CN106990159A - A kind of protein quantitation methods based on complete accurate same weight diethyl mark - Google Patents
A kind of protein quantitation methods based on complete accurate same weight diethyl mark Download PDFInfo
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Abstract
The present invention relates to a kind of based on the complete accurate protein quantitation methods with weight diethyl mark, first using two groups of different physiology or the overall albumen of pathological conditions as control group and disease group；All amino on control group protein and disease histone matter optional position lysine and N-terminal amino acid are carried out again it is complete with weight diethyl mark, be respectively labeled as N (13CH3 13CHD)2With N (CD3CH2)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, obtain the relative scale of ID and each protein of disease group relative to control group of protein, the up-regulation of all albumen or downward situation i.e. under disease conditions, up-regulation or the albumen for lowering multiple maximum are the albumen related to disease generation, development.Compared with prior art, the inventive method labeling effciency is high, and the degree of accuracy is high, it is adaptable to the overall protein-based quantitative analysis in high-resolution tandem mass spectrometry.
Description
Technical field
The present invention relates to a kind of quantitative approach of overall protein molecule, more particularly, to one kind based on complete accurate with weight diethyl
The protein quantitation methods of disjunction mark note, belong to the technical fields such as systems biology, the proteomics related to biological mass spectrometry.
Background technology
In recent years, the high-quality measurement accuracy with efficient dissociative pattern, high-quality resolution mass spectrograph (such as Orbitrap mass
Instrument) it is developed rapidly and popularizes；This cause the mass spectrographic cascade mass spectrometry of protein and high confidence level Qualitative Identification into
In order to possible.Protein research and analysis need the high quantitative approach of the degree of accuracy simultaneously under different physiology or pathological conditions.Same position
Element mark is quantitative because can effective conquering method and systematic error, it is typically more accurate than non-scalar quantity.In isotope marks
In, generally there is isotope, with weight and accurate same weight Three models；Standard is with molality formula because using different heavy labels, matter
The relative peak intensities that amount only has the same fragment ion of nuance (mDa) carry out relative quantification, can not only be to greatest extent
Conquering method and systematic error, and can be effectively with the interference from common imprison precursor ion under molality formula.
Internal isotope marks based on SILAC1-3With the external same heavy label based on TMT4, due to targeting amino acid
Non-fully mark (such as the non-fully replacement in SILAC of lysine or arginine of weight) and non-targeted amino acid is non-selective
Mark (the TMT marks of such as threonine), its application is received than larger limitation.(isotope, same to weight, standard are same for dimethyl
Weight) mark because its reagent is easy to get, labeling effciency is high, has obtained extensive research at the quantitative aspect of proteins and peptides and has answered
With.China scientist is also made that huge contribution in terms of this quantitative technique, has developed a variety of various forms of dimethyl
Mark5-8；Wherein applicant first will be complete accurate with weight dimethyl mark (universal pseudo-isobaric
Dimethylation) it is incorporated into the ammonia on the amino and lysine residue in the quantitative analysis of whole protein, N-terminal amino acid
" cooking-pot type " all mark of base effectively avoids extra pre- protection and its related side effect of the latter, substantially increases quantitative analysis
Efficiency and the degree of accuracy8.The whole protein database search engine ProteinGoggle (http of applicant oneself exploitation://
proteingoggle.tongji.edu.cn/)9-11Automatically optional position can be added to using dimethyl group as dynamic embellishment
On lysine, so that all mark is possibly realized.ProteinGoggle is the isotope profile fingerprint invented based on applicant
Alignment algorithm (isotopic envelope fingerprinting, iEF)12Exploitation；IEF algorithms are directly in initial data
Carry out the search of matching ion and the identification of protein.The algorithm, can without carrying out " removing isotope " pretreatment to experimental data
To save the corresponding time；According to the deviation for whether having isotopic peak missing and isotopic peak relative intensity in each ion to reason
Think of non-ideal experimental data to be distinguished well, it is ensured that the confidence level of Identification of Fusion Protein；According to the same position of known overlapping ion
The overlapping experimental data of plain peak relative intensity relation pair is effectively parsed.Entirety is successfully developed based on algorithm applicant
Albumen database search engine ProteinGoggle, is mixed in single standard protein (ubiquitin, myoglobins) and overall protein group
In the Qualitative Identification of compound (histone H 4 family posttranslational modification isomers, Escherichia coli), show higher confidence level and
Preferable application prospect.In terms of quantitative analysis, ProteinGoggle have in it and unique potential advantages；One side
Fingerprint comparison of the face based on isotope profile and wherein each isotope can quickly and accurately search for isotope or same heavy label
Quota ion pair；On the other hand, in search procedure in each ion the laboratory strength of each isotopic peak be recorded with it is defeated
Go out, can be for directly calculating relative quantification ratio.
In accurate same weight dimethyl mark is quantitative, the a1 ions of first amino acid of N-terminal are generally used because of peak intensity height
Make quantitative fragment ion.Plus the 28Da of dimethyl group, the mass charge ratio range of a1 ions is between 58-187Da；A1 ions
It is minimum fragment ion in second order mses.In order to ensure preferably to detect a1, typically to set one it is smaller than a1 ion
The second order mses lowest detection quality of (such as 10Da).Due to ion trap can only to the fragment within 15 times of lowest detection quality from
Son is imprisoned well, and this make it that the detection range of fragment ion is more much smaller than usually used 2000；That is, most
The detection of the a1 ions at low quality end causes the ion at high-quality end to imprison and detect well, so as to protein
Identification produces large effect.
Bibliography
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characterization of SILAC-labeled proteins.Journal of the American Society
for Mass Spectrometry 18,2058-2064,doi:10.1016/j.jasms.2007.09.001(2007).
2 Collier,T.S.,Hawkridge,A.M.,Georgianna,D.R.,Payne,G.A.&Muddiman,
D.C.Top-down identification and quantification of stable isotope labeled
proteins from Aspergillus flavus using online nano-flow reversed-phase liquid
chromatography coupled to a LTQ-FTICR mass spectrometer.Analytical chemistry
80,4994-5001,doi:10.1021/ac800254z(2008).
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4 Hung,C.W.&Tholey,A.Tandem mass tag protein labeling for top-down
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ac402834w(2013).
6 Wu,Y.et al.Five-plex isotope dimethyl labeling for quantitative
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7 Yang,S.J.et al.A novel quantitative proteomics workflow by isobaric
terminal labeling.Journal of proteomics 75,5797-5806,doi:10.1016/
j.jprot.2012.07.011(2012).
8 Fang,H.Q.et al.Intact Protein Quantitation Using Pseudoisobaric
Dimethyl Labeling.Analytical chemistry 88,7198-7205,doi:10.1021/
acs.analchem.6b01388(2016).
9 Xiao,K.J.,Yu,F.&Tian,Z.X.Top-down protein identification using
isotopic envelope fingerprinting.Journal of proteomics 152,41-47,doi:10.1016/
j.jprot.2016.10.010(2017).
10 Xiao,K.J.et al.Accurate and Efficient Resolution of Overlapping
Isotopic Envelopes in Protein Tandem Mass Spectra.Sci Rep-Uk 5,doi:Artn
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12 Xiao open victory, Shen Yun, Wang Yue, Tian Zhixin.A kind of method of protein post-translational modification positioning.Application number：
201510197719.3, the date of application：04/21/2015；Application publication number：CN104820011A, data of publication of application：08/05/
2015。
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201510125438.7, the date of application：03/20/2015；Application publication number：CN104765984A, data of publication of application：07/08/
2015。
14 Tian Zhixin.A kind of analytic method of the overlapping isotope profile of biological mass spectrometry.Application number：201410593905.4,
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Please the date：05/11/2012；Publication No.：CN103389335A, date of publication:11/23/2013.
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 a kind of labeling effciency is high, secondary
Reaction is less, the high kind of the degree of accuracy is based on the complete accurate protein quantitation methods with weight diethyl mark.
This method is based under the different physiology or pathological conditions lysine residue and N-terminal amino on overall protein sequence
Complete accurate same the weight diethyl mark and high resolution mass spectrum and cascade mass spectrometry of amino on acid, diethyl is included by calculating
The relative intensity of fragment ion so as to calculate albumen under different physiology or pathological conditions relative intensity change (up-regulation or under
Adjust).
The quality of a1 ions is increased 28Da (being marked relative to dimethyl) by the present invention using diethyl mark, can be preferably
Take into account the complete detection of minimum quality end a1 ions and the ion at high-quality end in ground.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of protein quantitation methods based on complete accurate same weight diethyl mark, comprise the following steps：
(1) using two groups of different physiology or the overall albumen of pathological conditions as control group and disease group；
(2) by the control group protein after reduction and alkylation and disease histone matter optional position lysine and N-terminal ammonia
All amino on base acid carry out it is complete with weight diethyl mark, be respectively labeled as-N (13CH3 13CHD)2With-N (CD3CH2)2, its matter
Amount difference is 11.68mDa；
(3) carry out high resolution mass spectrum by equal proportion mixing with two histones after heavy label and cascade mass spectrometry obtains one
Data group；
(4) 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.
In step (2), all amino acetaldehyde on a histone matter optional position lysine and N-terminal amino acid
(13CH3 13) and deuterated sodium cyanoborohydride (NaBD CHO3CN) mark, another histone matter optional position lysine and N-terminal amino
All amino acetaldehyde (CD on acid3) and sodium cyanoborohydride (NaBH CHO3CN) mark.
The course of reaction and condition being marked are as follows：
Albumen after reduction and alkylation is re-dissolved in after sodium acetate buffer solution, adds 4% (v/v) just prepared
Acetaldehyde solution；The sodium cyanoborohydride solution of 600mM new preparation, after reacting at room temperature 1 hour, reaction are added under agitation
It is quenched with 4% (v/v) ammoniacal liquor.
Described sodium acetate buffer solution is 100mmol/L, and pH is 5-6.
Control group protein is reduced with disease histone matter with dithiothreitol (DTT).
Control group protein is alkylated with disease histone matter with iodoacetamide.
Equal proportion mixing described in step (3) refers to：According to weight, volume or the mole of control group and disease group with 1:1
Ratio is mixed.
In step (4) 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 to the complete accurate with weight diethyl mark quantitative analysis of polypeptide.Standard is same to retry agent
To that can also be other combinations, for example13CH3CHO+NaBH3CN and CH3CHO+NaBD3CN；Diethyl at least one after mark
It is right13C-D.
Compared with prior art, analytic method of the invention is based on the mass spectrographic original second order mses, is wrapped by calculating
Containing standard with the average relative intensity of the fragment ion of heavy label group so as to calculate labelled protein in different physiology or pathological conditions
Under relative intensity.The quantitative approach of the present invention is entered to all amino on protein optional position lysine and N-terminal amino acid
Row is accurate with heavy label and to carrying out isotope wheel comprising the accurate fragment ion with heavy label group in corresponding high-resolution tandem mass spectrometry
Wide fingerprint comparison data parsing in situ, labeling effciency is high, and the degree of accuracy is high, it is adaptable to overall protein-based in high-resolution tandem mass spectrometry
Quantitative analysis.
Brief description of the drawings
Fig. 1, the complete accurate same weight diethyl disjunction mark based on all amino on protein optional position lysine and N-terminal amino acid
The quantitative global analysis flow chart of note.
The complete accurate same weight diethyl disjunction mark of all amino in Fig. 2, protein molecule optional position lysine and N-terminal amino acid
Remember schematic diagram.Control histidine tag for L (=- N (13CH3 13CHD)2, scheme A)；Disease histidine tag is H (=- N
(CD3CH2)2, scheme B)
Embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment
A kind of protein quantitation methods based on complete accurate same weight diethyl mark, using the arrangement flow shown in Fig. 1, including
Following steps：
(1) using two groups of different physiology or the overall albumen of pathological conditions as control group and disease group, revived with two sulphur
Sugar alcohol is reduced to control group protein with disease histone matter, with iodoacetamide to control group protein and disease histone
Matter is alkylated；
(2) by the control group protein after reduction and alkylation and disease histone matter optional position lysine and N-terminal ammonia
All amino on base acid carry out it is complete with weight diethyl mark, be respectively labeled as-N (13CH3 13CHD)2With-N (CD3CH2)2, such as scheme
Shown in 2, its mass discrepancy is 11.68mDa；
In step (2), all amino acetaldehyde on a histone matter optional position lysine and N-terminal amino acid
(13CH3 13) and deuterated sodium cyanoborohydride (NaBD CHO3CN) mark, another histone matter optional position lysine and N-terminal amino
All amino acetaldehyde (CD on acid3) and sodium cyanoborohydride (NaBH CHO3CN) mark.The course of reaction and bar being marked
Part is as follows：Albumen after reduction and alkylation is re-dissolved in after sodium acetate buffer solution, adds 4% (v/v) just prepared
Acetaldehyde solution；The sodium cyanoborohydride solution of 600mM new preparation, after reacting at room temperature 1 hour, reaction are added under agitation
It is quenched with 4% (v/v) ammoniacal liquor.Described sodium acetate buffer solution is 100mmol/L, and pH is 5-6.
(3) 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；
(4) data group is entered using the overall albumen database search engine ProteinGoggle introduced in background technology
Qualitative, the quantitative data library searching of row, obtains ID and each protein of disease group the comparing relative to control group of protein
Example, i.e., the up-regulation of all albumen or downward situation under disease conditions, up-regulation or the maximum albumen of downward multiple are and the disease
Occur, develop related albumen.
Above-mentioned analytic method is based on the mass spectrographic original second order mses, by calculating comprising standard with the broken of heavy label group
The average relative intensity of piece ion is so as to calculate relative intensity of the labelled protein under different physiology or pathological conditions.The present invention's
Quantitative approach carries out standard to all amino on protein optional position lysine and N-terminal amino acid with heavy label and to corresponding high
Differentiate comprising the accurate fragment ion progress isotope profile fingerprint comparison data parsing in situ with heavy label group in tandem mass spectrometry,
Labeling effciency is high, and the degree of accuracy is high, it is adaptable to the overall protein-based quantitative analysis in high-resolution 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 (8)
1. it is a kind of based on the complete accurate protein quantitation methods with weight diethyl mark, it is characterised in that to comprise the following steps：
(1) using two groups of different physiology or the overall albumen of pathological conditions as control group and disease group；
(2) by the control group protein after reduction and alkylation and disease histone matter optional position lysine and N-terminal amino acid
On all amino carry out it is complete with weight diethyl mark, be respectively labeled as-N (13CH3 13CHD)2With-N (CD3CH2)2, its is of poor quality
Different is 11.68mDa；
(3) carry out high resolution mass spectrum by equal proportion mixing with two histones after heavy label and cascade mass spectrometry obtains a data
Group；
(4) 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, development.
2. according to claim 1 a kind of based on the complete accurate protein quantitation methods with weight diethyl mark, its feature exists
In, in step (2), all amino acetaldehyde on a histone matter optional position lysine and N-terminal amino acid13CH3 13CHO and
Deuterated sodium cyanoborohydride NaBD3CN is marked, all amino on another histone matter optional position lysine and N-terminal amino acid
Use acetaldehyde CD3CHO and sodium cyanoborohydride NaBH3CN is marked.
3. according to claim 2 a kind of based on the complete accurate protein quantitation methods with weight diethyl mark, its feature exists
In the course of reaction and condition being marked are as follows：
Albumen after reduction and alkylation is re-dissolved in after sodium acetate buffer solution, adds 4% (v/v) acetaldehyde just prepared
Solution；The sodium cyanoborohydride solution of 600mM new preparation is added under agitation, and after reacting at room temperature 1 hour, reaction is used
4% (v/v) ammoniacal liquor is quenched.
4. according to claim 3 a kind of based on the complete accurate protein quantitation methods with weight diethyl mark, its feature exists
In described sodium acetate buffer solution is 100mmol/L, and pH is 5-6.
5. according to claim 1 a kind of based on the complete accurate protein quantitation methods with weight diethyl mark, its feature exists
In being reduced with dithiothreitol (DTT) to control group protein with disease histone matter.
6. according to claim 1 a kind of based on the complete accurate protein quantitation methods with weight diethyl mark, its feature exists
In being alkylated with iodoacetamide to control group protein with disease histone matter.
7. according to claim 1 a kind of based on the complete accurate protein quantitation methods with weight diethyl mark, its feature exists
In the equal proportion mixing described in step (3) refers to：According to weight, volume or the mole of control group and disease group with 1:1 ratio is entered
Row mixing.
8. according to claim 1 a kind of based on the complete accurate protein quantitation methods with weight diethyl mark, its feature exists
In in step (4), qualitative, quantitative data library searching is carried out to data group with ProteinGoggle.
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| CN108693294A (en) * | 2018-05-22 | 2018-10-23 | 复旦大学 | A variety of amino metabolins based on N- ethylization methods synchronize quantitative analysis method |
| 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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Application publication date: 20170728 |