CN113466354A - N-glycopeptide terminal sialic acid alpha 2,6 and alpha 2,3 connection isomerism relative quantitative method - Google Patents
N-glycopeptide terminal sialic acid alpha 2,6 and alpha 2,3 connection isomerism relative quantitative method Download PDFInfo
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Abstract
The invention belongs to the technical field of analytical chemistry, and particularly relates to a relative quantitative method for N-glycopeptide terminal sialic acid alpha 2,6 and alpha 2,3 connection isomerism. According to the invention, liquid chromatography and ion mobility mass spectrometry are combined, a complex N-glycopeptide sample is subjected to liquid chromatography separation, then parent ion selection is carried out according to specific retention time and specific mass-to-charge ratio, collision induced dissociation is carried out under specific conditions, and all generated fragments are subjected to two-dimensional separation in an ion mobility pool and then enter a mass spectrometry detector for detection; containing terminal sialic acid alpha 2,6 and alpha 2,3 isomers B3 +The fragments are separated in a mobility tube and finally the a 2,6 and a 2,3 isomers B are obtained according to their terminal sialic acids3 +Debris mobility separation peakThe peak areas of N-glycopeptides were used to make relative quantification of the terminal sialic acid α 2,6 and α 2,3 linkage isomers of N-glycopeptides. The method is efficient, rapid and environment-friendly, and has wide application prospect in the fields of proteomics, glycochemistry and biology.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a relative quantitative method for N-glycopeptide terminal sialic acid alpha 2,6 and alpha 2,3 connection isomerism.
Background
Glycosylation is an important protein posttranslational modification (PTM) widely present in organisms, and has a very important influence on the chemical and biological properties of its protein. Sialic acid is a family of acidic nine-carbon sugars on human glycoproteins that are linked to galactose (Gal) residues by α 2, 3-or α 2,6 glycosidic linkages, and that serve as both regulatory factors for attachment proteins and as recognition targets for other glycoproteins, thereby modulating a variety of physiological and pathological processes. The analysis method of sialic acid connection isomerism is various, the traditional spectroscopy method mainly comprises a nuclear magnetic resonance method and an infrared absorption spectroscopy, and the two methods are distinguished by the spectroscopy characteristics of different connection sialic acids; firstly, based on a nuclear magnetic resonance method, the complete structure of the analyzed sugar chain can be deduced by analyzing the spectrogram of each element; but has disadvantages in that quantitative analysis cannot be achieved while requiring a relatively large amount of a sample of high purity; ② the infrared absorption spectrum of the sugar chain is generally difficult to directly analyze, and the corresponding structure needs to be confirmed by comparing with a standard sample. Although the method has low requirements on the sample amount, the method has higher requirements on the sample purity; meanwhile, this method is difficult to analyze the structure of a complex sugar chain. In addition, lectins have also been used for analysis of sialic acid linked isomerism. Among the commonly used lectins, the Huai-Sophora lectin (MAL I) selectively recognizes α 2, 3-linked sialic acid, whereas the Soy lectin (SNA) selectively recognizes α 2, 6-linked sialic acid. The lectin method can be quickly used for analyzing the content of different connecting sialic acids in a certain sample, semi-quantitative comparison is carried out among samples, but site, glycoform and structural information thereof cannot be accurately obtained, and the performance in the aspect of sensitivity is also deficient. Although the identification and quantification of sialylated α 2,3-/α 2, 6-linked N-glycan chain isomerism based on Mass Spectrometry (MS) has been rapidly developed at present, the separation analysis of N-glycopeptide sialyl α 2,3/α 2, 6-linked isomerism remains technically very challenging. The sialic acid alpha 2, 3-/alpha 2, 6-linked isomerism of glycopeptides is reported to have differences in the relative intensities of several characteristic fragments in tandem mass spectrometry, and the linkage of sialic acid in parent ions can be calculated. However, this method cannot be used for quantitative analysis of the sialic acid α 2,3-/α 2, 6-linked isomerism of glycopeptides. PGC separation at high column temperatures and reverse phase chromatographic separation also enable separation of partial glycopeptide sialic acid linkage isomers, but are limited for complex glycan samples. Therefore, a convenient, quick and environment-friendly quantitative method is lacked for the relative quantitative analysis of the terminal sialic acid alpha 2, 3/ alpha 2,6 linkage isomerism of the complex N-glycopeptide.
Disclosure of Invention
The invention aims to provide a high-throughput, efficient and accurate relative quantitative method for the sialic acid alpha 2,6 and alpha 2,3 isomers at the tail end of the complete N-glycopeptide aiming at the current situation that the relative quantitative method for the sialic acid alpha 2,6 and alpha 2,3 isomers at the tail end of the complete N-glycopeptide is short.
The invention uses a method combining liquid phase separation and ion mobility mass spectrometry gas phase grading technology to process complete N-glycopeptide, selects specific complete N-glycopeptide ions under specific liquid chromatogram retention time and specific m/z, performs Collision Induced Dissociation (CID) in a mass spectrum collision pool, and adopts a data-dependent property spectrum scanning mode without dynamic exclusion to perform mass spectrum data acquisition, thereby realizing full acquisition of target parent ions; after completion of the mass spectrometric data acquisition, the terminal sialic acid α 2,6 and α 2,3 isomers B assigned to the intact N-glycopeptide3 +The mobility Arrival Time Distribution (ATDs) areas of the fragments were compared to achieve relative quantification of the terminal sialic acid α 2,6 and α 2,3 isomers of different intact N-glycopeptides. The method comprises the following specific steps:
(1) separating the N-complete glycopeptide after the enzymolysis and enrichment of the protein sample so as to reduce the complexity of the N-glycopeptide in the sample;
here, the separation of N-intact glycopeptides may be performed by reverse phase liquid chromatography, hydrophilic liquid chromatography, ion exchange liquid chromatography, hydrophobic liquid chromatography or capillary electrophoresis;
(2) carrying out mass spectrum gas phase separation and mass spectrum detection on the separated N-glycopeptide; the mass spectrometry gas phase separation specifically comprises the following steps:
1) selecting parent ions: carrying out gas phase parent ion selection according to a specific m/z and a window of +/-2 Da on the parent ion at a specific chromatographic fractionation retention time;
2) mass spectrum collection, namely fragmenting the selected target complete N-glycopeptide parent ions by 15-50 eV fragmentation energy, and sending all fragmented ions into an ion mobility mass spectrum ion mobility drift tube for separation and analysis, wherein the collection mode of the mass spectrum is set to be an HD-MS/MS mode; finally, acquiring mass spectrum data of fragment ion signals after ion mobility separation;
the mass spectrometer used for mass spectrometry is one or more of a quadrupole mass spectrometer, an ion trap mass spectrometer, a time-of-flight mass spectrometer and an ion mobility mass spectrometer;
(3) after completion of the mass spectrometric data acquisition, the terminal sialic acid α 2,6 and α 2,3 isoforms B belonging to the same intact N-glycopeptide3 +Fragment ([ NeuAc alpha 2-3/alpha 2-6Gal beta 1-4 GlcNAc)]+, m/z 657.24) to obtain α 2,6 and α 2,3 isomers B3 +Relative quantitative ratios of fragments to achieve relative quantification of terminal sialic acid α 2,6 and α 2,3 linkage isomerism of N-intact glycopeptides.
The method provided by the invention can be used for relatively quantifying the connection isomerism of the terminal sialic acid alpha 2,6 and alpha 2,3 of the complete N-glycopeptide in different biological protein samples under different physiological or pathological states, including tissues, cells, different subcellular organelles and body fluid.
Compared with the prior art, the invention has the following advantages:
(1) based on the liquid phase separation and mass spectrum fractionation combined technology, the relative quantification of N-glycopeptide terminal sialic acid alpha 2,6 and alpha 2,3 connection isomerism is realized. The method can be used for high-throughput, efficient and accurate relative quantification of N-glycopeptide terminal sialic acid alpha 2,6 and alpha 2,3 connection isomerism;
(2) the quantitative accuracy is high. The method can collect the mobility separation of all fragment ions based on the fragment full mobility scanning of the target complete N-glycopeptide, and aims at the complete N-glycopeptideOf terminal sialic acid of alpha 2,6 and alpha 2,3 isomers B3 +Fragment ([ NeuAc alpha 2-3/alpha 2-6Gal beta 1-4 GlcNAc)]+, m/z 657.24) was quantitatively analyzed for mobility Arrival Time Distributions (ATDs);
(3) environment optimization, no need of chemical reaction and higher accuracy.
Drawings
FIG. 1 is a flow chart of the relative quantitative work flow of the present invention for achieving N-glycopeptide terminal sialic acid α 2,6 and α 2,3 isomeric ligation based on the combination of liquid phase separation and ion mobility mass spectrometry gas fractionation technology.
FIG. 2 shows the ratios of standard N-glycopeptides of the present invention, alpha 2,3-SGP and B corresponding to alpha 2,6-SGP3 +The mobility arrival time profile after the fragment mobility separation.
FIG. 3 is a diagram of B of the standard N-glycopeptide of the present invention, α 2,3-SGP and α 2,6-SGP3Graph of mobility arrival time distribution peak area versus theoretical ratio for + chips.
FIG. 4 is a comprehensive characterization of the terminal sialic acid α 2,3 and α 2,6 isomeric linkages of haptoglobin N-glycopeptides of the present invention.
Detailed Description
Example 1:
the chemicals and solvents used in the examples were all analytical grade.
(1) Standard N-glycopeptide sample preparation: standard intact N-glycopeptides with terminal sialic acids alpha 2,6 and alpha 2,3 attached (alpha 2,6-SGP and alpha 2, 3-SGP) were dissolved in 0.1% TFA according to a certain mass to obtain two standard stock solutions with final concentrations of 8.7. mu.M. The standard glycopeptides α 2,6-SGP and α 2,3-SGP were mixed at different mixing ratios (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1) in absolute concentration (87 nM) to prepare analytical samples.
(2) Liquid phase separation of mixed standard glycopeptides: the online LC adopts an M-Class nano-LC system of Waters company: phase A is 0.1% FA aqueous solution; phase B was 0.1% TFA in ACN. The N-glycopeptide is loaded by adopting on-column at the flow rate of 300 nL/min for 4 min. Then the mixture enters an analytical column for separation, and a chromatographic column adopts 15 cm C18Column (NanoE MZ PST CSH 130C181.7 μ 75 μm × 150 mm). A gradient of 60min was used, starting from 1% of phase B, starting from 35% of phase B after 40min, increasing to 80% of phase B after 5min, and then adjusting to 1% of phase B.
(3) Mass spectrum gas phase classification and mass spectrum detection: the retention time of standard glycopeptide alpha 2,6-SGP and alpha 2,3-SGP is 14.75-15.25 min, and the selection range is +/-2 Da by respectively carrying out parent ion selection on two channels of m/z 956.08 (3 +) and m/z1433.35 (2 +) in quadrupole mass spectrometry. Performing Collision Induced Dissociation (CID) of parent ions according to different fragmentation energies (15, 20, 25, 30, 35, 40, 45 and 50 eV), the fragments produced being separated in an ion mobility cell (IM); and then detecting in a mass spectrometer detector (LC-CID-IM-MS). The spray voltage of the ion mobility mass spectrum is 2.2 kV, the voltage of a taper hole is 30V, the temperature of a heating capillary is 100 ℃, and data are collected by adopting a non-data-dependent mode HD-MS/MS. The mobility traveling wave velocity is 950 m/s, the wave height is set at 40V, N2The drift gas flow rate was 90 ml/min. Data acquisition Primary scan range 100-. Lockspray Mass LE (556.2771 Da) data acquisition software for MassLynx 4.1.
As shown in FIG. 2, the α 2,6 and α 2,3 isomers B of α 2,6-SGP and α 2,3-SGP3+ fragment ([ NeuAc α 2-3/α 2-6Gal β 1-4 GlcNAc)]+, m/z 657.24) was 236A, respectively2And 246A2The corresponding arrival times are 5.9 ms and 6.4 ms, respectively. The ATDs obtained in different proportions have different distribution trends.
(4) And (3) data analysis: after the completion of the mass spectrometric data acquisition, the α 2,6 and α 2,3 isomers B produced by α 2,6-SGP and α 2,3-SGP with the same retention time and mass to charge ratio were analyzed using MassLynx 4.1 software3+ fragment ([ NeuAc α 2-3/α 2-6Gal β 1-4 GlcNAc)]+, m/z 657.24) to extract their mobility arrival time distribution maps (ATDs), and peak area calculation is performed according to their mobility separation peaks; the areas of the peaks were then compared to give α 2,6 and α 2,3 isomers B3+ fragment relative quantitative ratio, which is ascribed to the relative proportions of the α 2,6 and α 2,3 isomeric linkages contained in α 2,6-SGP and α 2, 3-SGP.
In FIG. 3, as described aboveThe analysis method calculates that the actual proportion of the alpha 2,3-SGP and alpha 2,6-SGP mixed terminal sialic acid alpha 2,6 and alpha 2,3 isomeric connection is consistent with the theoretical proportion. 2+And 3+ Alpha 2,6 and alpha 2,3 isomers B produced by charged SGP3+ fragment R has higher quantitative accuracy and linear relation in the relative quantification of terminal sialic acid alpha 2,6 and alpha 2,3 isomeric connection2The values were 0.992 and 0.997, respectively. Thus, the present invention contains terminal sialic acid α 2,6 and α 2,3 isomer B as an intact N-glycopeptide3 +Fragment ([ NeuAc alpha 2-3/alpha 2-6Gal beta 1-4 GlcNAc)]+, m/z 657.24) to achieve relatively high accuracy in the relative quantification of the α 2,6 and α 2,3 isomers of terminal sialic acids of intact N-glycopeptides.
Example 2:
the relative proportion of terminal sialic acid alpha 2,3 and alpha 2,6 isomeric connection of complete N-glycopeptide contained in haptoglobin is analyzed by using purified haptoglobin (Hp) in serum as an analyte and through the established relative quantitative method for terminal sialic acid alpha 2,6 and alpha 2,3 isomeric connection of the N-glycopeptide.
(1) Purification of serum haptoglobin: serum samples were centrifuged at 12000 × g for ten minutes and subsequently purified using HiTrap columns. Haptoglobin on the binding column was eluted with elution buffer (pH 3.0, 100 mM Glycine, 0.5M NaCl) and concentrated by acetone precipitation.
(2) Enzymolysis and N-glycopeptide purification of haptoglobin: mu.l of haptoglobin at a concentration of 1mg/mL was taken, 10. mu.l of 200mM Dithiothreitol (DTT) solution was added thereto, and the mixture was reacted at 56 ℃ for 1 hour to denature and reduce the protein. Then, 10ul of 400mM iodoacetamide solution (IAA) was added thereto, and the mixture was reacted at room temperature for 30 min with exclusion of light to conduct protein alkylation. Finally, 4 mu g of trypsin is added for carrying out enzymolysis on the protein, and after the enzymolysis is carried out for 12 hours at 37 ℃, 4 mu g of Glu-C is added for digestion and the enzymolysis is continued for 12 hours. Finally, 10. mu.L of formic acid solution was added to terminate the enzymatic reaction.
(3) HILIC enriched N-glycopeptide: first, equilibrate HILIC column with 500. mu.L of equilibration solution (80% ACN/0.1% TFA) 3 times; after the haptoglobin enzymolysis peptide segment dissolved in the balance liquid is loaded for 3 times, the HILIC column enriched with glycopeptide is washed for 3 times by the balance liquid; finally, the N-glycopeptide enriched in HILIC was eluted 2 times with 300. mu.L of 0.1% TFA, and the eluates were combined and dried by vacuum centrifugation for further use.
(4) LC-MS/MS analysis: the online LC adopts an M-Class nano-LC system of Waters company: phase A is 0.1% FA aqueous solution; phase B was 0.1% TFA in ACN. Loading the complete N-glycopeptide by using on-column at a flow rate of 0.3 muL/min for 4 min; then the column was separated by a 15 cm C18 column (NanoE MZ PST CSH 130C 181.7. mu.75. mu. m x 150 mm): a gradient of 60 minutes was used, starting from 1% of phase B, starting from 35% of phase B after 40 minutes, increasing to 80% of phase B after 5 minutes, and then adjusting to 1% of phase B.
(5) N-glycopeptide data retrieval: by adopting ByonicTMThe software (version 2.16.11, Protein Metrics, San Carlos, Calif.) searched the raw data file for the haptoglobin sequence (P00738), setting the mass errors of the parent and child ions at 20 ppm and 0.05 Da respectively. The zero deletion cleavage site was digested with trypsin/Glu-C. The fixed modification is carbamoylation (C) and the variable modification is oxidation (M) and deamidation (N). Results were filtered at 1% FDR and Byonic scored>A manual verification is performed at a confidence threshold of 150. The retrieved highly reliable N-glycopeptides are used for the relative quantitative study of the terminal sialic acid alpha 2,6 and alpha 2,3 linkage isomers of N-glycopeptides of the present invention.
(6) N-glycopeptide terminal sialic acid alpha 2,6 and alpha 2,3 linkage isomerism relative quantitative method analysis: chromatographic conditions consistent with LC-MS/MS were selected. As shown in FIG. 1, at a specific retention time, each complete N-glycopeptide of haptoglobin is selected and subjected to Collision Induced Dissociation (CID) in a quadrupole according to its m/z, all fragments generated by parent ions are subjected to mobility separation in an ion mobility cell (IM), and finally enter a mass spectrometer for detection and signal collection. The spraying voltage is 2.2 kV, the taper hole voltage is 30V, the heating capillary is 100 ℃, and data are acquired in a non-data-dependent mode/data-dependent mode. The mobility traveling wave velocity is 950 m/s, the wave height is set at 40V, N2The drift gas flow rate was 90 ml/min. The CID energy of the glycopeptide adopts gradient energy from 15V to 50V. The data acquisition primary scanning range is 100-2000 m/z, the resolution is 20K,HDMS/MS scanning mode. Lockspray Mass LE (556.2771 Da) data acquisition software for MassLynx 4.1.
(7) The α 2,6 and α 2,3 isoforms B of each intact N-glycopeptide were analyzed using MassLyn 4.1 software3+ fragment ([ NeuAc α 2-3/α 2-6Gal β 1-4 GlcNAc)]+, m/z 657.24) to extract the mobility Arrival Time Distribution (ATDs); and according to its alpha 2,6 and alpha 2,3 isomers B3The relative proportion of peak areas of the distribution time of the fragments realizes the relative quantitative comprehensive characterization of the terminal sialic acid alpha 2,6 and alpha 2,3 isomeric connection of the complete N-glycopeptide in the serum haptoglobin. As shown in FIG. 4, the present invention achieves relative quantification of N-glycopeptide terminal sialic acid α 2,6 and α 2,3 isomeric linkages contained in different N-glycosylation sites of serum haptoglobin. Provides wide application prospect for medical diagnosis and application of subsequent haptoglobin.
Claims (4)
1. A relative quantitative method of N-glycopeptide terminal sialic acid alpha 2,6 and alpha 2,3 connection isomerism is characterized in that a method combining liquid phase separation and ion mobility mass spectrometry gas fractionation technology is used for processing complete N-glycopeptide, specific complete N-glycopeptide ions are subjected to parent ion selection under specific liquid chromatogram retention time and specific m/z, collision induced dissociation is carried out in a mass spectrum collision pool, mass spectrum data acquisition is carried out by adopting a data-dependent property spectrum scanning mode without dynamic exclusion, and full acquisition of target parent ions is realized; after completion of the mass spectrometric data acquisition, the terminal sialic acid α 2,6 and α 2,3 isomers B assigned to the intact N-glycopeptide3 +Comparing the mobility arrival time distribution areas of the fragments, thereby realizing the relative quantification of sialic acid alpha 2,6 and alpha 2,3 isomers at the tail ends of different complete N-glycopeptides; the method comprises the following specific steps:
(1) separating the N-complete glycopeptide after the enzymolysis and enrichment of the protein sample so as to reduce the complexity of the N-glycopeptide in the sample;
(2) carrying out mass spectrum gas phase separation and mass spectrum detection on the separated N-glycopeptide; the mass spectrometry gas phase separation specifically comprises:
1) selecting parent ions: carrying out gas phase parent ion selection according to a specific m/z and a window of +/-2 Da on the parent ion at a specific chromatographic fractionation retention time;
2) mass spectrum collection, namely fragmenting the selected target complete N-glycopeptide parent ions by 15-50 eV fragmentation energy, and sending all fragmented ions into an ion mobility mass spectrum ion mobility drift tube for separation and analysis, wherein the collection mode of the mass spectrum is set to be an HD-MS/MS mode; finally, acquiring mass spectrum data of fragment ion signals after ion mobility separation;
(3) after completion of the mass spectrometric data acquisition, the terminal sialic acid α 2,6 and α 2,3 isoforms B belonging to the same intact N-glycopeptide3 +Fragment ([ NeuAc alpha 2-3/alpha 2-6Gal beta 1-4 GlcNAc)]+, m/z 657.24) to obtain α 2,6 and α 2,3 isomers B3 +Relative quantitative ratios of fragments to achieve relative quantification of terminal sialic acid α 2,6 and α 2,3 linkage isomerism of N-intact glycopeptides.
2. The method for the relative quantification of terminal sialic acid α 2,6 and α 2,3 linkage isomers of N-glycopeptides of claim 1, wherein the N-intact glycopeptides after enzymatic enrichment of the protein sample in step (1) are separated by reverse phase liquid chromatography, hydrophilic liquid chromatography, ion exchange liquid chromatography, hydrophobic liquid chromatography or capillary electrophoresis.
3. The method for the relative quantification of N-glycopeptide terminal sialic acid alpha 2,6 and alpha 2,3 linkage isomers according to claim 1, wherein the mass spectrometer used for the mass spectrometric detection in step (2) is one or more of a quadrupole mass spectrometer, an ion trap mass spectrometer, a time-of-flight mass spectrometer and an ion mobility mass spectrometer.
4. The method for the relative quantification of terminal sialic acids α 2,6 and α 2,3 linkage isomers of N-glycopeptides of claim 1, wherein the protein samples are protein samples of different organisms in different physiological or pathological states, including tissues, cells, different subcellular organelles or body fluids.
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