Synthesis And Applications Of New Tags In MALDI-MS-based Glycomics Analysis

Glycosylation is a complicated and varied but extremely important post-translational modification that regulates the physical,chemical,and biological properties of proteins.Abnormal expression of glycans is closely related to many diseases,including cancer,and may provide potential biomarkers for the diagnosis,treatment and prognosis of diseases.Glycans is synthesized in a non-template-driven way,and its structure is diverse and heterogeneous,which hinders its analysis.Among variety of glyco-analytical tools,matrix assisted laser desorption ionization mass spectrometry(MALDI-MS)is one of the most commonly used analytical platforms for glycan researches because of its great advantages in sensitivity,efficiency and anti-interference.In order to alleviate the limitations of MALDI-MS analysis caused by the low ionization efficiency of highly hydrophilic glycan and the easy degradation of acidic sugars in and out of source,chemical derivatization is usually required before glycan analysis.Chemical derivatization improves the ionization efficiency and detection sensitivity by modifying the chemical groups on the glycan to improve its stability,hydrophilicity and electrically charged properties.Common types of markers include amines,hydrazines,succinimides and so on,among which hydrazine and succinimide ester compounds can label glycans by a one-step reaction,and hydrazine-derived glycans also have the advantage of direct loadeding without purification.However,the hydrazine and succinimide reagents currently used in MALDI-MS based glycosomics analysis are very limited,and there is a great potential for improvement in their derivatization efficiency and mass spectral sensitivity enhancement.Therefore,in this work,hydrazine and succinimide ester reagents based on highly active precursors and their corresponding isotope reagents were developed and synthesized,and they were applied to the qualitative and quantitative analysis of glycan.The specific contents are as follows:Based on the existing commonly used hydrazine reagent structures for glycan labeling,this work combined the orbital frontier theory and density functional theory to calculate the hydrazine molecules containing different precursor structures to predict the reaction activity of each molecule,and precursor structure N,N,N-trimethylaminobenzene(TMBA)with potential high activity was screened out.The synthesis of TMBA-based hydrazine HTMBA adopted the synthesis route of TMBA-based succinimide ester molecule TEBA as the intermediate,which could avoid the de-methylation of quaternary ammonium group at high temperature,and it can be used for the efficient synthesis of multiple target compounds and their isotopes.At present,the derivatization conditions of reported hydrzines are generally 70?80?of heat and 1?4hours of incubation,while HTMBA can achieve complete glycan labeling by simple mixing at room temperature,and the MS sensitivity of HTMBA labeled glycan is?20times higher,which is about twice that of Girard's Reagent T.Based on this,a one-step HTMBA-on-Target Derivation(HOD)strategy was proposed in this paper,in which the matrix,sample and HTMBA were successively placed on the MALDI sample board.After natural drying,mass spectrometric analysis was performed immediately.The results showed that the glycan was completely labeled under this strategy,and the sensitivity was significantly improved.The results showed that 44glycan structures could be detected in 0.5?L human serum after methylamidation-HOD derivatization,which was 28 more than that of the methylamidated samples.The structure of O-acetylated sialylated glycans were detected in horse serum for the first time.The established method was applied to the pathological serum samples of patients with early oral cancer.By statistical analysis,5 glycans with potential diagnostic capacity were screened out.HOD combines the high activity of HTMBA with the high throughput of on-target derivatization strategy,and this"point-and-test"efficient derivation approach provides an efficient pathway for clinical analysis of large samples.The synthetic hydrazine isotopics d0/9-HTMBA was applied to MALDI-MS based quantitative glycosomics study.The d0/9-HTMBA based relative quantitative method required only a few simple mixes to complete the isotopic labeling of the glycans,and good linear correlation(R~2?0.999)and reproducibility(CV?10%)were obtained.The established method combined with methylamine derivation was used for quantitative analysis of altered N-glycans in serum of patients with multiple myeloma(MM)during treatment.It was found that the significantly abnormally expressed N-glycans,such as galactosylated,acidic fucosylated and polysialylated glycans might be closely related to the development of MM.Such applications are helpful to deepen the understanding of the mechanism of MM treatment and may provide theoretical basis for future clinical treatment and drug administration.In addition,TEBA showed high reactivity during the experiment,therefore its applications in glycan derivatization were further studied.Commonly used succinimide reagent usually needs about 40 minutes incubation at 40?to complete glycan derivatization,while TEBA can complete labeling of the glycans in 1 minute at room temperature,and the MS detection sensitivity of TEBA labeled glycan was significantly increased(?16 times).Importantly,TEBA can be combined with microwave-assisted enzyme digestion included"one-pot"method and protease E digestion strategy,and is expected to be used as a common derivative reagent to achieve efficient labeling and high sensitivity detection of glycoamine or N-glycan with amino acid residues.In conclusion,TMBA,a marker precursor with high activity,was designed with the assistance of theoretical calculation technology in this paper.The glycan derivatization activities of TMBA-based molecules HTMBA and TEBA were both higher than those of existing reagents.Among them,HTMBA of hydrazine molecule combined with the on-target derivation method allowed the efficient preparation and detection of glycan samples,and provided a new method for the high-throughput glycan analysis of large pathological samples.In addition,the d0/9-HTMBA isotope quantitative method based on MALDI-MS proposed in this paper is accurate and stable,which provides a new perspective for a deep understanding of the mechanism that how current treatments affecting diseases.The work carried out in this paper provides a theoretical basis and practical scheme for the efficient design and synthesis of glycan markers in the future,and the designed labeling reagents have wide application prospects in the high-throughput qualitative and quantitative analysis of glycan.