CN105785048B - Based on the nearly overall Protein quantitative analysis method with heavy label of light isotope - Google Patents
Based on the nearly overall Protein quantitative analysis method with heavy label of light isotope Download PDFInfo
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Abstract
The present invention relates to a kind of based on the nearly overall Protein quantitative analysis method with heavy label of light isotope.First using two groups of different physiology or the overall albumen of pathological conditions as control group and disease group, lysine ε amino and N-terminal amino all same heavy labels are carried out respectively, N (C are respectively labeled as5H11O2CH2)2With N (C5H11SCH2)2；High resolution mass spectrum is carried out by equal proportion mixing and cascade mass spectrometry obtains a data group with two histones after heavy label；Qualitative, quantitative data library searching is carried out to data group, the relative scale of ID and each protein of disease group relative to control group of protein is obtained, i.e., the up-regulation of all albumen or downward situation under disease conditions.Compared with prior art, not only labeling effciency is high for quantitative mark reagent of the invention, and the degree of accuracy is high；And it is cheap, it is easy to get, it is adaptable to overall protein-based quantifying in high-resolution cascade mass spectrometry.
Description
Technical field
It is near based on light isotope more particularly, to one kind the present invention relates to the quantitative analysis method of a kind of protein or polypeptide
With the overall Protein quantitative analysis method of heavy label.
Background technology
Over nearly 1 year, commercialized high-quality resolution, high-quality measurement accuracy mass spectrograph have rapid development, also
It is the appearance of Orbitrap mass spectrometer.Orbitrap mass spectrometer is with fourier transform ion cyclotron resonance mass spectrometer resolution ratio and precision phase
When；But price is relatively cheap, mass spectrum picking rate faster, dissociation efficiency it is higher.The mass spectrometric relative popularization is molecular weight phase
Solid foundation is provided to the mass spectrum and cascade mass spectrometry of larger overall albumen.In terms of overall protein quantification mark,
Internal mark based on SILAC and the labeled in vitro based on TMT, due to targetting the non-fully mark of amino acid (such as the bad ammonia of weight
Non-fully replacement in SILAC of acid or arginine) and the unselected marker of non-targeted amino acid (TMT of such as threonine is marked
Note), its application is received than larger limitation.Dimethyl (isotope, isotopic or with weight, isobaric) mark by
It is easy to get in its reagent, labeling effciency is high, extensive research and application has been obtained at the quantitative aspect of polypeptide；China scientist is at this
Huge contribution is also made that in terms of individual quantitative technique, has developed a variety of various forms of dimethyl marks；Wherein Chinese science
The N-terminal of Dalian Chemical Physics Research Institute of institute Zhang Yukui academician and the development of Zhang Lihua researcher seminar is with weight dimethyl mark and again
The guanidineization protection that denier university Yang Peng original professor and land hero teach the amino on the lysine residue of seminar's development combines
The quantitative mark of albumen is may be directly applied to, there is preferable prospect.
However, it is above-mentioned with weight dimethyl mark used in heavy isotope (13C and D) reagent is all very expensive, such as 1mL
20%13C formaldehyde (H13CHO price) is 4350 yuan (Cambridge Isotope Lab, product code CLM-806-PK), 1g
The price of deuterated sodium cyanoborohydride is 2377.25 yuan (Sigma, product code 190020-1G).Therefore, it is new and effective but honest and clean
The reagent of valency has wide prospect and market.
Applicant's early stage has done more work in terms of overall protein qualitative and quantitative analysis, there is preferable base
Plinth.
In terms of quantitative data excavation, it is biological big that publication No. discloses a kind of identification for CN103389335A Chinese patent
The analytical equipment and method of molecule.Publication No. discloses a kind of biological mass spectrometry database for CN 104765984A Chinese patent
The quick method set up with search.Publication No. discloses a kind of overlapping same position of biological mass spectrometry for CN104359967A Chinese patent
The analytic method of plain profile.Isotope profile fingerprint comparison algorithm (isotopic envelope are disclosed in above-mentioned patent
Fingerprinting, iEF), the search of matching ion and the identification of protein are carried out directly in initial data.The algorithm without
" removing isotope " pretreatment need to be carried out to experimental data, the corresponding time can be saved；According to whether there is same position in each ion
The deviation of plain peak missing and isotopic peak relative intensity is distinguished well to preferable and non-ideal experimental data, it is ensured that albumen
The confidence level of identification；Effectively solved according to the overlapping experimental data of isotopic peak relative intensity relation pair of known overlapping ion
Analysis.Overall albumen database search engine ProteinGoggle is successfully developed based on algorithm applicant, in single standard
Albumen (ubiquitin, myoglobins) and overall protein group mixture (histone H 4 family posttranslational modification isomers, large intestine bar
Bacterium) Qualitative Identification in, show higher confidence level and preferable application prospect.In terms of quantitative analysis,
ProteinGoggle have in it and unique potential advantages；On the one hand isotope profile and wherein each same position are based on
The fingerprint comparison of element can quickly and accurately search for isotope or the quota ion pair with heavy label；On the other hand, search procedure
In in each ion the laboratory strength of each isotopic peak be recorded and export, can be for directly calculating relative quantification ratio.
In terms of quantitative mark, publication No. discloses a kind of overall quantification of protein for CN105137088A Chinese patent
Analysis method.Publication No. discloses overall albumen under a kind of different physiology or pathological conditions for CN105242050A Chinese patent
The quantitative analysis method of matter, the patent carries out same heavy label to N-terminal amino, is respectively labeled as-N (CH2D)2With-N (13CH3)2.On
State the quantitative mark method that two parts of patents are all based on heavy isotope.Due to using heavy isotope (13C and D), therefore its expense is still
It is so very high.
The content of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide one kind is based on light isotope
The nearly overall Protein quantitative analysis method with heavy label.
The purpose of the present invention can be achieved through the following technical solutions：
It is a kind of based on the nearly overall Protein quantitative analysis method with heavy label of light isotope, comprise the following steps：
(1) using two groups of different physiology or the overall albumen of pathological conditions as control group and disease group, carry out respectively
The whole same heavy label of Lysine s-amino groups and N-terminal amino, is respectively labeled as-N (C5H11O2CH2)2With-N (C5H11SCH2)2, its list
The mass discrepancy of amino is 0.017759Da after individual mark；
(2) carry out high resolution mass spectrum by equal proportion mixing with two histones after heavy label and cascade mass spectrometry obtains one
Data group；
(3) qualitative, quantitative data library searching is carried out to data group, obtains the ID and disease group each protein of protein
Up-regulation or downward situation relative to all albumen under the relative scale of control group, i.e. disease conditions, up-regulation or downward multiple are most
Big albumen is the albumen related to disease generation, development.
Preferably, in step (1), control histone or disease histone, a histone matter sodium cyanoborohydride
(NaCNBH3) and 4- (methoxymethoxy) butyraldehyde (C5H11O2CHO) mark, another histone matter sodium cyanoborohydride
(NaCNBH3) and 5- (methyl mercapto) valerals (C5H11SCHO) mark.
Preferably, progress is as follows with the course of reaction and condition of heavy label in step (1)：Protein dissolution is in acetate buffer
After solution, 4- (methoxymethoxy) butyraldehyde or 5- (methyl mercapto) valeral are added, sodium cyanoborohydride is added under agitation
Solution, after reacting at room temperature 1 hour, is quenched with ammoniacal liquor.
Preferably, described sodium acetate buffer solution is 100mmol/L, and pH is 5-6.
Preferably, described 4- (methoxymethoxy) butyraldehyde or 5- (methyl mercapto) valerals liquid quality fraction are 4% (w/
w)。
Preferably, described ammonia spirit volume fraction is 4% (v/v).
Equal proportion mixing described in step (2) refers to：According to weight, volume or the mole of control group and disease group with 1:1
Ratio is mixed.
In step (3) to data group carry out qualitative, quantitative data library searching, obtain protein ID and disease group each
Protein is the ordinary skill in the art relative to the relative scale of control group, preferably uses the overall egg introduced in background technology
White database search engine ProteinGoggle, while other databases can also be used.
Method of the present invention is equally applicable in protein sequence Lysine s-amino groups and N-terminal amino, and other are based on just
Normal light isotope it is complete with weight chemical covalent mark and amino acid other particular functional groups based on the complete of normal light isotope
With weight chemical covalent mark.
Compared with prior art, analytic method of the invention is based on the mass spectrographic original second order mses, is contained by calculating
The average relative intensity of labelling groups fragment ion is relatively strong under different physiology or pathological conditions so as to calculate labelled protein
Degree.Same heavy label and its high score of the quantitative approach of the present invention to Lysine s-amino groups in overall protein sequence and N-terminal amino
The data parsing in situ of tandem mass spectrometry mark fragment ion isotope profile fingerprint comparison is distinguished, labeling effciency is high, and the degree of accuracy is high, is applicable
In the overall protein-based quantitative analysis in high-resolution tandem mass spectrometry.
Brief description of the drawings
Fig. 1, based on the nearly overall Protein quantitative analysis method flow diagram with heavy label of light isotope.
Fig. 2, protein molecule Lysine s-amino groups and N-terminal amino are complete with heavy label schematic diagram.
Embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment
It is a kind of based on the nearly overall Protein quantitative analysis method with heavy label of light isotope, comprise the following steps：
(1) using two groups of different physiology or the overall albumen of pathological conditions as control group and disease group, carry out respectively
The whole same heavy label of Lysine s-amino groups and N-terminal amino, one group with sodium cyanoborohydride (NaCNBH3) and 4- (methoxyl group methoxies
Base) butyraldehyde (C5H11O2CHO), sodium cyanoborohydride (NaCNBH is used for another group3) and 5- (methyl mercapto) valerals (C5H11SCHO)
(as shown in Figure 1)；Namely it is respectively labeled as-N (C5H11O2CH2)2With-N (C5H11SCH2)2(as shown in Figure 2), its single marking
The mass discrepancy of amino is 0.017759Da afterwards, and course of reaction and condition are as follows：Protein dissolution is in sodium acetate buffer solution
After (100mM, pH 5-6), 4% (w/w) just prepared aldehyde solution is added；600mM new preparation is added under agitation
Sodium cyanoborohydride solution.After room temperature reaction 1 hour, reaction is quenched with 4% (v/v) ammoniacal liquor；
(2) carry out high resolution mass spectrum by equal proportion mixing with two histones after heavy label and cascade mass spectrometry obtains one
Data group, equal proportion mixing refers to：According to weight, volume or the mole of control group and disease group with 1:1 ratio is mixed；
(3) qualitative, quantitative data library searching is carried out to data group, obtains the ID and disease group each protein of protein
Up-regulation or downward situation relative to all albumen under the relative scale of control group, i.e. disease conditions, up-regulation or downward multiple are most
Big albumen is the albumen related to disease generation, development.Qualitative, quantitative data library searching is carried out to data group, obtained
The ID and each protein of disease group of protein are the ordinary skill in the art relative to the relative scale of control group, are preferably made
With the overall albumen database search engine ProteinGoggle introduced in background technology, while other data can also be used
Storehouse.
The method of above example is equally applicable in protein sequence Lysine s-amino groups and N-terminal amino, and other are based on just
Normal light isotope it is complete with weight chemical covalent mark and amino acid other particular functional groups based on the complete of normal light isotope
With weight chemical covalent mark.
Above analytic method is based on the mass spectrographic original second order mses, by calculating the flat of the fragment ion containing labelling groups
Equal relative intensity is so as to calculate relative intensity of the labelled protein under different physiology or pathological conditions.The quantitative approach pair of the present invention
The same heavy label of Lysine s-amino groups and N-terminal amino and its high-resolution tandem mass spectrometry mark fragment ion in overall protein sequence
Isotope profile fingerprint comparison original position data parsing, labeling effciency is high, and the degree of accuracy is high, it is adaptable to overall protein-based in high-resolution
The quantitative analysis of tandem mass spectrometry.
The above-mentioned description to embodiment is understood that for ease of those skilled in the art and using invention.
Person skilled in the art obviously can easily make various modifications to these embodiments, and described herein general
Principle is applied in other embodiment without passing through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability
Field technique personnel are according to the announcement of the present invention, and not departing from improvement and modification that scope made all should be the present invention's
Within protection domain.
Claims (7)
1. it is a kind of based on the nearly overall Protein quantitative analysis method with heavy label of light isotope, it is characterised in that including following
Step：
(1) using two groups of different physiology or the overall albumen of pathological conditions as control group and disease group, carry out relying ammonia respectively
The whole same heavy label of sour epsilon-amino and N-terminal amino, is respectively labeled as-N (C5H11O2CH2)2With-N (C5H11SCH2)2, its single mark
The mass discrepancy of amino is 0.017759Da after note；
(2) carry out high resolution mass spectrum by equal proportion mixing with two histones after heavy label and cascade mass spectrometry obtains a data
Group；
(3) qualitative, quantitative data library searching is carried out to data group, the ID and each protein of disease group for obtaining protein are relative
Up-regulation or downward situation in all albumen under the relative scale of control group, i.e. disease conditions, up-regulation or downward multiple maximum
Albumen is the albumen related to disease generation.
2. it is according to claim 1 a kind of based on the nearly overall Protein quantitative analysis method with heavy label of light isotope,
Characterized in that, in step (1), control histone or disease histone, a histone matter sodium cyanoborohydride and 4- (methoxies
Ylmethoxy) butyraldehyde mark, another histone matter sodium cyanoborohydride and 5- (methyl mercapto) valeral mark.
3. it is according to claim 2 a kind of based on the nearly overall Protein quantitative analysis method with heavy label of light isotope,
Characterized in that, progress is as follows with the course of reaction and condition of heavy label in step (1)：Protein dissolution is in sodium acetate buffer solution
Afterwards, 4- (methoxymethoxy) butyraldehyde or 5- (methyl mercapto) valeral are added, sodium cyanoborohydride solution is added under agitation,
After room temperature reaction 1 hour, it is quenched with ammoniacal liquor.
4. it is according to claim 3 a kind of based on the nearly overall Protein quantitative analysis method with heavy label of light isotope,
Characterized in that, described sodium acetate buffer solution is 100mmol/L, pH is 5-6.
5. it is according to claim 3 a kind of based on the nearly overall Protein quantitative analysis method with heavy label of light isotope,
Characterized in that, described 4- (methoxymethoxy) butyraldehyde or 5- (methyl mercapto) valerals liquid quality fraction are 4% (w/w).
6. it is according to claim 3 a kind of based on the nearly overall Protein quantitative analysis method with heavy label of light isotope,
Characterized in that, described ammonia spirit volume fraction is 4% (v/v).
7. it is according to claim 1 a kind of based on the nearly overall Protein quantitative analysis method with heavy label of light isotope,
Characterized in that, the equal proportion mixing described in step (2) refers to：According to weight, volume or the mole of control group and disease group with
1:1 ratio is mixed.
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| CN108088945B (en) * | 2016-11-21 | 2020-11-17 | 中国科学院大连化学物理研究所 | Absolute quantification method based on dimethylation multiple markers and characteristic fragment ions |
| CN111208299B (en) * | 2018-11-21 | 2021-05-28 | 中国科学院大连化学物理研究所 | Qualitative and quantitative analysis method for cross-linked peptide fragments |
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| US20100159501A1 (en) * | 2008-12-24 | 2010-06-24 | Florida State University Research Foundation | Method for quantification of cellular sphingolipids |
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| WO2005005990A2 (en) * | 2003-04-14 | 2005-01-20 | Novartis Ag | Inverse labeling method for the rapid identification of marker target proteins |
| TW200512459A (en) * | 2003-09-23 | 2005-04-01 | Univ Nat Cheng Kung | Global analysis for protein expression kit and protein qualitative and quantitative method thereof |
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| WO2008150091A2 (en) * | 2007-06-05 | 2008-12-11 | The Industry & Academic Cooperation In Chungnam National University (Iac) | Novel analytical method for protein |
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| CN105137088A (en) * | 2015-08-27 | 2015-12-09 | 同济大学 | Whole-body-protein quantitative analysis method |
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