CN105527334A

CN105527334A - Method for increasing oligosaccharide ionization efficiency

Info

Publication number: CN105527334A
Application number: CN201410521819.2A
Authority: CN
Inventors: 陆豪杰; 焦竞; 张莹
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2014-09-30
Filing date: 2014-09-30
Publication date: 2016-04-27
Anticipated expiration: 2034-09-30
Also published as: CN105527334B

Abstract

The invention belongs the field of biochemical analysis, and discloses a method for increasing oligosaccharide ionization efficiency, and relates to a method for increasing oligosaccharides ionization in a matrix auxiliary laser desorption flight time mass spectrum (MALDI-TOF MS). The method employs 6-HYNIC as matrix analysis oligosaccharides, and optionally increases the ionization efficiency of oligosaccharide, and inhibits ionization of other analyte such as a peptide segment. The 6-HYNIC has a structure shown in a formula (I); the 6-HYNIC as the matrix for analysis of oligosaccharide has the advantages of high sensitivity, uniform crystallization, strong salt resistance performance, and abundant tandem mass spectrometry fragment information. The method employs 6-HYNIC as the matrix for analysis of oligosaccharide, and optionally increases the ionization efficiency of oligosaccharide, and inhibits ionization of other analyte such as a peptide segment. The method has the advantages of simple operation, simple steps, economy, time saving, and no requirement of salt removal.

Description

A kind of method improving oligosaccharides Ionization Efficiency

Technical field

The invention belongs to biochemical analysis field, relate to a kind of Ionized method of oligosaccharides, be specifically related to a kind of method improving oligosaccharides Ionization Efficiency, especially a kind of oligosaccharides that improves is in the ionized method of substance assistant laser desorpted flight time mass spectrum (MALDI-TOFMS); The method adopts 6-buzane niacinamide (HYNIC) as substrate assay oligosaccharides, optionally improves the Ionization Efficiency of oligosaccharides, and inhibits other analyte as the ionization of peptide section.

Background technology

Oligosaccharides is the information substance that in body, a class is important, has many biological functions, is generally formed by connecting by glycosidic bond by 3-10 monosaccharide molecule.Described oligosaccharides has important physiological function, and as the ego defense system of active cell, determine cell recognition, gather and receptor acting etc., its many biological function determines it and has a wide range of applications at field of medicaments.

Described substance assistant laser desorpted flight time mass spectrum (MALDI-TOFMS) comes out for 20 end of the centurys and the mass-spectrometric technique developed rapidly, is widely used in biochemical field.Described MALDI-TOFMS because of quick, sensitive, can the advantages such as structural information be provided, become glucide analyze important tool.In described MALDI-TOFMS analyzes, matrix plays extremely important effect; Wherein, described matrix is the small organic molecule of a class energy absorbing laser, is mixed in by sample in matrix, and under the irradiation of laser, sample desorb also ionizes, then through flying time mass spectrum analysis.But the water wettability of native oligosaccharides is strong, and lacks proton acceptor, and Ionization Efficiency is low in the gas phase, hinders the research of described MALDI-TOFMS to oligosaccharides.At present, be improve the Ionization Efficiency of oligosaccharides in mass spectrum, the various trial that had researcher to carry out, derives as carried out permethylated derivative or reduction amination the hydrophobicity or charged character that promote oligosaccharides, thus improves the Mass Spectrometer Method signal of oligosaccharides.Described new medium is as binary substrate, and ionic liquid matrix, inorganic matrix etc. are also in the news successively can improve the detection sensitivity of oligosaccharides.Compared with derivative method, directly utilize matrix detection to have easy and simple to handle, save reagent, without the need to purifying, the advantages such as spectrum elucidation is convenient.

But up to now, there is not yet about adopting 6-buzane niacinamide (HYNIC) as substrate assay oligosaccharides, improving the report of oligosaccharides in the ionized method of substance assistant laser desorpted flight time mass spectrum.

Summary of the invention

The object of the present invention is to provide a kind of method improving oligosaccharides Ionization Efficiency, especially a kind of oligosaccharides that improves is in the ionized method of substance assistant laser desorpted flight time mass spectrum (MALDI-TOFMS); The method adopts 6-buzane niacinamide (HYNIC) as substrate assay oligosaccharides, optionally improves the Ionization Efficiency of oligosaccharides, and inhibits other analyte as the ionization of peptide section.

Specifically, raising oligosaccharides provided by the invention is in the ionized method of substance assistant laser desorpted flight time mass spectrum, it is characterized in that, adopt 6-buzane niacinamide (HYNIC) as the matrix of oligosaccharides in maldi analysis, its step comprises:

(1), after sample solution point target is dried, described matrix 6-buzane niacinamide (HYNIC) solution point is dried on corresponding target spot;

(2) target plate is sent into described MALDI-TOF-MS to analyze.

In method of the present invention, described 6-buzane niacinamide (HYNIC) has the structure of formula (I),

In method of the present invention, described host solvents system is methyl alcohol and acetic acid;

In method of the present invention, described acetic acid volume fraction is 1.5-5%, and in one embodiment of the present of invention, preferred acetic acid volume fraction is 2%;

In method of the present invention, the final concentration of described 6-buzane niacinamide (HYNIC) is 1.5-3mg/mL, is preferably 2mg/mL in one embodiment of the present of invention.

Invention has been contrast experiment, it comprises the steps:

(1) with Fructus Hordei Germinatus seven sugar (DP7), glucosan 1000 is standard oligosaccharide, standard Core fucose (NA2F), and the sialylated N sugar (A1) of standard is standard N-sugar chain, test the effect of described HYNIC as matrix, and contrast with traditional matrices DHB;

(2) with the potpourri of standard section of synthesized peptide and DP7, the selectivity ionising effect that ribonuclease B sugar chain and peptide section potpourri are HYNIC described in model measurement, and contrast with traditional matrices DHB;

(3) take DP7 as standard oligosaccharide, non-volatile salt sodium chloride is the brine tolerance that described HYNIC tested by solvent, and contrasts with traditional matrices DHB;

(4) be that standard oligosaccharide utilizes tandem mass spectrometry to test the impact of described HYNIC for its fragmentation pattern with DP7, and contrast with traditional matrices DHB;

(5) discharge human seroglycoid matter N-sugar chain as actual sample using desaccharification chain enzyme PNGaseF, test the feasibility that described HYNIC matrix is analyzed for actual sample.

Experimental result shows, and compared with prior art, the present invention optionally can improve the Ionization Efficiency of oligosaccharides to the inventive method, and inhibits other analyte as the ionization (as shown in Table 1 and Table 2) of peptide section; And there is the features such as highly sensitive, crystallization is even, brine tolerance is strong, tandem mass spectrometry patch information is abundant.

The mass-to-charge ratio information of the different polymerization degree oligosaccharides of the 50pmol glucosan 1000 that table 1 adopts HYNIC and DHB to detect respectively

Table 2 adopt HYNIC be matrix carry out the sugared tandem spectrogram of Fructus Hordei Germinatus seven detect in the information of transannular fracture

The method of the invention compared with prior art, has the following advantages:

(1) described HYNIC can increase substantially signal to noise ratio (S/N ratio) and the signal intensity of oligosaccharides mass spectrophotometry as matrix;

(2) described HYNIC optionally improves the Ionization Efficiency of oligosaccharides as matrix, simultaneously the Ionization Efficiency of peptide for inhibiting section;

(3) described HYNIC is even as matrix crystallization, improves the signal stabilization of oligosaccharides detection and repeatability;

(4) described HYNIC improves oligosaccharides second order ms signal to noise ratio (S/N ratio) as matrix, has enriched cracked information, is conducive to sugar chain structure and resolves.

Accompanying drawing explanation

Fig. 1 is the structural representation of HYNIC;

The DP7 of Fig. 2 to be applied sample amount be 1pmol, 1pmol methylated DP7,5pmolNA2F and 5pmolA1 adopt the mass spectrogram that DHB (picture top) and HYNIC (picture bottom) is matrix respectively; Wherein, ● represent sugar chain and add sodium peak, ■ represents sugar chain and adds potassium peak;

Fig. 3 applied sample amount is the mass spectrogram that the glucosan 1000 of 100pmol adopts respectively and DHB (a) and HYNIC (b) is matrix; Wherein, ● represent sugar chain and add sodium peak, ■ represents sugar chain and adds potassium peak;

When Fig. 4 to be standard peptide section and DP7 mol ratio be 1:1 (a, b) and 1:10 (c, d), adopt the mass spectrogram that DHB (a, c) and HYNIC (b, d) is matrix respectively; Wherein, ● represent sugar chain and add sodium peak, ■ represents sugar chain and adds potassium peak.* representative peptide Duan Feng;

Fig. 5 is the mass spectrogram that the ribonuclease B sugar chain of 10pmol equivalent and peptide section potpourri adopt that DHB (a) and HYNIC (b) is matrix respectively; Wherein, ● represent sugar chain and add sodium peak, * representative peptide Duan Feng;

The DP7 of Fig. 6 to be applied sample amount be 1pmol adopts HYNIC (redness) and DHB (black) for upshift signal repeatability schematic diagram respectively;

Fig. 7 applied sample amount is that Fructus Hordei Germinatus seven sugar of 1pmol does not adopt the mass spectrum imaging figure that HYNIC (a) and DHB (b) is matrix;

The DP7 of Fig. 8 to be applied sample amount be 1pmol adopts DHB (left side) and HYNIC (right side) to take sodium chloride as the mass spectrogram that non-volatile solvents carries out brine tolerance test respectively;

Fig. 9 is the mass spectrogram of the human serum sugar chain signal adopting HYNIC matrix to detect;

Fructus Hordei Germinatus seven sugar of Figure 10 to be applied sample amount be 1pmol does not adopt the tandem mass spectrometry figure that HYNIC (A) and DHB (B) is matrix.

Embodiment

Embodiment 1

1. get 1 μ LDP7 (1pmol/L) respectively with 1 μ L10mg/mLDHB matrix solution (50% acetonitrile, 0.1% trifluoroacetic acid) and 1 μ L2mg/mLHYNIC (98% methyl alcohol, 2% acetic acid) mixing, point target carries out MALDI-TOFMS analysis, result as shown in Figure 2, comparison diagram 2 (a) is known with Fig. 2 (b), adopt HYNIC can obtain higher-quality DP7 mass spectrogram as matrix, and its signal to noise ratio (S/N ratio) improves 10 times; Other analyte specific implementation process and DP7 similar;

2. get standard peptide section and DP7 respectively according to mol ratio 1:1 and 1:10 mixing, potpourri is respectively got 1 μ L point target and is carried out MALDI-TOFMS analysis, result as shown in Figure 4, when adopting HYNIC matrix to analyze, sugar chain DP7 signal intensity is higher, and standard peptide segment signal is suppressed; And DHB matrix effect is contrary;

3. take 0.5mg ribonuclease B (RnaseB), be dissolved in 50mM ammonium bicarbonate (pH8.0), boiling water bath heats 10 minutes; Add 10 μ g trypsase (trypsin), boil after 37 DEG C of reactions cooling in 12 hours and make trypsin inactivation obtain RnaseB peptide section; Add 0.5 μ LPNGaseF after cooling and discharge RnaseB sugar chain, 37 DEG C are reacted 24 hours, obtain the potpourri of RnaseB sugar chain and peptide section; Target is put by after mixture diluted to 10pmol, MALDI-TOFMS analysis is carried out respectively with DHB matrix and HYNIC matrix point target, result as shown in Figure 5, during the potpourri adopting DHB to measure as substrate assay 10pmol, only have peptide segment signal to be detected, and when adopting the potpourri of HYNIC as substrate assay equivalent, all 5 sugar chains are all detected, show that described HYNIC matrix optionally improves the Ionization Efficiency of sugar chain, and peptide for inhibiting segment signal.

Embodiment 2

Get 1pmol/ μ LDP71 μ L point target and carry out repeated experiment; On target spot, mass spectrophotometry is carried out in random selecting 12 positions, the intensity of 12 mass spectrograms is compared; As shown in Figure 6, when adopting HYNIC as matrix, signal intensity and stability are all better than DHB matrix to result.

For verifying the homogeneity of matrix crystallization further, 1pmol/ μ LDP71 μ L point target also carries out mass spectrum imaging analysis, and as shown in Figure 7a, described DHB crystal region is mainly positioned at target spot periphery to result, and target spot center almost nodeless mesh and cause signal intensity low; And HYNIC crystallization (as shown in Figure 7b) is positioned at whole target area scope and crystallization is even.

Embodiment 3

Get and respectively get 1 μ L point target after DP7 mixes with the non-volatile salt NaCl (25mM to 200mM) of variable concentrations and carry out MALDI-TOFMS analysis, result as shown in Figure 8, than described DHB matrix, described HYNIC matrix has stronger brine tolerance, and DP7 signal intensity is higher.

Embodiment 4

Healthy Human Serum heat denatured 5 minutes in 8mol/L urea and 200mMTris-HCl damping fluid, dithiothreitol (DTT) (DTT) is added in sample solution, make final concentration be 10mM, be placed in 37 DEG C of constant temperature, oscillating reactions 60min, carry out the reduction of protein disulfide bond; After having reacted, in solution, add IAA, make final concentration be 30mM, hatch 60min under room temperature, lucifuge condition, realize the alkylation of sulfydryl; Described DTT and IAA and denaturant are removed by ultrafiltration step; Desaccharification chain reaction is carried out, this process lasts 24 hours subsequently to adding 1 μ LPNGaseF in sample; Final N-sugar chain is separated by ultrafiltration step and the albumen in serum.; Get 1 μ L point target and utilize HYNIC to carry out MALDI-TOFMS analysis as matrix, result as shown in Figure 9, detects more than 40 kinds of sugar chains.

Embodiment 5

Get 1 μ LDP7 (100ng/ μ L) and 1 μ LDHB matrix and 1 μ LHYNIC matrix, point target carries out MALDI-TOFMS/MS analysis (result as shown in Figure 10), chooses parent ion m/z1175.4 and carries out MALDI-TOFMS/MS analysis (result is as Figure 10); Compared with described DHB matrix (as shown in fig. lob), described HYNIC matrix (as shown in Figure 10 a) is more conducive to the MS/MS collection of illustrative plates signal to noise ratio (S/N ratio) of oligosaccharides and signal intensity significantly strengthens, and different fragmentation patterns is conducive to sugar chain structure resolves.

The advantages such as the result of above-described embodiment shows, when described 6-buzane niacinamide is as substrate assay oligosaccharides, has highly sensitive, and crystallization is even, and brine tolerance is strong, and tandem mass spectrometry patch information is abundant; Due to without the need to the step such as derivative, separation, desalination, sample need not carry out purifying, avoids sample loss, and described 6-buzane niacinamide is as reaching 1amol to the detection limit of standard oligosaccharide glucose seven glycan during matrix; When analyzing sugar chain and peptide section potpourri, described 6-buzane niacinamide alternative improves the Ionization Efficiency of sugar chain and the Ionization Efficiency of peptide for inhibiting section; In addition, in second order ms figure, the fragment signal intensity obtained by described 6-buzane niacinamide improves and has part transannular fracture information, is conducive to the parsing of sugar chain structure; In actual sample human serum, described 6-buzane niacinamide is as detecting during matrix that more than 40 plant N-sugar chain altogether; Compared with prior art, the method for the invention has that easy and simple to handle, step is simple, economy saves time, without the need to the feature of desalination.

Claims

1. improve a method for oligosaccharides Ionization Efficiency, it is characterized in that, adopt 6-buzane niacinamide (HYNIC) as the matrix of oligosaccharides in described maldi analysis, its step comprises:

(2) target plate is sent into described substance assistant laser desorpted flight time mass spectrum (MALDI-TOF-MS) to analyze;

Described host solvents system is methyl alcohol and acetic acid, and acetic acid volume fraction is 1.5-5%;

Described 6-buzane niacinamide (HYNIC) final concentration is 1.5-3mg/mL.

2., by method according to claim 1, it is characterized in that, described 6-buzane niacinamide (HYNIC) has the structure of formula (I),

3., by method according to claim 1, it is characterized in that, described acetic acid volume fraction is 2%.

4., by method according to claim 1, it is characterized in that, described 6-buzane niacinamide (HYNIC) final concentration is 2mg/mL.