CN110487888A - Combined matrix DHB/DHBH is in MALDI mass spectrum to the application in reduction sugar detection - Google Patents
Combined matrix DHB/DHBH is in MALDI mass spectrum to the application in reduction sugar detection Download PDFInfo
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Abstract
The invention discloses DHBH (2,5- dihydroxybenzoyl hydrazine) (DHB/DHBH) is combined as novel reaction type matrix and DHB (2,5- dihydroxy-benzoic acid) that application in high-throughput qualitative and quantitative detection is carried out to reduced sugar.In MALDI mass spectrum, the target plate derivatization strategy based on DHB/DHBH be can be achieved to reduced sugar high throughput qualitative and quantitative analysis.DHBH has hydrazide group active group, is easy to react with reduced sugar hemiacetal as response type matrix, is remarkably improved ionization efficiency；Meanwhile DHBH as matrix can assisted carbohydrate ionization, therefore excessive DHBH without separation.DHB is organic acid, can greatly be catalyzed into the progress of hydrazone reaction；DHB can assist its ionization as the most common matrix of carbohydrate simultaneously.DHB/DHBH is applicable in the detection of acid sugar and neutral sugar as matrix.
Description
Technical field
The present invention relates to sugared biochemical analysis fields, and in particular to 2,5- dihydroxybenzoyl hydrazines (DHBH) and 2,5- dihydroxy
The combined matrix of benzoic acid (DHB) is in MALDI mass spectrum to the application in reduction sugar detection.
Background technique
Glucide is the third class large biological molecule of life entity, has highly important biological function, is mainly used as
Energy source, structural material and functional materials.Glucide is in many life processes, such as fertilization, growth, differentiation, development, letter
Number transduction and immune response etc. play an important role.Glycosylation modified is most extensive, most complicated and important protein
One of posttranslational modification type.50% or more protein can occur it is glycosylation modified, and more and more researches show that, glycosyl
The generation, development and transfer process for changing modification in disease, especially tumour play a crucial role.Glycosylated exception with
Kinds cancer generation is closely related, and sugar chain molecule is high to establish in the tumor markers screening of research object on using glycoprotein
The method for qualitative and quantitative detection of flux has great importance.
Mass spectrum is one of the important tool for detecting large biological molecule, and high sensitivity, amount of samples is few, it can be achieved that high-throughput
Detection.Wherein, MALDI (Matrix-Assisted Laser Desorption/Ionization, it is substance assistant laser desorpted
Ionization) mass spectrum is easy to operate with its, and map easily parses, and it is raw to be widely used in low abundance endogenous suitable for the features such as complex sample
The detection of object macromolecular.However, the ionization inefficient of glucide, this is largely compared with other biological macromolecular
On limit MALDI technology to the sensitive analysis of the substance.In addition, in glucide detection, common MALDI matrix
DHB (2,5-dihydroxybenzoic acid) and the cocrystallization of analyte are uneven, are unfavorable for quantitative analysis.For the electricity for improving glucide
Efficiency of ionization need to usually perform the derivatization processing before Mass Spectrometer Method to it.Currently used derivatization mode specifically includes that
(1) methylation method, such reaction is derived for hydroxyl, but derivatization reagent is more toxic, and the reaction time is long, and is difficult to protect
Demonstrate,prove hydroxyl total overall reaction；(2) reduction amination；(3) at hydrazone reaction.Reduction amination and at hydrazone reaction for reducing end hemiacetal carry out
It is derivative, but need to carry out the purifying of desalination and sample after deriving and etc., process is cumbersome, easily causes the loss of sample.As it can be seen that at present
Derivatization method there are still deficiency, still need to improve.
For the limitation for overcoming the above method, studies have reported that the target plate derivatization strategy of reduced sugar, i.e., sample is in target
Point sample is synchronous with deriving on plate carries out, and simplifies operating procedure while improving reduced sugar ionization efficiency, has been compatible with MALDI
High pass flow characteristic.The reduced sugar target plate derivatization reagent and response type matrix registered mainly have aniline, 3- aminoquinoline, ammonia
Base pyrazine, phenylhydrazine, 2- hydrazino pyridine and girard reagent T etc..Here derivatization reagent refer to as ionization label can be with reduction
Sugar is reacted, but itself is not protruded as the characteristic of matrix；And response type matrix then refer to can not only be used for ionization label, together
When with excellent medium characteristics.It is said on stricti jurise, aniline, Aminopyrazine, phenylhydrazine, 2- hydrazino pyridine and girard reagent T
Belong to derivatization reagent.And girard reagent T, per se with positive charge, excessive derivatization reagent necessarily will cause sample letter
Number inhibition.3- aminoquinoline belongs to response type matrix, but it is relatively slow with reducing sugar reaction, and the schiff bases generated is unstable
It is fixed, limit further extensive use.
In view of the above problems, the MALDI target plate derivatization strategy of design and exploitation based on novel reaction type matrix is to realization
Reduced sugar high throughput qualitative and quantitative detection has important value.
Summary of the invention
The present invention provides DHBH (2,5- dihydroxybenzoyl hydrazines) as novel reaction type matrix and DHB (2,5- dihydroxies
Yl benzoic acid) it is combined the application of (DHB/DHBH) in reduction sugar detection.In MALDI mass spectrum, based on DHB/DHBH's
Target plate derivatization strategy can be achieved to reduced sugar high throughput qualitative and quantitative analysis.DHBH has hydrazide group active group, as anti-
It answers type matrix to be easy to react with reduced sugar hemiacetal, is remarkably improved ionization efficiency；Meanwhile DHBH can assist sugar as matrix
The ionization of class compound, therefore excessive DHBH is without separation.DHB is organic acid, can greatly be catalyzed into hydrazone reaction into
Row；DHB can promote its ionization as the most common matrix of carbohydrate simultaneously.DHB/DHBH is as matrix to acid sugar and neutral sugar
Detection be applicable in.In addition, organic acid (DHB) and organic base (DHBH), which are applied in combination, can significantly improve the sample crystallization uniformity,
Improve the accuracy of quantitative detection.Shown in DHBH structural formula of the present invention such as formula (1):
The DHBH (2,5- dihydroxybenzoyl hydrazine) and the combined matrix of DHB (2,5-dihydroxybenzoic acid) exist
To the application in reduction sugar detection in MALDI mass spectrum.DHBH is response type matrix, has hydrazide group active reactive group, can be with
The reaction of reduced sugar hemiacetal.DHB is catalyst, can significantly promote DHBH and reduced sugar at hydrazone reaction.DHB and DHBH is sugar
The efficient matrix of substance detection.
Described is detected as to reduced sugar progress high-throughput qualitative and quantitative detection reduced sugar, and the application specifically includes
Following steps:
1, preparation of reagents
DHB methanol solution is prepared, DHB is 2,5-dihydroxybenzoic acid；Using DHBH as solute, DHBH 2,5- dihydroxy benzenes
Formylhydrazine, DHB methanol solution are solvent, prepare DHB/DHBH mixed-matrix methanol solution；Prepare the aqueous solution of reduced sugar to be measured；
2, point sample
The aqueous solution and DHB/DHBH mixed-matrix methanol solution for taking isometric reduced sugar to be measured respectively, successively put
MALDI polishes the same loading wells of target plate, is directly mixed on MALDI polishing target plate, the target plate after obtaining point sample.
3, target plate derivative reaction
Target plate after point sample places 20~40min at 60~70 DEG C, and sample spot is completely dried, and carries out MALDI mass spectrum point
Analysis.
In step 1, the methanol for dissolved matrix is to analyze pure or chromatographic grade solvent；
The solvent for preparing the aqueous solution of reduced sugar to be measured is ultrapure water or distilled water；
The concentration of DHBH is 0.05~0.15mol/L in the DHB/DHBH mixed-matrix methanol solution；
The molar concentration rate of DHB and DHBH is 1: 1~4: 1 in the DHB/DHBH mixed-matrix methanol solution, into one
Step preferably 3: 1；
In step 2, the point sample volume of the aqueous solution of reduced sugar to be measured is 0.5~1.0 μ L；
The point sample volume of DHB/DHBH mixed-matrix solution is 0.5~1.0 μ L；
The mixing of the aqueous solution and DHB/DHBH mixed-matrix solution of reduced sugar to be measured is using liquid-transfering gun in MALDI polishing target
Plate carries out repeating to inhale making a call to 10~20 times；
In step 3, the target plate after point sample places 30min at 65 DEG C,
The detection pattern that the MALDI mass spectral analysis uses is positive ion mode or negative ion mode；
The detection pattern that the MALDI mass spectral analysis uses is reflective-mode or linear model.
Heretofore described " MALDI mass spectrum " refers to Matrix-assisted laser desorption ionization (MALDI-
TOF-MS), referred to as MALDI mass spectrum.The working principle of MALDI is with laser irradiation sample and substrate formed cocrystallization, base
Matter absorbs the process that energy transmission ionizes it to sample molecule from laser.MALDI is a kind of Soft ionization techniques, is suitable for
The measurement of large biological molecule.
It, can be with it while heretofore described " response type matrix " is referred to as the ionization of conventional substrate assisted Reduction sugar
Fast reaction occurs for reducing end hemiacetal, and covalent bond ionizes label, improves ionization efficiency.
Heretofore described " target plate derivatization strategy " refers to the progress synchronous with deriving of the point sample of sample, pole on target plate
The earth simplifies operating procedure, has been compatible with the high pass flow characteristic of MALDI.
Heretofore described " acid sugar " refers to the reduced sugar of carboxyl (- COOH) acidic substituent, such as contains saliva
The glycan molecule of liquid acid.
Heretofore described " neutral sugar " refers to the reduced sugar without containing acidic substituent.
Heretofore described derivatization efficiency=derivatization product peak intensity/(derivatization product peak intensity+not derivative
Peak intensity)
Compared with prior art, the invention has the following beneficial effects:
1, DHBH has similar skeleton structure with DHB, and laser energy effectively can be absorbed and be transmitted, and is applicable in very much
In the matrix as glucide.
2, in DHB/DHBH mixed-matrix, DHBH is response type matrix, has hydrazide group active reactive group, can be with reduction
Sugared hemiacetal reaction.DHB is acidic catalyst, can significantly promote DHBH and reduced sugar at hydrazone reaction.DHB and DHBH is sugar
The efficient matrix of substance detection is suitable for target plate derivatization and carries out high-throughput detection to reduced sugar there is no need to separate.
3, due to multiple-effects such as derivative, catalysis and promotion ionization, DHB/DHBH mixed-matrix has excellent matrix special
Property, it is remarkably improved the ionization efficiency and detection sensitivity of reduced sugar, expands the quality testing range of reductive polysacchride.
4, DHB and DHBH are used in mixed way, and belong to the combination of organic acid and organic base, it is uniform to can obviously improve sample crystallization
Degree, improves the Stability and veracity (RSD < 10%) of quantitative detection.DHB/DHBH mixed-matrix is suitable for acid sugar and neutrality
The qualitative and quantitative detection of sugar, neutral sugar have good linear relationship, R in 0.025~20pmol/ μ L range2> 0.995；Acid
Property sugar 0.2~20pmol/ μ L range have good linear relationship, R2> 0.999.
Detailed description of the invention
Fig. 1 is the synthesis flow schematic diagram of DHBH；
Fig. 2 is the NMR spectra and high-resolution ESI mass spectrogram of DHBH；Fig. 2A DHBH's1H H NMR spectroscopy；Fig. 2 B DHBH's13C
H NMR spectroscopy；Fig. 2 C DHBH negative ion mode high-resolution ESI mass spectrogram；
Fig. 3 is the uv absorption spectra of same molar ratio DHB and DHBH；
Fig. 4 is the MALDI mass spectrogram for detecting maltose (G6) and maltodextrin 2000 (D2000)；Fig. 4 A DHB conduct
The MALDI mass spectrogram of matrix detection G6；MALDI mass spectrogram of Fig. 4 B DHBH as matrix detection G6；Fig. 4 C DHB is as matrix
Detect the MALDI mass spectrogram of D2000；MALDI mass spectrogram of Fig. 4 D DHBH as matrix detection D2000；
Fig. 5 is the reduced sugar target plate derivatization schematic diagram based on DHB/DHBH；
Fig. 6 is organic acid matrix and influence of the DHBH difference molar concentration ratio to G6 derivatization efficiency；
Fig. 7 is the cocrystallization aspect graph under organic acid matrix and DHBH difference molar concentration ratio；
Fig. 8 be respectively with DHB, CHCA/DHBH and DHB/DHBH be matrix detection different molecular weight reduced sugar MALDI matter
Spectrogram；Fig. 8 A detects maltotriose；Fig. 8 B detects D2000；Fig. 8 C detects D 5000；Fig. 8 D detects D12000；
Fig. 9 is the MALDI mass spectrogram that small molecule carbohydrate is detected by matrix of DHB/DHBH；Fig. 9 A DHB is matrix
Detect the MALDI mass spectrogram of glucose；Fig. 9 B-9F DHB/DHBH is that matrix detects glucose (9B), sucrose (9C), sugarcane respectively
The MALDI mass spectrogram of fruit trisaccharide (9D), Nystose (9E) and sugarcane sugar (9F)；
The reproducibility of signal that Figure 10 is respectively with DHB, CHCA/DHBH and DHB/DHBH when be matrix detection G6 and to NA2,
The range of linearity of NA2F and A1 detection；45 detection peak intensity normalization figures of the same loading wells of Figure 10 A；The same loading wells of Figure 10 B
The RSD value of 45 detection peak intensities；Figure 10 C DHBH is the MALDI mass spectrum imaging figure that matrix detects G6；Figure 10 D positive ion mode
Detect the range of linearity of NA2；The range of linearity of Figure 10 E positive ion mode detection NA2F；The line of Figure 10 F negative ion mode detection A1
Property range；
Figure 11 is to carry out the MALDI matter after target plate derivatization to haemocyanin reproducibility N- sugar chain by matrix of DHB/DHBH
Spectrogram；Figure 11 A positive ion mode detects neutral sugar；Figure 11 B negative ion mode detects acid sugar；After the normalization of Figure 11 C content not
With the disease group and health group proportion grading of reduced sugar.
Specific embodiment
The following examples will be further described the present invention, but the present invention is not limited to the following embodiments.
Sample as used in the following examples, reagent etc., unless otherwise specified, commercially obtain.Lead to below
Crossing specific embodiment, the present invention will be described, but the present invention is not limited thereto.
The concrete model of Matrix-assisted laser desorption ionization instrument used in following embodiments is
UlrafleXtremeTMMALDI-TOF/TOF MS (Bruker Daltonic, Germany), laser is using 355nm wavelength
Nd:YAG laser.Neutral sugar is detected in positive ion mode, and acid sugar is detected in negative ion mode；Molecular weight is less than 5000Da's
Sugar is detected in reflective-mode, and sugar of the molecular weight greater than 5000Da is detected in linear model.Cation linear model parameter: accelerate electricity
Pressure, 25.00kV；Postpone extraction voltage, 23.20kV；Time, 400ns are drawn in delay；Lens voltage, 7.50kV；Frequency,
2000Hz.Cation reflective-mode parameter: acceleration voltage, 25.00kV；Postpone extraction voltage, 22.30kV；The time is drawn in delay,
130ns；Reflector voltage 1,26.50kV；Reflector voltage 2,13.50kV；Lens voltage, 7.50kV；Frequency, 1000Hz.It is negative
Ion reflections mode parameter: acceleration voltage, 20.00kV；Postpone extraction voltage, 17.75kV；Time, 100ns are drawn in delay；Instead
Emitter voltage 1,21.10kV；Reflector voltage 2,10.70kV；Lens voltage, 8.50kV；Frequency, 2000Hz.Target plate used is
384 polishing plates (384 polished steel of MTP), MASS SPECTRAL DATA ANALYSIS are soft using Bruker Flexanalysis 3.4
Part.
The use of logogram word or foreign language term below is through the present invention:
DHBH 2,5- dihydroxybenzoyl hydrazine；
DHB 2,5-dihydroxybenzoic acid；
CHCA alpha-cyano -4- hydroxycinnamic acid；
ESI electrospray ionization mass spectrum；
D maltodextrin；
RSD relative standard deviation；
Min minutes；
μ L microlitre；
NMR nuclear magnetic resonance；
G6 maltose；
MeOH methanol；
V volts；
Hz hertz；
PA proton affinity；
M.P. fusing point；
LOD detection limit；
Rt room temperature；
S/N signal-to-noise ratio；
Normal health group；
CRC colorectal cancer group；
DMSO dimethyl sulfoxide；
HRMS high resolution mass spectrum；
2AB 2- aminobenzamide；
PNGase F Peptide N-glycosidase F；
TFA trifluoroacetic acid；
The synthesis and characterization of embodiment 1:DHBH
1, the synthesis of DHBH
The 2,5-dihydroxybenzoic acid methyl esters and 10mL methanol of 0.168g (0.001mol) are sequentially added in round-bottomed flask,
5min mixing is stirred at room temperature.Then the hydrazine hydrate solution of 0.2mL (about 0.004mol) 85% is slowly added dropwise, 25 DEG C of room temperature stirred
Night, until raw material fully reacting generates brown precipitate.
2, DHBH is isolated and purified
Rotary evaporation removes excessive solvent and hydrazine hydrate at 25 DEG C of room temperature, obtains sticky solid.It is then respectively adding excess
Methanol and 1mL deionized water redissolve product, are recrystallized using Rotary Evaporators to it.Controlled at 25 DEG C of room temperature,
And slow down solvent rotary evaporation speed, until remaining about 3mL solvent in flask.Precipitating is water-soluble with the methanol of percentage by volume 50%
Liquid is dried in vacuo at washing 3 times, 25 DEG C of room temperature, can obtain 0.1g brown ceramic powder, as DHBH.
3, the characterization of DHBH
The characterization of DHBH uses NMR, high-resolution ESI, ultraviolet absorption spectrum instrument and melting point apparatus etc..
In embodiment 1, Fig. 1 is the synthesis flow schematic diagram of DHBH, and it is room that synthesis and purification process, which need strict temperature control,
Temperature 25 DEG C or be lower than 25 DEG C of room temperature, prevent the generation of by-product.Fig. 2 is DHBH's1H H NMR spectroscopy (2A),13C H NMR spectroscopy (2B) and
High-resolution ESI negative ion mode mass spectrogram (2C), as a result as follows:1H NMR (600MHz, DMSO) δ 11.52 (s, 1H), 9.84
(s, 1H), 8.96 (s, 1H), 7.18 (d, J=2.6Hz, 1H), 6.83 (dd, J=8.7,2.5Hz, 1H), 6.72 (d, J=
8.8Hz, 1H), 4.58 (s, 2H)；13C NMR (151MHz, DMSO) δ 167.39 (s), 151.71 (s), 149.24 (s), 120.87
(s), 117.62 (s), 114.95 (s), 112.94 (s)；HRMS-ESI:167.0482 [M-H]-.Spectral Signal is clean, and and its
Structural information corresponds, it is seen that the isolation and purification method in through this embodiment can get the target product of high-purity.Fig. 3 is
The uv absorption spectra of same molar ratio DHB and DHBH, DHBH is similar to the UV absorption peak shape of DHB, but the suction of DHBH
Receipts intensity is higher, so DHBH itself meets the primary condition as matrix absorption energy and transmitting energy.DHB is molten in table 1
Point is 195~196 DEG C, and DHBH fusing point is 208~210 DEG C, it is seen that DHBH meets the requirement of high vacuum working environment in mass spectrum.
Embodiment 2:DHBH itself detects glucide as MALDI matrix
(1) the D2000 sugar juice of the G6 and 0.1mg/mL of 20pmol/ μ L are prepared, can be saved in 4 DEG C of refrigerators；
(2) the DHBH methanol solution in 0.1mol/L embodiment 1 is prepared, can be saved in 4 DEG C of refrigerators；
(3) the DHB methanol solution of 0.01,0.025,0.05,0.1,0.2,0.3 and 0.4mol/L is prepared respectively, it can be at 4 DEG C
Refrigerator saves；
(4) the DHBH methanol solution in the G6 liquid glucose or D2000 liquid glucose and 1 μ L step 2 in 1 μ L step 1 is taken respectively, successively
Point is in the same loading wells of polished steel target plate.It directly repeats to inhale in MALDI target plate using liquid-transfering gun and make a call to 10 times, mixed
It is even.It is spontaneously dried at 15 DEG C；
(5) as control, the 0.1mol/L in the G6 liquid glucose or D2000 liquid glucose and 1 μ L step 2 in 1 μ L step 1 is taken respectively
DHB methanol solution is successively put in the same loading wells of polished steel target plate.Using liquid-transfering gun directly in MALDI target plate weight
It relapses and makes a call to 10 times, mixed.It is spontaneously dried at 15 DEG C；
(6) target plate is sent into MALDI mass spectrograph, and cation reflective-mode is selected to carry out data acquisition.
In embodiment 2, Fig. 4 is the MALDI mass spectrogram for detecting G6 and D2000 by matrix of DHB and DHBH respectively.
Corresponding 1013.317Da [G6+Na]+Peak, 1661.528Da are corresponding [G10+Na]+Peak, it is seen then that the cryogenic conditions of the present embodiment control
Under, for DHBH not with reducing sugar reaction, this is used as the characteristic of matrix convenient for investigation DHBH itself.Fig. 4 B signal-to-noise ratio is higher than Fig. 4 A, and
The signal-to-noise ratio of Fig. 4 D is higher than Fig. 4 C.As it can be seen that DHBH itself is used as MALDI matrix suitable with DHB performance, it is ideal detection carbohydrate
The matrix of substance.
Embodiment 3:DHBH and DHB is combined, and constructs reduced sugar target plate derivatization strategy
1, organic acid matrix and the molar concentration ratio of DHBH optimize in mixed-matrix
(1) DHB/DHBH mixed-matrix is prepared, using the various concentration DHB methanol solution prepared in embodiment 2 as solvent, with
DHBH is solute, prepares the DHBH mixed solution of 0.1M respectively.As 0.01: 0.1,0.025: 0.1,0.05: 0.1,0.1:
0.1,0.2: 0.1,0.3: 0.1,0.4: 0.1 DHB/DHBH mixed-matrix solution；
(2) 0.1,0.05,0.025,0.01M CHCA methanol solution is prepared respectively, can be saved in 4 DEG C of refrigerators；
(3) prepare CHCA/DHBH mixed-matrix, 0.01: 0.1,0.025: 0.1,0.05: 0.1 and 0.1: 0.1 CHCA/
Preparation method in step (1) can be used in DHBH mixed-matrix, i.e., using various concentration CHCA methanol solution in step (2) as solvent,
Using DHBH as solute, the DHBH mixed solution of 0.1M is prepared respectively.0.2: 0.1,0.3: 0.1,0.4: 0.1 CHCA/DHBH is mixed
It closes matrix and can be used and first remove the method that solvent is redissolved, i.e., it will be in step (2) in 0.1M CHCA methanol solution and embodiment 2
0.1M DHBH methanol solution according to volume ratio be 2: 1,3: 1 and 4: 1 to be separately added into 1.5mL centrifuge tube, traditional vacuum removal is molten
Agent is then respectively adding the methanol solvate of 1 volume, due to acid ion alkali ion pairing effect, can obtain 0.2: 0.1,0.3 respectively:
0.1,0.4: 0.1 CHCA/DHBH mixed-matrix solution；
(4) the DHB/DHBH mixed-matrix solution in the G6 liquid glucose and 1 μ L step 1 in 1 μ L embodiment 2 is taken respectively, successively
Point is in the same loading wells of polished steel target plate.It directly repeats to inhale in MALDI target plate using liquid-transfering gun and make a call to 10 times, mixed
It is even.30min is reacted at 65 DEG C；
(5) as control, take CHCA/DHBH mixed-matrix in G6 liquid glucose and the 1 μ L step 3 in 1 μ L embodiment 2 molten respectively
Liquid is successively put in the same loading wells of polished steel target plate.It directly repeats to inhale in MALDI target plate using liquid-transfering gun and makes a call to 10
It is secondary, it is mixed.30min is reacted at 65 DEG C；
(6) target plate is sent into MALDI mass spectrograph, and cation reflective-mode is selected to carry out data acquisition.
In embodiment 3, Fig. 5 is the reduced sugar target plate derivatization schematic diagram based on the combined matrix of DHB/DHBH, wherein
DHBH is response type matrix, has hydrazide group active reactive group, can react with reduced sugar hemiacetal.DHB is catalyst, can be shown
Write catalysis DHBH and reduced sugar at hydrazone reaction.DHB and DHBH is the efficient matrix of glucide detection, without separation, is fitted
For target plate derivatization strategy, it can be achieved that being detected to reduced sugar high throughput.Fig. 6 is organic acid matrix and DHBH difference molar concentration
Influence of the ratio to G6 derivatization efficiency.It is derivative when the molar concentration rate of DHB and DHBH is 3: 1 using DHB/DHBH as matrix
Change efficiency highest, can reach 95% or more；Using CHCA/DHBH as matrix, when its molar concentration rate is 1: 4, derivatization efficiency
Highest can reach 80% or more.As it can be seen that the target plate derivatization efficiency based on DHB/DHBH is higher than CHCA/DHBH.
2, organic acid matrix is compared with the cocrystallization form of DHBH difference molar concentration ratio
(1) the DHB/DHBH mixed-matrix solution in the G6 liquid glucose and 1 μ L step 1 in 1 μ L embodiment 2 is taken respectively, successively
Point is in the same loading wells of polished steel target plate.It directly repeats to inhale in MALDI target plate using liquid-transfering gun and make a call to 10 times, mixed
It is even.30min is reacted at 65 DEG C；
(2) as control, take CHCA/DHBH mixed-matrix in G6 liquid glucose and the 1 μ L step 3 in 1 μ L embodiment 2 molten respectively
Liquid is successively put in the same loading wells of polished steel target plate.It directly repeats to inhale in MALDI target plate using liquid-transfering gun and makes a call to 10
It is secondary, it is mixed.30min is reacted at 65 DEG C；
(3) target plate is sent into MALDI mass spectrograph, cocrystallization form.
In embodiment 3, Fig. 7 is the cocrystallization aspect graph under organic acid matrix and DHBH difference molar concentration ratio.DHB/
In the combined matrix of DHBH, as DHB ratio increases, cocrystallization becomes more uniform；And the combined matrix of CHCA/DHBH
In, as CHCA ratio increases, the cocrystallization uniformity is relatively poor without too big variation.In conjunction with molar concentration rate each in Fig. 6
The derivatization efficiency of the lower G6 of example, it is known that the mixed-matrix derivatization based on DHB/DHBH is high-efficient, and cocrystallization is more evenly, optimal to rub
Your concentration ratio is DHB: DHBH=3: 1.
Embodiment 4: the qualitative ability for investigating each matrix detection different molecular weight reduced sugar
1, DHB/DHBH detects reproducibility widow polysaccharide as matrix
(1) the maltotriose aqueous solution of 20pmol/ μ L is prepared, can be saved in 4 DEG C of refrigerators；
(2) the D12000 sugar juice of the D5000 and 0.5mg/mL of 0.5mg/mL are prepared respectively, can be saved in 4 DEG C of refrigerators；
(3) respectively prepare 20000fmol/ μ L NA2, NA2F and A1 standard sugar juice, after be diluted to 10000,2000,
1000,200,100,50,25fmol/ μ L obtain NA2, NA2F and the A1 liquid glucose of each gradient；
(4) it takes respectively in the D2000 liquid glucose in 1 μ L embodiment 2 or the maltotriose solution in step 1 or step 2
The methanol solution of DHB/DHBH (molar concentration rate 3: 1) in D5000 liquid glucose or D12000 liquid glucose and 1 μ L embodiment 3, successively
Point is in the same loading wells of polished steel target plate.It directly repeats to inhale in MALDI target plate using liquid-transfering gun and make a call to 10 times, mixed
It is even.30min is reacted at 80 DEG C, guarantees that reduced sugar reacts completely；
(5) as control, the D2000 liquid glucose in 1 μ L embodiment 2 or maltotriose solution or step in step 1 are taken respectively
The 0.3M DHB methanol solution in D5000 liquid glucose or D12000 liquid glucose and 1 μ L embodiment 2 in rapid 2, successively puts in polished
The same loading wells of steel target plate.It directly repeats to inhale in MALDI target plate using liquid-transfering gun and make a call to 10 times, mixed.It is natural at room temperature
Dry 30min；
(6) as control, the D2000 liquid glucose in 1 μ L embodiment 2 or maltotriose solution or step in step 1 are taken respectively
The methanol of the CHCA/DHBH (molar concentration rate 1: 4) in D5000 liquid glucose or D12000 liquid glucose and 1 μ L embodiment 3 in rapid 2
Solution is successively put in the same loading wells of polished steel target plate.It directly repeats to inhale in MALDI target plate using liquid-transfering gun and makes a call to 10
It is secondary, it is mixed.30min is reacted at 80 DEG C, guarantees that reduced sugar reacts completely；
(7) target plate is sent into MALDI mass spectrograph, and cation reflective-mode or linear model is selected to carry out data acquisition.
In embodiment 4, Fig. 8 is the MALDI matter of different substrates qualitative detection maltotriose, D2000, D5000 and D12000
Spectrogram.The detection of Fig. 8 A maltotriose, 527.158Da are corresponding [G3+Na]+Peak, 677.198Da are corresponding [G3+DHBH+Na]+Peak.
Using DHB/DHBH as the signal-to-noise ratio highest of matrix；The detection of Fig. 8 B D2000,1013.222Da are corresponding [G6+Na]+Peak,
Corresponding 1163.320Da [G6+DHBH+Na]+Peak, the corresponding saccharide residue of 162Da.It is bright as the signal-to-noise ratio of matrix using DHB/DHBH
It is aobvious to be higher than other matrix；The detection of Fig. 8 C D5000,4092.320Da are corresponding [G25+Na]+Peak, the corresponding [G25+ of 4242.360Da
DHBH+Na]+Peak, the corresponding saccharide residue of 162Da.It is higher than other matrix by the peak intensity of matrix of DHB/DHBH, and peak shape is in
The normal distribution of standard；The detection of Fig. 8 D D12000 is apparently higher than other matrix by the peak intensity of matrix of DHB/DHBH.Table 1
In using DHB as matrix detect glycoprotein on neutral sugar molecules NA2 and NA2F detection limit be respectively 500fmol and 200fmol, and
The detection limit of NA2 and NA2F can be down to 10fmol when using DHB/DHBH as matrix；Meanwhile DHB/DHBH is suitable for acid sugar A1's
Detection, detection are limited to 100fmol or less.As it can be seen that it is best as matrix detection effect using DHB/DHBH, carbohydrate object can be significantly improved
The ionization efficiency of matter improves detection sensitivity, the detection suitable for oligosaccharides and polysaccharide etc..Table 1: the proton affinity of matrix,
Fusing point and the detection of NA2, NA2F and A1 is limited.
Table 1
2, DHB/DHBH detects glucose and sugarcane oligosaccharides as matrix
(1) glucose, sucrose, ketose, Nystose and the sugarcane sugar aqueous solution of 20pmol/ μ L are prepared respectively,
It can be saved in 4 DEG C of refrigerators；
(2) glucose, sucrose, ketose, Nystose or the sugarcane sugar solution and 1 μ L in 1 μ L step 1 are taken respectively
The methanol solution of DHB/DHBH (molar concentration rate 1: 1) in embodiment 3, successively puts same in polished steel target plate
Loading wells.It directly repeats to inhale in MALDI target plate using liquid-transfering gun and make a call to 10 times, mixed.It spontaneously dries at room temperature；
(3) as control, the 0.1M DHB methanol solution in the glucose and 1 μ L embodiment 2 in 1 μ L step 1 is taken respectively,
It successively puts in the same loading wells of polished steel target plate.It directly repeats to inhale in MALDI target plate using liquid-transfering gun and make a call to 10 times, into
Row mixes.It spontaneously dries at room temperature；
(4) target plate is sent into MALDI mass spectrograph, and cation reflective-mode is selected to carry out data acquisition.
In embodiment 4, Fig. 9 is different substrates qualitative detection glucose, sucrose, ketose, Nystose and sugarcane fruit five
The MALDI mass spectrogram of sugar.Fig. 9 A is the MALDI mass spectrogram that glucose is detected using DHB as matrix, matrix peak serious interference, Portugal
The more difficult detection of grape sugar.Glucose is detected by matrix of DHB/DHBH in Fig. 9 B, complete derivative can occur for drying process at room temperature,
Corresponding 353.096Da [G1+DHBH+Na]+Peak.Fig. 9 C, Fig. 9 D, in Fig. 9 E and Fig. 9 F, it is non-also by matrix detection of DHB/DHBH
Raw sugar such as sucrose, ketose, Nystose and sugarcane sugar, with [M+Na]+Peak is detected, and almost without the dry of matrix peak
It disturbs.As it can be seen that the inspection when the molar ratio of DHB in DHB/DHBH and DHBH is 1 or smaller, suitable for small molecule carbohydrate
It surveys, the interference of matrix peak is small, and signal-to-noise ratio is high.
Embodiment 5: quantitative expedition is using DHB/DHBH as the stability of substraturn approach and the range of linearity
(1) NA2 the and A1 sugar juice with 2AB label that compound concentration is 100fmol/ μ L respectively, can be in -20 DEG C of refrigerators
It saves；
(2) the G6 liquid glucose in 1 μ L embodiment 2 is taken respectively, in the DHB matrix solution or embodiment 3 in 1 μ L embodiment 2
DHB/DHBH mixed-matrix solution or CHCA/DHBH mixed-matrix solution are successively put in polished steel target plate same point
Sample hole.It directly repeats to inhale in MALDI target plate using liquid-transfering gun and make a call to 10 times, mixed, react 30min at 65 DEG C.Target plate is sent into
MALDI mass spectrograph, the same loading wells of cation reflective-mode sample 45 times；
(3) DHB/DHBH mixed-matrix solution in G6 liquid glucose and the 1 μ L embodiment 3 in 1 μ L embodiment 2 is taken respectively, successively
Point is in the same loading wells of polished steel target plate.It directly repeats to inhale in MALDI target plate using liquid-transfering gun and make a call to 10 times, mixed
It is even, 30min is reacted at 65 DEG C.MALDI imaging uses cation reflective-mode, and resolution ratio is 200 μm, and each pixel 500 opens spectrum
Figure, laser intensity 50%.
(4) the NA2 liquid glucose of various concentration in 1 μ L embodiment 4 is taken respectively, and the NA2 internal standard of 2AB label is had in 1 μ L step 1
Solution, DHB/DHBH mixed-matrix solution in 1 μ L embodiment 3, successively puts in the same loading wells of polished steel target plate.Make
It directly repeats to inhale in MALDI target plate with liquid-transfering gun and make a call to 10 times, mixed, react 30min at 65 DEG C.Target plate is sent into MALDI matter
Spectrometer, cation reflective-mode acquire data；
(5) the NA2F liquid glucose of various concentration in 1 μ L embodiment 4 is taken respectively, in 1 μ L step 1 in the NA2 with 2AB label
Solution is marked, DHB/DHBH mixed-matrix solution in 1 μ L embodiment 3 is successively put in the same loading wells of polished steel target plate.
It directly repeats to inhale in MALDI target plate using liquid-transfering gun and make a call to 10 times, mixed, react 30min at 65 DEG C.Target plate is sent into MALDI
Mass spectrograph, cation reflective-mode acquire data；
(6) the A1 liquid glucose of various concentration in 1 μ L embodiment 4 is taken respectively, and the A1 internal standard with 2AB label is molten in 1 μ L step 1
Liquid, DHB/DHBH mixed-matrix solution in 1 μ L embodiment 3, successively puts in the same loading wells of polished steel target plate.It uses
Liquid-transfering gun directly is repeated to inhale and be made a call to 10 times in MALDI target plate, is mixed, reacts 30min at 65 DEG C.Target plate is sent into MALDI mass spectrum
Instrument, anion reflective-mode acquire data.
In embodiment 5, the reproduction of signal when Figure 10 is is matrix detection reduced sugar with DHB, CHCA/DHBH and DHB/DHBH
Property and the range of linearity.In Figure 10 A and 10B, the relative standard deviation that G6 is detected by matrix of DHB/DHBH is 10.0%, hence it is evident that low
In control group matrix.In Figure 10 C, using DHB/DHBH as in the MALDI image of matrix detection G6, G6 ionic strength is in entire point
The target plate hole of sample is more stable.As it can be seen that the stability of method is good when detecting G6 as matrix using DHB/DHBH.Figure 10 D and 10E be with
The NA2 of 2AB label is internal standard, is investigated respectively to the range of linearity of NA2 and NA2F under positive ion mode, find they
0.025~20pmol/ μ L is in good linear relationship, R2> 0.995；Figure 10 F is the A1 that is marked using 2AB as internal standard, bear from
The linearly interval range that A1 is detected under subpattern is in good linear relationship, R in 0.2~20pmol/ μ L2> 0.999.As it can be seen that
Acid sugar and neutral sugar can be analyzed respectively by matrix of DHB/DHBH, the target plate derivatization strategy based on DHB/DHBH can
It realizes and the high throughput of reducing sugar is detected.
Reduction N- sugar chain in embodiment 6:DHB/DHBH qualitative and quantitative detection serum
The preparation method that N- reduced sugar uses standard glycoprotein N- sugar is made in the present invention by oneself, with sugared in Serum In Patients With Colorectal Carcinoma
It is illustrated for albumen.
The present invention prepares N- reduced sugar using PNGase F enzyme process, and steps are as follows:
(1) it digests.1 μ L 10X glycoprotein denaturation buffer is added in 10 μ L serum, 100 DEG C of heating 10min become albumen
Property, after be cooled to room temperature.2 μ L 10X GlycoBuffer, 2 reaction buffer, 2 μ L 10%NP-40,4 μ L ddH are added2O, 1
μ L PNGase F, mixes, and 37 DEG C of incubations are digested for 24 hours.
(2) it purifies.The sample centrifugation that enzymatic hydrolysis is obtained, successively uses C18 solid phase extraction column and graphitized carbon Solid Phase Extraction
Pillar purifies the reproducibility N- sugar of dissociation.C18 solid phase extraction column is for removing impurity peptide, albumen and other pollutions
Object, purification process are as follows: 4mL methanol is for activating pillar, and 4mL ultrapure water is for balancing pillar.Pillar is added in Aspirate supernatant
It is interior, collect eluent with ultrapure water elution pillar, each 1mL points for 3 times.Then, using graphitized carbon solid phase extraction column to salt
It cleans with small molecule polar compound, detailed process is as follows: successively using the acetonitrile solution pair of 3mL acetonitrile and 1mL 50%
Pillar is activated, and the balance of pillar is carried out with 3mL ultrapure water.The liquid glucose loading that C18 purification process is collected, 5mL ultrapure water
Elution removal of impurities.Successively neutral sugar, the acetonitrile water containing 0.05%TFA of 2mL 60% are eluted with the acetonitrile solution of 2mL 40%
Solution elutes acidic polysaccharose, collects elution liquid glucose respectively, and room temperature in vacuo centrifugation removal solvent redissolves in 50 μ L ultrapure waters, can
It is saved in -20 DEG C of refrigerators；
(3) the neutral liquid glucose in 1 μ L step 2 is taken respectively, the NA2 inner mark solution that 2AB is marked in 1 μ L embodiment 5,1 μ L reality
DHB/DHBH mixed-matrix solution in example 3 is applied, is successively put in the same loading wells of polished steel target plate.It is straight using liquid-transfering gun
It connects to repeat to inhale in MALDI target plate and make a call to 10 times, mixed, react 30min at 65 DEG C.Target plate is sent into MALDI mass spectrograph, cation
Reflective-mode acquires data；
(4) the acid liquid glucose in 1 μ L step 2 is taken respectively, and the A1 inner mark solution that 2AB is marked in 1 μ L embodiment 5,1 μ L is implemented
DHB/DHBH mixed-matrix solution in example 3 is successively put in the same loading wells of polished steel target plate.It is direct using liquid-transfering gun
It repeats to inhale in MALDI target plate and make a call to 10 times, mixed, react 30min at 65 DEG C.Target plate is sent into MALDI mass spectrograph, and anion is anti-
Emission mode acquires data；
(5) internal standard method carries out instrument quality calibration.The a variety of reproducibility N- sugar data detected are normalized,
Each reproducibility N- sugar is then acquired in the relative ratio of cancer group and health group.
In embodiment 6, Figure 11 is that cancer group and health group haemocyanin reproducibility N- sugar are detected by matrix of DHB/DHBH
MALDI mass spectrogram.11 kinds of neutral sugar can be detected under Figure 11 A positive ion mode；Acid can be detected under Figure 11 B negative ion mode
Property sugar 11 kinds.Figure 10 C is the cancer group and health group proportion grading of different reduced sugars after content normalization.The result shows that cancer
Group has 12 kinds of sugar to have an apparent content ascendant trend, including 6 kinds of neutral sugars and with 6 kinds of acid sugars.As cancer markers high pass
Screening method is measured, which may provide important data supporting and theoretical direction for the quick diagnosis of clinical disease.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, In
Under the premise of not departing from design, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to protection model of the invention
It encloses.
Claims (10)
- The combined matrix of 1.2,5- dihydroxybenzoyl hydrazine and 2,5- dihydroxy-benzoic acid examines reduced sugar in MALDI mass spectrum Application in survey.
- 2. application according to claim 1, which is characterized in that shown in 2, the 5- dihydroxybenzoyl hydrazine such as formula (1):
- 3. application according to claim 1, which is characterized in that described to be detected as carrying out high pass to reduced sugar to reduced sugar Qualitative and quantitative detection is measured, the application specifically comprises the following steps:1, preparation of reagentsDHB methanol solution is prepared, DHB is 2,5-dihydroxybenzoic acid；Using DHBH as solute, DHBH 2,5- dihydroxybenzoyl Hydrazine, DHB methanol solution are solvent, prepare DHB/DHBH mixed-matrix methanol solution；Prepare the aqueous solution of reduced sugar to be measured；2, point sampleThe aqueous solution and DHB/DHBH mixed-matrix methanol solution for taking isometric reduced sugar to be measured respectively, successively put in polishing target The same loading wells of plate is directly mixed, the target plate after obtaining point sample on polishing target plate.3, target plate derivative reactionTarget plate after point sample places 26646min at 66666 DEG C, and mixing sample spot is completely dried, and carries out MALDI mass spectral analysis.
- 4. application according to claim 3, which is characterized in that in step 1, the DHB/DHBH mixed-matrix methanol is molten The concentration of DHBH is 6.6566.15mol/L in liquid.
- 5. application according to claim 3, which is characterized in that in step 1, the DHB/DHBH mixed-matrix methanol is molten The molar concentration rate of DHB and DHBH is 1:164:1 in liquid.
- 6. application according to claim 3, which is characterized in that in step 2, the point sample volume of reduction sugar aqueous solution to be measured is 6.561.6μL。
- 7. application according to claim 3, which is characterized in that in step 2, the point sample body of DHB/DHBH mixed-matrix solution Product is 6.561.6 μ L.
- 8. application according to claim 3, which is characterized in that in step 2, the aqueous solution and DHB/DHBH of reduced sugar to be measured The mixing of mixed-matrix solution carries out repeating to inhale making a call to 16626 times using liquid-transfering gun in polishing target plate.
- 9. application according to claim 3, which is characterized in that in step 3, the target plate after point sample is placed at 65 DEG C 36min。
- 10. application according to claim 3, which is characterized in that in step 3, the inspection of the MALDI mass spectral analysis use Survey mode is positive ion mode or negative ion mode or reflective-mode or linear model.
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