CN105527334B - A method of improving oligosaccharides Ionization Efficiency - Google Patents

Abstract

The invention belongs to biochemical analysis fields, are related to a kind of method improving oligosaccharides Ionization Efficiency, especially a kind of to improve the oligosaccharides method ionized in substance assistant laser desorpted flight time mass spectrum (MALDI TOF MS)；This method is used as substrate assay oligosaccharides using 6 buzane niacinamides (HYNIC), selectively improves the Ionization Efficiency of oligosaccharides, and inhibits the ionization of other analytes such as peptide fragment.Heretofore described 6 buzane niacinamide has the structure of formula (I)；When the 6 buzane niacinamide is as substrate assay oligosaccharides, have many advantages, such as that high sensitivity, crystallization are uniform, salt tolerance is strong, tandem mass spectrometry fragment information is abundant；Method of the present invention, using the 6 buzane niacinamide as substrate assay oligosaccharides, the Ionization Efficiency of oligosaccharides can be selectively improved, and inhibit the ionization of other analytes such as peptide fragment, had the advantages that easy to operate, step is simple, economy is time saving, be not necessarily to desalination.

Description

A method of improving oligosaccharides Ionization Efficiency

Technical field

The invention belongs to biochemical analysis fields, are related to a kind of method of oligosaccharides ionization, and in particular to a kind of raising oligosaccharides The method of Ionization Efficiency, especially a kind of raising oligosaccharides is in substance assistant laser desorpted flight time mass spectrum (MALDI-TOF MS) ionized method；This method is used as substrate assay oligosaccharides using 6- buzanes niacinamide (HYNIC), selectively carries The Ionization Efficiency of high oligosaccharides, and inhibit the ionization of other analytes such as peptide fragment.

Background technology

Oligosaccharides is internal a kind of important information substance, has many biological functions, generally by 3-10 monosaccharide molecule It is formed by glucosides key connection.The oligosaccharides has important physiological function, such as the ego defense system of active cell, determines thin Born of the same parents identify, gather and receptor acting etc., and many biological functions determine that it has a wide range of applications in field of medicaments.

The substance assistant laser desorpted flight time mass spectrum (MALDI-TOF MS) is to come out and send out rapidly at the end of the 20th century The mass-spectrometric technique that exhibition is got up, is widely used in biochemical field.The MALDI-TOF MS because it is quick, sensitive, knot can be provided The advantages that structure information, it has also become the important tool of glucide analysis.In MALDI-TOF MS analyses, matrix plays pole Its important role；Wherein, the matrix is a kind of small organic molecule that can absorb laser, and sample is mixed in matrix, Under the irradiation of laser, sample is desorbed and is ionized, using flying time mass spectrum analysis.But the hydrophily of native oligosaccharides is strong, and Lack proton acceptor, Ionization Efficiency is low in the gas phase, hinders researchs of the MALDI-TOF MS to oligosaccharides.Currently, being Ionization Efficiency of the oligosaccharides in mass spectrum is improved, has researcher to carry out various trials, such as carries out permethylated derivative or reduction Ammonification derives the hydrophobicity for promoting oligosaccharides or electrification property, to improve the Mass Spectrometer Method signal of oligosaccharides.The new medium is such as Binary substrate, ionic liquid matrix, inorganic matrix etc. are also reported and the detection sensitivity of oligosaccharides can be improved successively.With derivative method It compares, directly utilizes matrix detection with the advantages that easy to operate, saving reagent is not necessarily to purifying, and spectrum elucidation facilitates.

But it is so far, there is not yet related few as substrate assay oligosaccharides, raising using 6- buzanes niacinamide (HYNIC) Report of the sugar in the ionized method of substance assistant laser desorpted flight time mass spectrum.

Invention content

The purpose of the present invention is to provide a kind of method improving oligosaccharides Ionization Efficiency, especially a kind of raising oligosaccharides exists The ionized method of substance assistant laser desorpted flight time mass spectrum (MALDI-TOF MS)；This method uses 6- buzane Buddhist nuns gram Amide (HYNIC) is used as substrate assay oligosaccharides, selectively improves the Ionization Efficiency of oligosaccharides, and inhibits other analytes Such as the ionization of peptide fragment.

Specifically, raising oligosaccharides provided by the invention is ionized in substance assistant laser desorpted flight time mass spectrum Method, which is characterized in that use matrix of the 6- buzanes niacinamide (HYNIC) as oligosaccharides in MALDI analysis, step Including：

(1) after the drying of sample solution point target, by matrix 6- buzanes niacinamide (HYNIC) the solution point in corresponding target spot Upper drying；

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

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

In the method for the present invention, the host solvents system is methanol and acetic acid；

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

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

The present invention has carried out contrast experiment comprising following step：

(1) sugared (DP7) with malt seven, glucan 1000 is standard oligosaccharide, standard Core fucose (NA2F), standard saliva It is standard N- sugar chains to be acidified N sugared (A1), tests effects of the HYNIC as matrix, and compared with traditional matrices DHB；

(2) with the mixture of standard section of synthesized peptide and DP7, ribonuclease B sugar chain is model measurement with peptide fragment mixture The selective ionising effect of the HYNIC, and compared with traditional matrices DHB；

(3) using DP7 as standard oligosaccharide, non-volatile salt sodium chloride is that solvent tests the salt tolerance of the HYNIC, and with biography System matrix DHB is compared；

(4) using DP7 as standard oligosaccharide influences of the HYNIC for its fragmentation pattern is tested using tandem mass spectrometry, and with Traditional matrices DHB is compared；

(5) to go sugar chain enzyme PNGase F to discharge human seroglycoid's matter N- sugar chains as actual sample, described in test HYNIC matrix is used for the feasibility of actual sample analysis.

Experimental result shows that compared with prior art, the present invention can selectively improve the ion of oligosaccharides to the method for the present invention Change efficiency, and inhibits the ionization (as shown in Table 1 and Table 2) of other analytes such as peptide fragment；And with high sensitivity, crystallization Uniformly, the features such as salt tolerance is strong, tandem mass spectrometry fragment information is abundant.

The matter lotus of the different polymerization degree oligosaccharides for the 50pmol glucans 1000 that HYNIC and DHB are detected is respectively adopted in table 1 Compare information

Table 2 uses HYNIC to carry out the information that transannular is broken in the sugared tandem spectrogram detection of malt seven for matrix

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

(1) HYNIC can increase substantially the signal-to-noise ratio and signal strength of oligosaccharides mass spectral analysis as matrix；

(2) HYNIC optionally improves the Ionization Efficiency of oligosaccharides as matrix, while inhibiting the ion of peptide fragment Change efficiency；

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

(4) HYNIC improves oligosaccharides second order ms signal-to-noise ratio as matrix, enriches fragmentation information, is conducive to sugar Chain structure parses.

Description of the drawings

Fig. 1 is the structural schematic diagram of HYNIC；

Fig. 2 is the DP7 that applied sample amount is 1pmol, the DP7 that 1pmol methylates, and 5pmol NA2F and 5pmol A1 are respectively adopted DHB (picture top) and HYNIC (picture lower part) is the mass spectrogram of matrix；Wherein, ● represent sugar chain and sodium peak, ■ is added to represent sugar chain Add potassium peak；

The mass spectrum that the glucan 1000 that Fig. 3 applied sample amounts are 100pmol is respectively adopted with DHB (a) and HYNIC (b) are matrix Figure；Wherein, ● represent sugar chain and sodium peak, ■ is added to represent sugar chain and add potassium peak；

Fig. 4 is standard peptide fragment and DP7 molar ratios are 1:1 (a, b) and 1:When 10 (c, d), be respectively adopted DHB (a, c) and HYNIC (b, d) is the mass spectrogram of matrix；Wherein, ● represent sugar chain and sodium peak, ■ is added to represent sugar chain and add potassium peak.* peptide fragment peak is represented；

Fig. 5 is that DHB (a) and HYNIC (b) is respectively adopted in the ribonuclease B sugar chain of 10pmol equivalents and peptide fragment mixture For the mass spectrogram of matrix；Wherein, ● represent sugar chain and sodium peak, * is added to represent peptide fragment peak；

Fig. 6 is that HYNIC (red) and DHB (black) is respectively adopted as upshift signal repeatability in the DP7 that applied sample amount is 1pmol Schematic diagram；

It is the mass spectrum imaging figure of matrix that HYNIC (a) and DHB (b), which is respectively adopted, in seven sugar of malt that Fig. 7 applied sample amounts are 1pmol；

Fig. 8 be the DP7 that applied sample amount is 1pmol be respectively adopted DHB (left side) and HYNIC (right side) with sodium chloride be it is non-volatile Property solvent carry out salt tolerance test mass spectrogram；

Fig. 9 is the mass spectrogram of the human serum sugar chain signal detected using HYNIC matrix；

Figure 10 is the tandem mass spectrometry that seven sugar of malt that applied sample amount is 1pmol is respectively adopted that HYNIC (A) and DHB (B) is matrix Figure.

Specific implementation mode

Embodiment 1

1. take 1 μ L DP7 (1pmol/L) respectively with 1 μ L10mg/mL DHB matrix solutions (50% acetonitrile, 0.1% trifluoro second Acid) and 1 μ L2mg/mL HYNIC (98% methanol, 2% acetic acid) mixing, point target carries out MALDI-TOF MS analyses, as a result such as Fig. 2 Shown, comparison diagram 2 (a) is with Fig. 2 (b) it is found that can get higher-quality DP7 mass spectrograms, and its letter as matrix using HYNIC It makes an uproar than improving 10 times；Other analyte specific implementation process are similar with DP7；

2. taking standard peptide fragment and DP7 respectively according to molar ratio 1:1 and 1：10 mixing, mixture respectively take 1 μ L point targets to carry out MALDI-TOF MS analyses, the results are shown in Figure 4, when being analyzed using HYNIC matrix, sugar chain DP7 signal strength highers, and mark Quasi- peptide segment signal is suppressed；And DHB matrix effects are opposite；

3. weighing 0.5mg ribonuclease Bs (Rnase B), it is dissolved in 50mM ammonium hydrogen carbonate (pH8.0), boiling water bath adds Heat 10 minutes；10 μ g trypsase (trypsin) are added, being boiled after 37 DEG C of reactions cooling in 12 hours makes trypsin inactivation obtain Rnase B peptide fragments；0.5 μ L PNGase F release Rnase B sugar chains are added after cooling, 37 DEG C are reacted 24 hours, and Rnase is obtained The mixture of B sugar chains and peptide fragment；Put target after mixture is diluted to 10pmol, use respectively DHB matrix and HYNIC matrix point target into Row MALDI-TOF MS analysis, the results are shown in Figure 5, when using mixtures of the DHB as substrate assay 10pmol amounts, only peptide Segment signal is detected, and when mixtures of the use HYNIC as substrate assay equivalent, all 5 sugar chains are all detected, table The bright HYNIC matrix optionally improves the Ionization Efficiency of sugar chain, and peptide for inhibiting segment signal.

Embodiment 2

1pmol/ μ L DP71 μ L point targets are taken to carry out repeated experiment；12 positions are randomly selected on target spot carries out mass spectrum Analysis, the intensity of 12 mass spectrograms is compared；The results are shown in Figure 6, when using HYNIC as matrix, signal strength and Stability is superior to DHB matrix.

Further to verify the uniformity of matrix crystallization, 1pmol/ μ L DP71 μ L point targets simultaneously carry out mass spectrum imaging analysis, tie Fruit is as shown in Figure 7a, the crystal regions DHB be predominantly located at target spot periphery, and target center almost nodeless mesh and cause signal strong It spends low；And HYNIC crystallization it is (as shown in Figure 7b) be located at entire target area range and crystallization uniformly.

Embodiment 3

1 μ L point targets are respectively taken to carry out MALDI- after taking DP7 to be mixed with the non-volatile salt NaCl of various concentration (25mM to 200mM) TOF MS analyses, the results are shown in Figure 8, and compared with the DHB matrix, the HYNIC matrix has stronger salt tolerance, DP7 letters Number intensity higher.

Embodiment 4

Healthy Human Serum heat denatured 5 minutes in 8mol/L urea and 200mM Tris-HCl buffer solutions, it is molten to sample Dithiothreitol (DTT) (DTT) is added in liquid, makes final concentration of 10mM, is placed in 37 DEG C of constant temperature, oscillating reactions 60min, carries out albumen two The reduction of sulfide linkage；After the completion of reaction, IAA is added into solution, final concentration of 30mM, room temperature is made to be incubated under the conditions of being protected from light 60min realizes the alkylation of sulfydryl；The DTT and IAA and denaturant are removed by ultrafiltration step；Then to being added 1 in sample μ L PNGase F carry out desaccharification chain reaction, this process continues 24 hours；Final N- sugar chains pass through the egg in ultrafiltration step and serum It is white to separate.；It takes 1 μ L point targets and carries out MALDI-TOF MS analyses as matrix using HYNIC, the results are shown in Figure 9, detects More than 40 kinds sugar chains.

Embodiment 5

1 μ L DP7 (100 ng/ μ L) and 1 μ L DHB matrix and 1 μ L HYNIC matrix, point target is taken to carry out MALDI-TOF MS/MS analyzes (the results are shown in Figure 10), chooses parent ion m/z1175.4 and carries out MALDI-TOF MS/MS analyses (result such as figure 10)；With the DHB discrete phases ratio (as shown in fig. lob), the HYNIC matrix is more advantageous to oligosaccharides (as shown in Figure 10 a) MS/MS collection of illustrative plates signal-to-noise ratio is significantly increased with signal strength, and different fragmentation patterns is conducive to sugar chain structure parsing.

Above-described embodiment the result shows that, when the 6- buzanes niacinamide is as substrate assay oligosaccharides, have sensitivity The advantages that height, crystallization is uniform, and salt tolerance is strong, and tandem mass spectrometry fragment information enriches；Due to being not necessarily to derivative, separation, desalination, Sample need not be purified, and sample loss be avoided, to standard oligosaccharide glucose when the 6- buzanes niacinamide is as matrix The detection limit of seven glycan is up to 1amol；In analyzing sugar chain and peptide fragment mixture, the 6- buzanes niacinamide alternative carries The Ionization Efficiency of high sugar chain and the Ionization Efficiency for inhibiting peptide fragment；In addition, in second order ms figure, by the 6- buzanes Buddhist nun gram The fragment signal strength that amide obtains improves and has part transannular fracture information, is conducive to the parsing of sugar chain structure；In practical sample In product human serum, more than 40 kind N- sugar chains are detected when the 6- buzanes niacinamide is as matrix altogether；Compared with prior art, originally Invention the method has the characteristics that easy to operate, step is simple, economy is time saving, is not necessarily to desalination.

Claims (4)

1. a kind of method improving oligosaccharides Ionization Efficiency, which is characterized in that using 6- buzanes niacinamide (HYNIC) as few Matrix of the sugar in MALDI analysis, step include：

(1) after the drying of sample solution point target, 6- buzanes niacinamide (HYNIC) the solution point is dried on corresponding target spot；

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

The dicyandiamide solution of the matrix is methanol and acetic acid, acetic acid volume fraction 1.5-5%；

The final concentration of 1.5-3mg/mL of the 6- buzanes niacinamide (HYNIC).

2. method as described in claim 1, which is characterized in that the wherein described 6- buzanes niacinamide (HYNIC) has formula (I) Structure,

3. method as described in claim 1, which is characterized in that the wherein described acetic acid volume fraction is 2%.

4. method as described in claim 1, which is characterized in that the wherein described 6- buzanes niacinamide (HYNIC) is final concentration of 2mg/mL。