| Glycosylation is one of the most common and complex forms of protein post-translational modification.The complex diversity and micro heterogeneity of its structure make the sugar chain have more branch structure,connection mode and spatial conformation.The changes of glycochain structure and expression are closely related to human immunity,inflammation,tumor,metabolism and other diseases.Although great progress has been made in the qualitative study of sugar chain structure,there is no more effective quantitative analysis method due to complicated and various structures of sugar chain,and the difficulty in synthesizing standard products.In view of this,we have developed an isotope labeled mass spectrometry derivatization reagent that can target the labeled sugar chain,and developed and established a highly sensitive and selective relative quantitative analysis method of sugar chain by using ultra-high performance liquid-high resolution mass spectrometry(UHPLC-HRMS).In this study,we focused on benzoyl chloride(d0/d5-bzc)with stable isotope labeling as the parent.First,we introduced the structure of alanine(OTZD),synthesized the intermediate of mass spectrometry derivatization reagent d0/d5-BOC,and then introduced the N-hydroxysuccinimide(NHS)structure,which can activate carboxyl group,a stable isotope mass spectrometry derivatization reagent(R)2,5-dioxopyrrolidin-1-yl-3-benzoyl-2-oxithiazolidine-4-carboxylate(d0-DPBOC),(R)-2,5-dioxopyrrolidin-1-yl-3-(2,3,4,5,6-pentadeuterio-benzoyl-2-oxothiazolidine-4carboxylate(d5-DPBOC),which can target to recognize amino functional groups and can be used for relatively quantitative analysis of complex sugar chains in biological samples,which can detect stable characteristic fragment ions d0(m/z 77.2 and 105.2)/d5(m/z 82.3 and 110.3)in LC-MS.D-glucosamine was used as the model monosaccharide to explore the derivatization conditions of d0-DPBOC.The results showed that the molecular weight of the product was m/z 415.10 under SIM condition,and the ionic strength of fragment ion m/z 77.2 was the highest when CE=70 eV,and the detection limit(S/N=3)was 10 attomole.In addition,the non-specific pronase e enzyme can hydrolyze the N-/O-linked glycochain enzyme on glycoprotein to glycosamine(N-glycan-Asn,O-glycan-Ser/Thr)with an amino acid active group.In this study,SGP(sialylglycopeptide)was cut into glycosamines with one asparagine residue by Pronase E enzyme and relative quantitative analysis of SGP by isotope mass spectrometry.The results showed that the actual value of the detected glycochain was consistent with the theoretical value in different molar ratio(d0/d5=4:1;2:1;1:1;1:2;1:4).The results showed that in the range of d0/d5=0.029-20 and d0/d5=0.029-20,the linear relationship of SGP were great(R2>0.9994).And the detection limit(S/N=3)was 13-15 fmol.In this study,a stable isotope labeled mass spectrometry derivatization reagent d0/d5-DPBOC which can effectively label the sugar chain in glycoprotein with benzoyl chloride as the parent structure was developed.A new high sensitivity isotope mass spectrometry derivatization reagent is provided for the relative quantitative analysis of the sugar chain in glycoprotein in the field of biological analysis.An effective and reliable analysis method for the development of differential glycomics and the screening of sugar chain markers are established. |
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