity during electrospray ionization (ESI). Therefore, we developed a highly accurate, large-scale absolute quantification method for proteomics based on peptide sensitization. This method utilizes derivatized sensitizers with positively charged or proton-affinity structures to chemically derivatize enzymatically digested peptides, improving the ionization efficiency of low-mass-spectrum responsive peptides, thereby reducing the intensity differences between equimolar peptides and enhancing the accuracy of large-scale quantification. Furthermore, by using dynamic range standards (UPS2) or standard proteins with known concentrations as internal standards, we established a highly accurate, large-scale absolute quantification method for proteomics based on peptide sensitization. Summary of the Invention

[0004] A highly accurate, large-scale absolute quantification method for proteomics based on peptide sensitization has the advantages of high sensitivity, high accuracy and high throughput, and can perform large-scale accurate quantification of proteomics in routine biological samples.

[0005] To achieve the above objectives, the technical solution adopted by this invention is: a highly accurate, large-scale absolute quantification method for proteomics based on peptide sensitization, comprising the following steps:

[0006] 1. Protein extraction from biological samples: The lysis buffer may be one or a mixture of several of the following: sodium dodecyl sulfate (SDS), urea, guanidine hydrochloride, and NP-40; the extraction method may be one or a combination of two of the following: ultrasonication and grinding. The biological sample may be one or more of the following: cells, microorganisms, biological tissues, or body fluids.

[0007] 2. After denaturing, reducing, and alkylating the protein sample to be tested, add enzyme at a certain enzyme / protein ratio, incubate overnight, and after desalting, obtain the enzymatically digested peptide fragments of the sample. The protease used has one or more cleavage sites at the lysine carboxyl group, arginine carboxyl group, aspartic acid carboxyl group, glutamic acid carboxyl group, aspartic acid amino group, or non-specific cleavage sites.

[0008] Standard proteins from different species than the biological sample and whose enzymatically digested peptides are not contained in the sample (as internal standards) are usually added after the sample protein extraction. The amount of standard protein added is 1 millimole (mmole) to 1 femtomoles (fmole), which participate in subsequent processing to obtain the enzymatically digested peptides.

[0009] 3. Derivatize and label the peptides obtained from the enzymatic digestion of the sample and internal standard in step 2. The mass ratio of sample to derivatizing sensitizer is (1:5 to 1:20), and the labeling time is 1 to 24 hours. The derivatizing sensitizer used consists of three parts: the host structure, the functional structure, and the reactive group. The functional structure acts as the "connecting arm" between the host structure and the reactive group, and the three are chemically bonded to form a molecule. The host structure is a quaternary ammonium salt, pyranonium salt, or phosphine salt containing one or more charges; or one or more of pyridine, imidazole, pyran, piperazine, and guanidine salts containing proton affinity structures; the functional structure is one or more of a C1-C10 alkyl carbon chain or a structure with 1 to 3 benzene rings; the reactive group is a group that reacts with amino or carboxyl groups. Groups that react with amino groups include N-hydroxysuccinimide groups, N-hydroxyphthalimide groups, and C1-C10 acyl halide groups, while groups that react with carboxyl groups are mainly C1-C10 amino groups.

[0010] Preferably, the main structure of the derivatization sensitizer used has a single or multiple charge structure. When the main single or multiple charge structure is highly hydrophilic, the reactive group is preferably a hydrophobic group, including N-hydroxyphthalimide groups, etc.; when the main single or multiple charge structure is highly hydrophobic, the reactive group is preferably a hydrophilic group, including N-hydroxysuccinimide groups, C1-C5 amino groups, etc. This structural combination helps in the initial purification of the derivatization sensitizer, and at the same time, it can effectively improve the ionization efficiency of peptides in subsequent liquid chromatography-mass spectrometry analysis without interfering with large-scale mass spectrometry detection. 4. Mass spectrometry data are acquired from the labeled sample in step 3 using a liquid chromatography-mass spectrometry system. After derivatization, due to the different physicochemical properties of the peptides, the ionization efficiency of low ionization efficiency peptides is significantly improved, and their mass spectrometry signal intensity is greatly increased, while the mass spectrometry signal intensity of high ionization efficiency peptides changes less, thereby reducing the difference in mass spectrometry response signals between peptides. Mass spectrometers used in liquid chromatography-mass spectrometry (LC-MS) include electrostatic orbital traps, ion traps, time-of-flight (TOF) tubes, or Fourier transform ion cyclotron resonance mass analyzers (FT-ICR); the mass spectrometry acquisition modes can be data-dependent acquisition (DDA) or data-independent acquisition (DIA).

[0011] 5. Construct a large-scale absolute proteomics quantification algorithm using the mass spectrometry signal intensity obtained above, add appropriate labeling reagents for modification, and obtain the signal response intensity information of all peptides corresponding to the protein through database retrieval (such as Skyline, MaxQuant, Spectronaut, DIANN software) and intensity extraction. The intensities of low ionization efficiency peptides and high ionization efficiency peptides belonging to the same protein are summed to obtain the intensity information corresponding to that protein.

[0012] 6. By multiplying the ratio of the intensity of the target protein and / or peptide in the sample to that of the internal standard by the absolute content of the known target protein and / or peptide in the internal standard, the absolute quantification of other target proteins and / or peptides to be analyzed in the sample can be obtained, thereby achieving absolute quantification of the sample proteome.

[0013] The quantitative method of this invention is applicable to large-scale absolute quantification of target sample proteomes, large-scale absolute quantification of target peptides, or absolute quantitative analysis of target proteins.

[0014] The present invention has the following advantages:

[0015] 1. High labeling efficiency and good labeling selectivity: Using optimized labeling conditions, the peptide labeling efficiency and labeling selectivity are both above 95%.

[0016] 2. High quantitative accuracy, enabling large-scale absolute quantitative analysis of proteomics.

[0017] 3. This invention provides a method that can be used for large-scale absolute quantitative detection of proteomics. Attached Figure Description

[0018] Figure 1 Working principle of a high-precision, large-scale absolute quantification method for proteomics based on peptide sensitization

[0019] Figure 2 a) Peak area of ​​the standard peptide before and after reaction with the derivatization sensitizer; b) Labeling efficiency of the derivatization sensitizer.

[0020] Figure 3 Peak areas of myoglobin peptides before and after reaction with derivatized sensitizers Detailed Implementation

[0021] The present invention will be further explained by selecting standard peptides, standard proteins, and complex biological samples through specific implementation methods.

[0022] Example 1

[0023] Weigh 1 mg each of standard peptides VPELDASK and WSFDGNAPDVK powder and prepare a 10 mg / mL solution using DMSO. Add the derivatizing sensitizer 1-(3-((2,5-dioxopyrrolidone-1-yl)oxy)-3-oxopropyl)pyridine bromide at a peptide-to-sensitizer mass ratio of 1:10. Adjust the pH to 8 with triethylamine and react at room temperature for 2 h. After lyophilization, reconstitute the sample with 0.1% formic acid solution and perform liquid chromatography-mass spectrometry (LC-MS) analysis.

[0024] Meanwhile, the standard peptide fragments were dissolved in 0.1% formic acid solution to a concentration of 0.5 mg / mL, which served as an unlabeled control group for direct liquid chromatography-mass spectrometry (LC-MS) analysis.

[0025] The liquid chromatography mobile phase A consisted of an aqueous solution containing 2% acetonitrile and 0.1% formic acid (by mass), and the mobile phase B consisted of an aqueous solution containing 98% ACN and 0.1% FA (by mass). A C18 reversed-phase capillary column (150 μm id × 15 cm, 1.9 μm) was used for separation. Samples were separated using a ReproSil-Pur C18-AQ mass spectrometer with a separation gradient (v / v) of 0 to 10 min at 2% B, followed by a 36 min linear gradient separation from 8% B to 28% B, and then an 8 min linear gradient separation from 28% B to 45% B, at a flow rate of 600 nL / min. Acquisition was performed using an LTQ OrbitrapVelos mass spectrometer in DDA mode with a Full MS resolution of 60,000, a mass range of 350–1500 m / z, an automatic gain control (AGC) value of 1e6, a maximum ion implantation time of 60 ms, and dynamic exclusion of 22 s.

[0026] Skyline analysis showed that the peak areas before and after VPELDASK labeling of the standard peptide were 1.43 × 10⁻⁶. 10 1.84×10 10 The peak areas before and after labeling the standard peptide WSFDGNAPDVK were 1.29 × 10⁻⁶. 10 1.4×10 10 ( Figure 2 a) According to the formula in the example below, the labeling efficiencies are calculated to be 98.5% and 97.7%, respectively. Figure 2 b) The labeling efficiency is over 95%.

[0027]

[0028] Y: Labeling efficiency (%); A: Peak area of ​​unlabeled peptides; B: Peak area of ​​unlabeled peptides in labeled samples.

[0029] Example 2

[0030] 2 mg of myoglobin (derived from equine skeletal muscle) standard was weighed and dissolved in 6 M guanidine hydrochloride solution (Sigma). Dithiothreitol (DTT) solution was added to a final concentration of 10 mM. After denaturation and reduction at 56 °C for 1 h, the mixture was cooled to room temperature, and iodoacetamide (IAA) solution was added to a final concentration of 25 mM. The mixture was then alkylated at room temperature in the dark for 30 min. Subsequently, guanidine hydrochloride was diluted to 0.5 mM with 50 mM phosphate buffer, and trypsin (enzyme to protein ratio 1:50) was added. The mixture was reacted at 37 °C for 16 h. After desalting using a desalting column, the sample was lyophilized to obtain peptides. The peptides were then prepared into a 10 mg / mL solution using DMSO. The derivatizing sensitizer 1-(3-(((1,3-dioxoisoindoline-2-yl)oxy)-3-oxopropyl)pyridine bromide was added at a peptide-to-sensitizer mass ratio of 1:10. The pH was adjusted to 8 with triethylamine, and the reaction was carried out at room temperature for 2 hours. After lyophilization, the sample was reconstituted with 0.1% formic acid solution and analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) (sampling conditions as in Example 1). The desalted, lyophilized, and unreacted myoglobin-digested peptide was reconstituted with 0.1% formic acid solution as a label-free control and analyzed by LC-MS / MS (sampling conditions as in Example 1). MaxQuant analysis showed the peak area changes of the myoglobin-digested peptide before and after labeling as follows: Figure 3 As shown, the peptide signal response exhibits a "centralization" trend. The standard deviation between labeled samples was calculated to be 4.64%, while the standard deviation between unlabeled samples was 3.02%, indicating that derivatization reduced the differences in signal response between peptides.

[0031] Example 3

[0032] 10 mg of *E. coli* cells were added to 1 mL of 7M urea lysis buffer (Sigma), and the mixture was ground and lysed for 10 min. The mixture was then centrifuged at 16000 x g for 15 min. The supernatant was collected and the protein concentration was determined using the BCA (Bicinchoninic Acid Assay) method, yielding a protein concentration of 1.27 mg / mL. 180 μg of *E. coli* protein lysis buffer was taken, and 50 picomolar (3.4 μg) bovine serum albumin (BSA) was added as an internal standard. Dithiothreitol (DTT) solution was then added to bring the final concentration to 10 mM. The mixture was denatured and reduced at 56 °C for 1 h, then cooled to room temperature. Iodoacetamide (IAA) solution was added to bring the final concentration to 25 mM, and the mixture was alkylated at room temperature in the dark for 30 min. The 7M urea solution was then diluted to 0.5 mM with 50 mM phosphate buffer, and trypsin (1:25 by mass) was added. The mixture was incubated overnight at 37 °C for 16 h. After desalting using a desalting column, the samples were lyophilized. A 10 mg / mL solution of the lyophilized peptides was prepared using DMSO. A derivatizing sensitizer, 1-(3-((2,5-dioxopyrrolidone-1-yl)oxy)-3-oxopropyl)-4-(trimethylammonium)pyridine bromide, was added at a peptide-to-sensitizer mass ratio of 1:10. The pH was adjusted to 9 with triethylamine, and the reaction was carried out at room temperature for 3 hours. The lyophilized samples were then reconstituted in 150 μL of 0.1% formic acid solution and analyzed using liquid chromatography-mass spectrometry (LC-MS / MS) (same acquisition conditions as in Example 1). The signal intensity of the proteins was obtained using MaxQuant database search analysis. Based on the BSA (protein number: P02769) content, the total protein content in 1 μL of *E. coli* sample was calculated to be 1.08 μg using the following formula. The content of each protein is shown in Table 1, and the protein number corresponds to its serial number in the Uniprot database.

[0033]

[0034] S: Protein content to be tested; A1: Sum of protein intensities to be tested; A2: Sum of internal standard intensities; I: Absolute content of internal standard.

[0035] Table 1. Protein content of Escherichia coli

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Claims

1. A method for large-scale absolute quantification of proteomics based on peptide sensitization, characterized by the following steps: include: (1) Add a standard protein (as an internal standard) of a different species from the biological sample and whose enzymatically digested peptides are not included in the enzymatically digested peptides of the biological sample to 1 microgram to 1 milligram of biological sample protein. The amount of standard protein added is 1 micromole to 1 femtomole. The standard protein participates in subsequent denaturation, reduction, alkylation, enzymatic digestion and desalting treatment to obtain the enzymatically digested peptides. (2) The above peptide samples were reacted with a derivatizing sensitizer to obtain labeled peptide samples. Mass spectrometry data were then acquired using a liquid chromatography-mass spectrometry system. Due to the different physicochemical properties of the peptides after derivatization, the ionization efficiency was low (signal response intensity is usually less than or equal to 10). 7 Increased ionization efficiency of peptides leads to increased mass spectrometry signal intensity, while high ionization efficiency (signal response intensity typically greater than 10) results in increased mass spectrometry signal intensity. 7 The changes in mass spectrometry signal intensity of peptides are relatively small, which leads to a reduction in the differences in mass spectrometry response signals between peptides. (3) Using the mass spectrometry signal intensity obtained above, a large-scale absolute proteome quantification algorithm is constructed. The signal response intensity information of all peptides corresponding to the protein is obtained by database retrieval and intensity extraction. The intensities of low ionization efficiency peptides and high ionization efficiency peptides belonging to the same protein are summed as the intensity information corresponding to the protein. The ratio of the intensity of the target protein and / or peptide in the sample to that of the internal standard is multiplied by the absolute content of the known target protein and / or peptide in the internal standard to obtain the absolute quantification of other target proteins and / or peptides to be analyzed in the sample, thereby realizing the absolute quantification of the sample proteome.

2. The method according to claim 1, characterized in that, The biological sample is one or more of the following: cells, microorganisms, biological tissues, or body fluids. Biological samples need to be digested with proteases. The proteases used must have one or more cleavage sites at the lysine carboxyl group, arginine carboxyl group, aspartic acid carboxyl group, glutamic acid carboxyl group, aspartic acid amino group, or non-specific cleavage sites.

3. The method according to claim 1, characterized in that, The derivatized sensitizer used consists of three parts: a host structure, a functional structure, and a reactive group; the functional structure acts as a "connecting arm" between the host structure and the reactive group, and the three are chemically bonded together to form a molecule; one or two reactive groups are connected to the host structure through one or two functional structures; The main structure is a quaternary ammonium salt, pyranonium salt, or phosphine salt containing one or more charges; or one or more of the following: pyridine, imidazole, pyran, piperazine, and guanidine salts containing a proton-affinity structure; The functional structure is one or more of the following: a C1-C10 alkyl carbon chain, or a 1-3 benzene ring structure; The reactive groups are those that react with amino and carboxyl groups. The groups that react with amino groups include N-hydroxysuccinimide groups, N-hydroxyphthalimide groups, and C1-C10 acyl halide groups. The groups that react with carboxyl groups are mainly C1-C10 amino groups.

4. The method according to claim 1 or 3, characterized in that, The specific operation method for the sample and the derivatized sensitizer is as follows: the desalted peptide sample is reacted with the derivatized sensitizer, the mass ratio of the sample to the derivatized sensitizer is (1:5 to 1:20), and the labeling time is 1 to 24 hours.

5. The method according to claim 3, characterized in that, The main structure of the derivatized sensitizer used is preferably a single or multiple charge structure. When the main single or multiple charge structure is highly hydrophilic, the reactive group is preferably a hydrophobic group, including N-hydroxyphthalimide group, etc. When the main body has a single or multiple charge structure with strong hydrophobicity, the reactive group is preferably a hydrophilic group, including one or more of N-hydroxysuccinimide groups, C1 to C5 amino groups, etc. This structural combination facilitates the purification of derivatized sensitizers in the early stages, while effectively improving peptide ionization efficiency during subsequent liquid chromatography-mass spectrometry analysis without interfering with large-scale mass spectrometry detection.

6. The method according to claim 1, characterized in that: The mass spectrometer used in the liquid chromatography-mass spectrometry (LC-MS) includes one or more of the following: an electrostatic orbital trap, an ion trap, a time-of-flight (TOF) tube, or a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer; the mass spectrometry acquisition mode can be one or two of the following: data-dependent acquisition (DDA) or data-independent acquisition (DIA).

7. The quantitative method according to claim 1, characterized in that: The quantitative method is suitable for large-scale absolute quantification of the target sample proteome, large-scale absolute quantification of target peptides, or absolute quantitative analysis of target proteins.

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