MULTIPLEX PROTEOME QUANTIFICATION METHOD BASED ON ISOBARIC DIMETHYL LABELING

Abstract

A multiplex proteome quantification method based on isobaric dimethyl labeling, which implements dimethyl labeling of peptide N-terminal in an acidic condition and C-terminal in an alkaline condition one after another by means of a property that a dimethylation reaction has different rates on an amino group at the peptide N-terminal and an amino group on a Lysine side chain at the peptide C-terminal in the acidic condition. Multiplex labeling of peptide samples is implemented by means of the organic combination of various isotope forms of a dimethyl labeling reagents. The mass-to-charge ratios in MS1 of peptides after multiplex labeling are completely the same, the mass-to-charge ratios of the fragment ions in MS2 are different, and multiplex quantitative analyses are carried out by extracting the intensity values of corresponding fragment ions in the MS2. The method has advantages of high quantitative accuracy, high precision, wide dynamic range and high-throughput of quantitative analysis, which can implement the simultaneous quantitative analysis of six protein samples, thereby greatly improving the throughput of the quantitative analysis of proteins and saving the analysis time.

Claims

1. A multiplex proteome quantification method based on isobaric dimethyl labeling, wherein a protein is cut into peptides using the protease, which cleaves at the carboxyl side of lysine; according to a property that a dimethyl reaction has different rates on an amino group at a peptide N-terminal and an amino group on a Lysine side chain at peptide C-terminal in the acidic condition, the dimethyl labeling of the peptide N-terminal and the peptide C-terminal is implemented in an acidic condition and an alkaline condition one after another; more than one or two of two to six samples are labeled by the combination of the dimethyl labeling reagents such as CH.sub.2O, NaBH.sub.3CN and corresponding isotopes thereof such as .sup.13CH.sub.2O, CD.sub.2O, .sup.13CD.sub.2O and NaBD.sub.3CN, to make the labeling of each peptide terminal different, and make the total mass-to-charge ratios of the peptides same, so the obtained MS1 of the peptides in a LC-MS has the same mass-to-charge ratio, and each fragment ion in an MS2 has difference in mass-to-charge ratio; and the multiplex quantitative analyses are carried out by the intensities of the fragment ions in the MS2.

2. The quantitative method according to claim 1, wherein the dimethyl labeling of the peptide N-terminal and the peptide C-terminal is implemented in the acidic condition and the alkaline condition one after another, and the sextuple labeling is implemented by the combination of the dimethyl labeling reagents such as CH.sub.2O, NaBH.sub.3CN and corresponding isotopes thereof; and the reagents for sextuple labeling are: Peptide N-terminal: .sup.13 CH.sub.2O+NaBH.sub.3CN-Peptide C-terminal: CD.sub.2O+NaBD.sub.3CN; Peptide N-terminal: CD.sub.2O+NaBH.sub.3CN-Peptide C-terminal: .sup.13CH.sub.2O+NaBD.sub.3CN; Peptide N-terminal: .sup.13CD.sub.2O+NaBH.sub.3CN-Peptide C-terminal: CH.sub.2O+NaBD.sub.3CN; Peptide N-terminal: CH.sub.2O+NaBD.sub.3CN-Peptide C-terminal: .sup.13CD.sub.2O+NaBH.sub.3CN; Peptide N-terminal: .sup.13CH.sub.2O+NaBD.sub.3CN-Peptide C-terminal: CD.sub.2O+NaBH.sub.3CN; Peptide N-terminal: CD.sub.2O+NaBD.sub.3CN-Peptide C-terminal: .sup.13CH.sub.2O+NaBH.sub.3CN.

3. The analysis method according to claim 1, wherein the sextuple labeling is simultaneously shown on each MS2, so the intensity values of the fragment ions of a, b and y which are simultaneously shown in the sextuple labeling of each labeled peptide on the MS2 are extracted and summed as an intensity of the labeled peptide; and the sextuple quantitative analysis is implemented by comparing the intensity of each of the sextuple labeling as the relative quantitative results.

4. The quantitative method according to claim 2, wherein protein samples are denatured, reduced, alkylated and then are incubated by a protease, which cleaves at the carboxyl side of lysine; for a protein sample after digestion, the dimethyl labeling is first selectively carried out on the amino group at the peptide N-terminal in the acidic condition; and then the dimethyl labeling is correspondingly carried out on the amino group on a Lysine side chain at the peptide C-terminal in the alkaline condition; the pH value of the acidic condition is controlled to be 2.0 to 5.0, and the labeling time is 5 to 120 min; and the pH value of the alkaline condition is controlled to be 7.5 to 12, and the labeling time is 5 to 120 min.

5. The quantitative method according to claim 1, wherein the mass spectrometry comprises Orbitrap, TOF or an FT-ICR.

6. The quantitative analysis according to claim 1, wherein comprising simultaneous quantitative analysis of more than one or more of two, three, four, five or six samples.

7. The quantitative analysis according to claim 1, wherein the protease, which cleaves at the carboxyl side of lysine, is a Lys-C protease.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0026] FIG. 1 is a schematic diagram of isobaric dimethyl labeling;

[0027] FIG. 2 is a theory mass spectrum diagram of the mixture of the labeling of six samples; and

[0028] FIG. 3 is a diagram of equivalently mixed quantitative results of six samples. The figure shows results obtained by taking the log 2 for the quantitative ratio after the six samples are mixed in a ratio of 1:1:1:1:1:1.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiment 1

[0029] 1. Protein Digestion

[0030] The protein samples are dissolved in 8 M urea solution, and the DTT is added to reach the final concentration of 10 mM, then the samples are denatured and reduced at 56 C. for 1 h, and the IAA is added to reach the final concentration of 20 mM, and the samples are kept in the dark at room temperature for 30 min, and then the urea solution is then diluted to 1 M. Finally, the Lys-C is added to the samples in a ratio of 1:50 (enzyme/protein, w/w), and the samples are digested at 37 C. overnight. The samples are desalted by C18 trap column and lyophilized, and then resolved with water.

[0031] 2. Dimethyl Labeling of Peptides

[0032] The sextuple dimethyl labeling is separately carried out on the peptides. As shown in Table 3, the specific labeling methods are as follows:

[0033] The first labeling: The peptide digests are loaded on a C18-trap column and firstly balanced by 1.5% (v/v) CH.sub.3COOH aqueous solution of the acid solution. The dimethyl labeling is selectively carried out on an amino group at peptide N-terminal in the acid solution, and the labeling solution is obtained by adding 50 L of 4% (v/v).sup.13CH.sub.2O and 50 L of 0.6 M (w/v) NaBH.sub.3CN to 1 mL 1.5% (v/v) CH.sub.3COOH aqueous solution for 20 min with the flow rate of 50 L/min. The trap column is then washed with 1.5% (v/v) CH.sub.3COOH aqueous solution of the acid solution and water, and then is balanced by the phosphate buffer with pH value of 8.0 of the alkaline solution. The dimethyl labeling is carried out on an amino group on a Lysine side chain at peptide C-terminal in the alkaline condition, and the labeling solution is obtained by adding 50 L of 4% (v/v) CD.sub.2O and 50 L of 0.6 M (w/v) NaBD.sub.3CN to 1 mL phosphate buffer with pH value of 8.0 for 10 min with the flow rate of 50 L/min. Finally, the trap column is washed with the phosphate buffer solution with pH value of 8.0 of the alkaline solution and water in sequence; and finally the sample is eluted with 80% (v/v) ACN from the trap column.

[0034] The second labeling: The labeling method is the same as the first labeling. The labeling solution in an acidic condition is changed to the solution obtained by adding 50 L of 4% (v/v) CD.sub.2O and 50 L of 0.6 M (w/v) NaBH.sub.3CN to 1 mL 1.5% (v/v) CH.sub.3COOH aqueous solution. The labeling solution in an alkaline condition is changed to the solution obtained by adding 50 L of 4% .sup.13CH.sub.2O and 50 L of 0.6 M (w/v) NaBD.sub.3CN to 1 mL phosphate buffer solution with pH value of 8.0.

[0035] The third labeling: The labeling method is the same as the first labeling. The labeling solution in an acidic condition is changed to the solution obtained by adding 50 L of 4% (v/v).sup.13CD.sub.2O and 50 L of 0.6 M (w/v) NaBH.sub.3CN to 1 mL 1.5% (v/v) CH.sub.3COOH aqueous solution. The labeling solution in an alkaline condition is changed to the solution obtained by adding 50 L of 4% (v/v) CH.sub.2O and 50 L of 0.6 M (w/v) NaBD.sub.3CN to 1 mL phosphate buffer solution with pH value of 8.0.

[0036] The fourth labeling: The labeling method is the same as the first labeling. The peptide digests are loaded on a C18-trap column and firstly balanced by 1.5% (v/v) CH.sub.3COOH D.sub.2O solution of the acid solution. The dimethyl labeling is selectively carried out on an amino group at peptide N-terminal in the acid solution, and the labeling solution is obtained by adding 50 L of 4% (v/v) CH.sub.2O and 50 L of 0.6 M (w/v) NaBD.sub.3CN to 1 mL 1.5% (v/v) CH.sub.3COOH D.sub.2O solution for 30 min with the flow rate of 25 L/min. The trap column is then washed with 1.5% CH.sub.3COOH aqueous solution of the acid solution and water in sequence, and then is balanced by the phosphate buffer solution with pH value of 8.0 of the alkaline solution. The dimethyl labeling is carried out on an amino group on a Lysine side chain at peptide C-terminal in the alkaline condition, and the labeling solution is obtained by adding 50 L of 4% (v/v).sup.13CD.sub.2O and 50 L of 0.6 M (w/v) NaBH.sub.3CN to 1 mL phosphate buffer solution with pH value of 8.0 for 10 min with the flow rate of 50 L/min. Finally, the trap column is washed with the phosphate buffer solution with pH value of 8.0 of the alkaline solution and water in sequence; and finally the sample is eluted with 80% (v/v) ACN from the trap column.

[0037] The fifth labeling: The labeling method is the same as the fourth labeling. The labeling solution in an acidic condition is changed to the solution obtained by adding 50 L of 4% (v/v).sup.13CH.sub.2O and 50 L of 0.6 M (w/v) NaBD.sub.3CN to 1 mL 1.5% (v/v) CH.sub.3COOH D.sub.2O solution. The labeling solution in an alkaline condition is changed to the solution obtained by adding 50 L of 4% (v/v) CD.sub.2O and 50 L of 0.6 M (w/v) NaBH.sub.3CN to 1 mL phosphate buffer solution with pH value of 8.0.

[0038] The sixth labeling: The labeling method is the same as the fourth labeling. The labeling solution in an acidic condition is changed to the solution obtained by adding 50 L of 4% (v/v) CD.sub.2O and 50 L of 0.6 M (w/v) NaBD.sub.3CN to 1 mL 1.5% (v/v) CH.sub.3COOH D.sub.2O solution. The labeling solution in an alkaline condition is changed to the solution obtained by adding 50 L of 4% (v/v).sup.13CH.sub.2O and 50 L of 0.6 M (w/v) NaBH.sub.3CN to 1 mL phosphate buffer solution with pH value of 8.0.

[0039] All the samples are lyophilized and then resolved to the mass concentration of 0.5 mg/mL with buffer solution A (containing aqueous solution of 2% (v/v) ACN and 0.1% (v/v) FA). The samples are mixed with fixed ratios to be for MS analysis.

[0040] 3. LC-MS Analysis.

[0041] Liquid chromatography condition: Mobile phases are buffer solution A (containing aqueous solution of 2% (v/v) ACN and 0.1% (v/v) FA) and buffer solution B (containing aqueous solution of 98% (v/v) water and 0.1% (v/v) FA). The gradient separation is performed using buffer solution B with a gradient of 0% for 10 min, the linear gradient separation is performed using buffer solution B with a gradient of 5% to 25% for 125 min, and then the linear gradient separation is performed using buffer solution B with a gradient of 25% to 35% for 10 min at a flow rate of 300 nL/min.

[0042] Mass acquisition condition: A Q-Exactive MS is used, and a data-dependent acquisition (DDA) mode is used. The resolution of Full MS is 70000 (m/z=200), and the mass range is from 350 to 1800 m/z. Ten most intensive ions are selected for MS/MS fragmentation. The dynamic exclusion time is 20 s. The activation type is HCD, a normalized collision energy is 28%, a isolation window is 2.0 Da, fixed first mass of the MS2 is 50.0 Da, and the resolution of MS/MS is 35000 (m/z=200). Each sample above is injected in parallel for three times.

[0043] 4. Data Analysis

[0044] The acquired raw files by MS use a MaxQuant (v1.2.2.5) software package, and use Andromeda as database search engine, to conduct database search. The fixed modification is set as cysteine carbamidomethylation; and the variable modification is set as acetyl (Protein N-term), oxidation (M), N-term and K respectively select the corresponding dimethyl labeling modifications. The human protein database is downloaded from ftp.uniprot.org on July, 2013. The mass tolerance of the MS1 is 6 ppm, and the mass tolerance of the MS2 is 20 ppm. The protease is Lys-C. Two missed cleavage sites are allowed. The FDRs less than 0.01 for proteins and peptides are required. For quantitative analysis, the identified peptides are theoretically fragmented and matched with the corresponding mgf files to extract all the eligible fragment ions. Then the ion pairs are matched, the appeared ion of each sample is kept, and then most of all the fragment ion intensities of the labeling are summed as the ion intensity of the peptide. The ion intensities of multiplex labeled peptides are compared in pairs as relative quantitative ratios of identified peptides of the spectra. The median of the spectra matching the same peptide is taken as a quantitative result of the peptide, and the medians of all identified peptides for the same protein are taken as a quantitative result of the protein. All the results of three replicates are combined, and the values of the same protein are averaged as the final quantitative result of the protein. All the analyses are completed by SPSS (v20) and Excel (v2010).

Method Evaluation:

[0045] 1. Six samples are mixed in a ratio of 1:1:1:1:1:1. The quantitative coverage is more than 95%. The simultaneous quantitative analysis of six protein samples can be implemented. The quantitative accuracy and precision results are shown in Table 1 and FIG. 3. The quantitative results are close to the theoretical values with high precision.

[0046] 2. Wide quantitative dynamic range: The samples are mixed in a ratio of 1:2:5:10:20:50. The results are shown in Table 2. This method can achieve dynamic ranges of 50-fold with good quantitative accuracy, and doesn't have underestimated effect based on a reporter ion quantitative method, which is due to the fact that the method is based on a quantitative method of multiple fragment ions of the MS2, thereby effectively reducing the quantitative interference of the co-eluted peptides.

Embodiment 2

[0047] The labeling process is carried out in a centrifuge tube. After the labeling in an acidic condition, the solvent is exchanged by a C18-trap column. Then the labeling in an alkaline condition is carried out after elution. The other processes are the same as those in Embodiment 1.

Embodiment 3

[0048] The labeling process in the acidic condition is carried out in a centrifuge tube. After the solvent is exchanged by the C18-trap column, the labeling in the alkaline condition is carried out on the trap column. The other processes are the same as those in Embodiment 1.

Embodiment 4

[0049] The dimethyl labeling is selectively carried out on the amino group at the peptide N-terminal in the acidic condition, and the labeling solution comprises 1% (v/v) CH.sub.3COOH aqueous solution, 4% (v/v) CH.sub.2O aqueous solution and isotopic forms thereof, and 0.6 M (w/v) NaBH.sub.3CN solution and isotopic forms thereof. The labeling time is 10 min. The other processes are the same as those in Embodiment 1.

Embodiment 5

[0050] The quadruple labeling is achieved by using the first four labeling methods in Embodiment 1. The other processes are the same as those in Embodiment 1.

TABLE-US-00001 TABLE 1 Quantitative accuracy and precision results of six equivalently mixed samples 30H/30L 32L/30L 32H/30L 34L/30L 34H/30L Average 0.92 1.20 0.96 0.98 1.03 RSD/% 14 16 14 14 14

TABLE-US-00002 TABLE 2 Quantitative results of dynamic range Theoretical ratio (Heavy/Light Labeling) 2 5 10 20 50 Experimental ratio 2.54 5.34 13.28 21.39 53.00 (Heavy/Light Labeling)

TABLE-US-00003 TABLE 3 Sextuple labeling methods Flow rate Flow rate Mass increase of Labeling and time for Labeling and time for both terminals Multi- Abbrev- reagent of labeling of reagent of labeling of (N-terminal- plicities iation N-terminal N-terminal K-terminal K-terminal K-terminal/Da) 1 2L-6H .sup.13CH.sub.2O + 20 min CD.sub.2O + 10 min 30.03801-34.06896 NaBH.sub.3CN 50 L/min NaBD.sub.3CN 50 L/min 2 4H-4L CD.sub.2O + .sup.13CH.sub.2O + 32.05641-32.05056 NaBH.sub.3CN NaBD.sub.3CN 3 6L-2H .sup.13CD.sub.2O + CH.sub.2O + 34.06312-30.04385 NaBH.sub.3CN NaBD.sub.3CN 4 2H-6L CH.sub.2O + 30 min .sup.13CD.sub.2O + 30.04385-34.06312 NaBD.sub.3CN 25 L/min NaBH.sub.3CN 5 4L-4H .sup.13CH.sub.2O + CD.sub.2O + 32.05056-32.05641 NaBD.sub.3CN NaBH.sub.3CN 6 6H-2L CD.sub.2O + .sup.13CH.sub.2O + 34.06896-30.03801 NaBD.sub.3CN NaBH.sub.3CN

[0051] The method has advantages of high accuracy, good precision, and wide dynamic range, which can simultaneously implement the simultaneous quantitative analysis of six protein samples, thereby greatly improving the throughput of the quantitative analysis of proteins and saving the analysis time.