CN105137088A - Whole-body-protein quantitative analysis method - Google Patents
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Abstract
The invention relates to a whole-body-protein quantitative analysis method. The whole-body-protein quantitative analysis method includes the steps that firstly, two sets of whole-body proteins under different physiological or pathological conditions serve as a control set and a disease set respectively, possibly-existing disulfide bonds are reduced, and meanwhile alkylated protection is carried out on sulfydryl; secondly, full same-weight chemical covalence marking is carried out on specific functional groups of amino acid of the two sets of alkylated proteins; thirdly, the two sets of proteins obtained after same-weight marking are mixed in equal proportions, high resolution mass spectrum and tandem mass spectrometry analysis is carried out, and a data set is obtained; finally, qualitative and quantitative database searching is carried out on the data set, and the ID of the proteins and the relative proportions of the proteins of the disease set relative to the control set are obtained; in other words, up-regulation or down-regulation conditions of all the proteins under the disease condition are obtained, wherein the protein with the largest up-regulation or down-regulation time number is the protein related to occurrence and development of a disease. Compared with the prior art, the method is high in marking efficiency and accuracy and suitable for high-resolution-tandem-mass-spectrometry-based quantitative analysis of the whole-body proteins.
Description
Technical field
The present invention relates to a kind of analytical approach of protein molecule, especially relate to a kind of overall Protein quantitative analysis method, relate generally to the technical fields such as the systems biology relevant to biological mass spectrometry, proteomics.
Background technology
Over nearly 1 year, business-like high-quality resolution, high-quality measuring accuracy mass spectrometer have had rapid development, the namely appearance of Orbitrap mass spectrometer.Orbitrap mass spectrometer is suitable with precision with fourier transform ion cyclotron resonance mass spectrometer resolution; But price is relatively cheap, mass spectrum picking rate is faster, dissociation efficiency is higher.This is mass spectrometric popularizes relatively as the mass spectrum of the relatively large overall albumen of molecular weight and cascade mass spectrometry provide solid foundation.In overall protein quantification mark, based on the body internal labeling of SILAC and the labeled in vitro based on TMT, due to target amino acid whose non-fully mark (as heavy lysine or the non-fully of arginine in SILAC substitute) and non-targeted amino acid whose unselected marker (TMT as threonine marks), its range of application receives larger restriction.Dimethyl (isotope, isotopic or same heavy, isobaric) marks because its reagent is easy to get, and labeling effciency is high, obtains investigation and application widely in polypeptide is quantitative; China scientist has also made huge contribution in this quantitative technique, has developed multiple multi-form dimethyl mark; Wherein the guanidine protection group of the Lysine s-amino groups of the same heavy dimethyl mark of the N end of Dalian Inst of Chemicophysics, Chinese Academy of Sciences Zhang Yukui academician and the development of Zhang Lihua researcher seminar and the former professor of Fudan University's poplar Peng and the development of land hero's teach problem group can directly apply to the quantitative mark of albumen altogether, has good prospect.
In overall Protein Data Bank new algorithm and engine development, Chinese patent CN103389335A, CN104359967A, CN104765984A disclose isotope profile fingerprint comparison algorithm (isotopicenvelopefingerprinting, iEF), in raw data, the coupling search of ion and the qualification of protein is directly carried out.This algorithm, without the need to carrying out " removing isotope " pre-service to experimental data, can save the corresponding time; According to whether there being the deviation of isotopic peak disappearance and isotopic peak relative intensity well to distinguish desirable and imperfect experimental data in each ion, ensure the confidence level of Identification of Fusion Protein; Isotopic peak relative intensity relation according to known overlapping ion is effectively resolved overlapping experimental data.Overall albumen database search engine ProteinGoggle is successfully developed based on this algorithm applicant, in the Qualitative Identification of single standard protein (ubiquitin, myoglobins) and overall Leaf proteins potpourri (histone H 4 family posttranslational modification isomeride, Escherichia coli), show higher confidence level and good application prospect.In quantitative test, ProteinGoggle have its inherence with the potential advantages of uniqueness; On the one hand based on isotope profile and wherein each isotopic fingerprint comparison can search for isotope or the quota ion pair with heavy label quickly and accurately; On the other hand, in search procedure, in each ion, the laboratory strength of each isotopic peak is recorded and exports, and can be used for directly calculating relative quantification ratio.
Summary of the invention
Object of the present invention be exactly provide to overcome defect that above-mentioned prior art exists a kind of labeling effciency high, without the need to protecting part functional group in advance, overall protein quantitation methods that accuracy is high.
Object of the present invention can be achieved through the following technical solutions:
A kind of overall Protein quantitative analysis method, comprises the following steps:
(1) using the overall protein of two groups of different physiology or pathological conditions as control group and disease group, and respectively the disulfide bond that may exist is reduced, alkylation protection is carried out to sulfydryl simultaneously;
(2) complete same heavy chemical covalent mark is carried out to the amino acid particular functional group of two groups of alkylating protein;
(3) high resolution mass spectrum is carried out and cascade mass spectrometry obtains a data group with two histone matter after heavy label by equal proportion mixing;
(4) qualitative, quantitative data library searching is carried out to data group, obtain the ID of protein and each protein of disease group relative scale relative to control group, the i.e. rise of all albumen or the situation of downward under disease conditions, raises or lowers the maximum albumen of multiple and be and occur to this disease, develop relevant albumen.
Preferably, with dithiothreitol (DTT), the disulfide bond that may exist is reduced in step (1), with iodoacetamide, alkylation protection is carried out to all sulfydryls.
Preferably, amino acid particular functional group comprises Lysine s-amino groups and N Amino End Group in step (2), and the method is equally applicable to the complete in heavy chemical covalent mark of other particular functional groups in protein sequence except Lysine s-amino groups and N Amino End Group simultaneously.
Preferably, in step (2), the amino acid particular functional group in a histone matter is labeled as-N (CH
2d)
2, the amino acid particular functional group in another histone matter be labeled as-N (
13cH
3)
2, mass discrepancy amino after single marking is 0.00584Da.Same, the inventive method is equally applicable to Lysine s-amino groups and other complete same heavy chemical covalent marks of N Amino End Group in protein sequence.
For being labeled as-N (CH
2d)
2with-N (
13cH
3)
2method, a histone matter formaldehyde and deuterated sodium cyanoborohydride mark, another histone matter use
13c labeled formaldehyde and sodium cyanoborohydride mark; Course of reaction and condition as follows: after alkylating protein is dissolved in sodium acetate buffer solution again, add 4% (v/v) formalin; Add the sodium cyanoborohydride solution of the new preparation of 600mM under agitation, room temperature reaction is after 1 hour, with the ammoniacal liquor cancellation of 4% (v/v).Wherein, described sodium acetate buffer solution is 100mmol/L, pH is 5-6.
Equal proportion mixing described in step (3) refers to: mix with 1:1 ratio according to the weight of control group and disease group, volume or molar weight.
In step (4), qualitative, quantitative data library searching is carried out to data group, ID and each protein of disease group of obtaining protein are the ordinary skill in the art relative to the relative scale of control group, the overall albumen database search engine ProteinGoggle introduced in preferred use background technology, also can use other databases simultaneously.
Compared with prior art, the present invention has the following advantages and beneficial effect:
1, analytic method of the present invention is based on described mass spectrographic original second order ms, by calculating the average relative intensity thus the relative intensity of calculating labelled protein under different physiology or pathological conditions that contain labelling groups fragmention.Quantivative approach of the present invention is to the same heavy label of Lysine s-amino groups in overall protein sequence and N Amino End Group and its high-resolution tandem mass spectrometry mark fragmention isotope profile fingerprint comparison original position Data Analysis, labeling effciency is high, accuracy is high, is applicable to overall protein-based in the quantitative test of high-resolution tandem mass spectrometry.
2, the inventive method only needs to carry out disulfide bond reduction to protein; alkylation protection is carried out with to sulfydryl; then carry out complete same heavy chemical covalent to mark; do not need to carry out guanidine protection to the epsilon-amino on lysine residue in protein sequence; method simple and fast, labeling effciency is high simultaneously, subsidiary reaction is few, accuracy is high.
Accompanying drawing explanation
Fig. 1 is the quantitative process flow diagram marked with heavy dimethyl based on protein lysine epsilon-amino and N Amino End Group.
Fig. 2 is that protein molecule Lysine s-amino groups and N Amino End Group mark schematic diagram with heavy dimethyl entirely.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
Embodiment
A kind of overall Protein quantitative analysis method, adopts overall flow as shown in Figure 1, comprises the following steps:
The overall albumen (for control group and disease group) of (1) two kind of different physiology or pathological conditions first unification dithiothreitol (DTT) reduces to the disulfide bond that may exist; Then with iodoacetamide, alkylation protection is carried out to all sulfydryls;
(2) two histone matter after alkylation use respectively formaldehyde and deuterated sodium cyanoborohydride,
13c labeled formaldehyde and sodium cyanoborohydride carry out whole same heavy label (as shown in Figure 2) to the Lysine s-amino groups in protein molecular and N Amino End Group; Namely be labeled as-N (CH respectively
2d)
2with-N (
13cH
3)
2, mass discrepancy amino after its single marking is 0.00584Da.Course of reaction and condition as follows: after the albumen after alkylation is dissolved in sodium acetate buffer solution (100mM, pH5-6) again, add 4% formalin just prepared; Add the sodium cyanoborohydride solution of the new preparation of 600mM under agitation.Room temperature reaction, after 1 hour, reacts the ammoniacal liquor cancellation with 4%;
(3) high resolution mass spectrum is carried out with two histones after heavy label in equal proportion mixing (mixing with 1:1 ratio according to the weight of control group and disease group, volume or molar weight) and cascade mass spectrometry obtains a data group;
(4) ProteinGoggle carries out qualitative, quantitative data library searching to data group, obtains the ID of protein and each protein of disease group relative scale relative to control group, i.e. the rise of all albumen or the situation of downward under disease conditions.Namely the albumen raised or lower multiple maximum occur to this disease, develop relevant albumen.
Above-mentioned analytic method, based on described mass spectrographic original second order ms, contains the average relative intensity of labelling groups fragmention by calculating thus calculates the relative intensity of labelled protein under different physiology or pathological conditions.This quantivative approach is to the same heavy label of Lysine s-amino groups in overall protein sequence and N Amino End Group and its high-resolution tandem mass spectrometry mark fragmention isotope profile fingerprint comparison original position Data Analysis, labeling effciency is high, accuracy is high, is applicable to overall protein-based in the quantitative test of high-resolution tandem mass spectrometry.
Above-mentioned is can understand and use invention for ease of those skilled in the art to the description of embodiment.Person skilled in the art obviously easily can make various amendment to these embodiments, and General Principle described herein is applied in other embodiments and need not through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, those skilled in the art, according to announcement of the present invention, do not depart from improvement that scope makes and amendment all should within protection scope of the present invention.
Claims (7)
1. an overall Protein quantitative analysis method, is characterized in that, comprise the following steps:
(1) using the overall protein of two groups of different physiology or pathological conditions as control group and disease group, and respectively the disulfide bond that may exist is reduced, alkylation protection is carried out to sulfydryl simultaneously;
(2) complete same heavy chemical covalent mark is carried out to the amino acid particular functional group of two groups of alkylating protein;
(3) high resolution mass spectrum is carried out and cascade mass spectrometry obtains a data group with two histone matter after heavy label by equal proportion mixing;
(4) qualitative, quantitative data library searching is carried out to data group, obtain the ID of protein and each protein of disease group relative scale relative to control group, the i.e. rise of all albumen or the situation of downward under disease conditions, raises or lowers the maximum albumen of multiple and be and occur to this disease, develop relevant albumen.
2. the overall Protein quantitative analysis method of one according to claim 1, is characterized in that, reduces, carry out alkylation protection with iodoacetamide to all sulfydryls in step (1) with dithiothreitol (DTT) to the disulfide bond that may exist.
3. the overall Protein quantitative analysis method of one according to claim 1, is characterized in that, in step (2), amino acid particular functional group comprises Lysine s-amino groups and N Amino End Group.
4. the overall Protein quantitative analysis method of one according to claim 1, is characterized in that, in step (2), the amino acid particular functional group in a histone matter is labeled as-N (CH
2d)
2, the amino acid particular functional group in another histone matter be labeled as-N (
13cH
3)
2, mass discrepancy amino after single marking is 0.00584Da.
5. the overall Protein quantitative analysis method of one according to claim 4, is characterized in that, a histone matter formaldehyde and deuterated sodium cyanoborohydride mark, another histone matter is used
13c labeled formaldehyde and sodium cyanoborohydride mark;
Course of reaction and condition as follows: after alkylating protein is dissolved in sodium acetate buffer solution again, add 4% (v/v) formalin; Add the sodium cyanoborohydride solution of the new preparation of 600mM under agitation, room temperature reaction is after 1 hour, with the ammoniacal liquor cancellation of 4% (v/v).
6. the overall Protein quantitative analysis method of one according to claim 5, is characterized in that, described sodium acetate buffer solution is 100mmol/L, pH is 5-6.
7. the overall Protein quantitative analysis method of one according to claim 1, is characterized in that, the mixing of equal proportion described in step (3) refers to: mix with 1:1 ratio according to the weight of control group and disease group, volume or molar weight.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105785048A (en) * | 2016-04-15 | 2016-07-20 | 同济大学 | Global protein quantitative analysis method based on light isotope and isobar labeling |
| CN105866429A (en) * | 2016-05-06 | 2016-08-17 | 同济大学 | Biomolecular marker based on isotope reagent and provided with electric charge itself and quantitative analysis method |
| CN106093224A (en) * | 2016-06-01 | 2016-11-09 | 同济大学 | A kind of polysaccharide Sync enrichment and the nearly quantitative analysis method with heavy label |
| CN106990159A (en) * | 2017-05-04 | 2017-07-28 | 同济大学 | A kind of protein quantitation methods based on complete accurate same weight diethyl mark |
| CN109580814A (en) * | 2018-12-06 | 2019-04-05 | 复旦大学 | The synchronous quantitative analysis method of a variety of amino metabolins based on N- alkylation process |
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| CN104076098A (en) * | 2013-03-29 | 2014-10-01 | 中国科学院大连化学物理研究所 | Protein quantitative method utilizing equiponderance dimethylation marking |
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2015
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Patent Citations (4)
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| CN101042375A (en) * | 2006-03-20 | 2007-09-26 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method and reagent kit for rare-earth element metal chelating making quantitative proteome |
| CN101339187A (en) * | 2008-08-15 | 2009-01-07 | 中国科学院上海有机化学研究所 | Methylsulfonyl miazines isotope labelling reagent, synthesis method and uses thereof |
| CN104076098A (en) * | 2013-03-29 | 2014-10-01 | 中国科学院大连化学物理研究所 | Protein quantitative method utilizing equiponderance dimethylation marking |
| CN105242050A (en) * | 2015-09-16 | 2016-01-13 | 同济大学 | Quantitative analysis method for intact protein under different physiological or pathological conditions |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105785048A (en) * | 2016-04-15 | 2016-07-20 | 同济大学 | Global protein quantitative analysis method based on light isotope and isobar labeling |
| CN105785048B (en) * | 2016-04-15 | 2017-07-28 | 同济大学 | Based on the nearly overall Protein quantitative analysis method with heavy label of light isotope |
| CN105866429A (en) * | 2016-05-06 | 2016-08-17 | 同济大学 | Biomolecular marker based on isotope reagent and provided with electric charge itself and quantitative analysis method |
| CN106093224A (en) * | 2016-06-01 | 2016-11-09 | 同济大学 | A kind of polysaccharide Sync enrichment and the nearly quantitative analysis method with heavy label |
| CN106990159A (en) * | 2017-05-04 | 2017-07-28 | 同济大学 | A kind of protein quantitation methods based on complete accurate same weight diethyl mark |
| CN109580814A (en) * | 2018-12-06 | 2019-04-05 | 复旦大学 | The synchronous quantitative analysis method of a variety of amino metabolins based on N- alkylation process |
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