| Background:Protein glycosylation plays a critical role in carcinogenesis and cancer progression,and it is necessary to deeply investigate changes in disease=related protein glycosylation to elucidate potential mechanisms in cancer development and clinical diagnosis and treatment.Haptoglobin(Hp)plays an important role in promoting and inhibiting inflammation in vivo,and its N-glycosylation is closely related to the pathological status of the body.This study aims to investigate the relationship between disease-specific haptoglobin(DSHp)glycosylation and different pathological states of various disease types,including female reproductive system(cervix,uterus,and ovary),breast,lung,digestive system(esophagus,stomach,liver,pancreas,colon,and rectum),thyroid,and prostate diseases,as well as the differences in inflammatory responses between benign diseases and cancer,and to screen potential biomarkers to distinguish between different types of cancer and benign diseases.Methods&Results1.Establishment of a high-throughput sample pretreatment and MALDI-MS analysis method for DSHp-β N-glycopeptides in serumEstablishing a high-throughput method for analyzing DSHp-β N-glycosylation in serum samples,our research group previously isolated immunoinflammation-related protein complexes(IIRPCs)from patient serum using non-denaturing polyacrylamide gel electrophoresis with a 4%-12%gradient.Separation of the DSHp-β chain from the complex was achieved using 12%SDS-PAGE.In this study,we utilized 3D printing technology to independently develop a high-throughput DTT/IAM reaction tank for opening disulfide bonds in the complex and a simple gel breaker for processing gel strips,improving experimental throughput and stability.We achieved specific,simple,and high-throughput enrichment and separation of DSHp-β N-glycopeptides using selfdeveloped magnetic polyaniline nanomaterials for the first time in a 96-well plate.Matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance mass spectrometry(MALDI-FTICR MS)was used for glycopeptide detection and identification,and a method suitable for rapid analysis of DSHp-β N-glycosylation in clinical serum samples of a large population was established.Using this method,a total of 95 DSHp-β N-glycopeptides were detected,including 55 at Asn207/211,19 at Asn241,and 21 at Asn184.The relative standard deviations(RSDs)of intra-day and inter-day precision for different N-glycopeptides were both less than 10%2.Exploring the inflammatory response differences in 13 types of cancer and their benign diseases based on glycosylation of DSHp.Using the aforementioned method,this study analyzed 9465 serum samples,including 5724 samples from 13 types of cancers(digestive system cancers:esophageal cancer,gastric cancer,liver cancer,pancreatic cancer,colon cancer,and rectal cancer;lung cancer;breast cancer;female reproductive system cancers:uterine cancer,cervical cancer,ovarian cancer;thyroid cancer,and prostate cancer)and 3741 samples from patients with benign diseases,to investigate the differences in DSHp glycosylation.Using LASSO regression and Fold change algorithms,DSHp differential Nglycopeptides were screened from different cancer and benign disease groups.Then,support vector machines(SVM)method was used to construct disease diagnostic models for distinguishing between cancer and benign diseases in different diseases,and finally,ROC analysis was performed to examine the diagnostic ability of DSHp differential Nglycopeptides for different diseases.At the overall research level,the disease diagnostic model constructed based on DSHp differential N-glycopeptides from different sites showed a poor ability to distinguish between cancer and benign diseases,with the area under the ROC curve(AUC)of 0.678.At the systemic level,the AUC of the disease diagnostic model for female reproductive system diseases(cervical,uterine,and ovarian)was 0.780,and the AUC of the diagnostic model for digestive system diseases(gastric,liver,pancreatic,colon,and rectal)was 0.781.In different disease groups,the disease diagnostic models constructed based on DSHp differential N-glycopeptides from different sites(A:N207/211,B:N241,and C:184)showed different abilities to distinguish between cancer and benign diseases.Among them,DSHp differential Nglycopeptides for cervical cancer and cervicitis,gastric cancer and chronic gastritis,as well as lung cancer and benign lung diseases,exhibited the best diagnostic ability to distinguish between cancer and benign diseases in their respective disease diagnostic models(cervix:A(G2N3F),A(G4NS),A(G4NFS),A(G7NF2),A(GS-N&G-NS),A(G2S2&G-N),A(GN2F&G2F),A(G7N2F2S5),A(G2N2&G4N3FS),B(G3NFS),B(G3NFS2),C(GN2F2),C(G6N2F5S),C(G4N3F4S2),C(G4N3F5S),and C(G6N3F4S),AUC=0.941;stomach:A(GF2S),A(GN3),A(G2S2),A(G3N3),A(G5F2),B(G3NFS),A(G2N2FS2),A(G7NF2),A(G-N&GS),A(G5N2F5),A(GN&G2S2),A(G4N6F2S),A(G2S2&GS),A(GN2F&G2F),A(G2S2&G2),A(G2F2S&GNFS),A(G2&G3NFS),A(G2S2&G2S),A(G5N2F4S4),B(G-NS),B(G2NS),B(GN2F2),and C(G3N2F3),AUC=0.918;lung:A(GF2),A(G2S2),A(G4NS),A(G5F2),A(G2N2FS2),A(G5N2F5),A(G2F2S&G-NFS),B(GO-N),B(G-N),B(G-NS),B(G2),B(G2S),B(GN2F2),B(G2S2),B(G3NS),B(GNFS2),B(G3NS2),B(G3NS3),and(G2S2),AUC=0.835).In addition,the diagnostic models of other disease groups also showed certain diagnostic capabilities(uterine:AUC=0.776;ovarian:AUC=0.747;breast:AUC=0.698;liver:AUC=0.778;pancreatic:AUC=0.645;colon:AUC=0.739;rectum:AUC=0.805).Conclusion:This study established a high-throughput mass spectrometry analysis method for DSHp N-glycopeptides in human serum,which meets the requirements for rapid analysis of serum samples in a large clinical population.By applying this method,potential biomarkers for distinguishing different pathological states of DSHp Nglycosylation were obtained in large serum sample populations,revealing differences in DSHp inflammatory response under different pathological states of various organ diseases and providing new insights into the differential inflammatory response specific to different diseases. |
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