Fundamental Study On Biomarker Candidates Of Early Diagnosis Of Diabetes

Diabetes mellitus is a common clinical metabolic disease. Recently, theincidence of diabetes showed rapidly growth trend. Not only the soaring rates ofdiabetes, the complications also perform great harm to the patient. Right now,commonly used in clinical diagnostic criteria of diabetes include: blood glucose,glucose tolerance test and glycosylated hemoglobin (HbA1c). But these diagnosismethods are taken when the symptomsalready manifested. If diabetes can bediagnosed in the early asymptomatic stage, it is quite significant in prevention andtreatment of diabetes.The mainly crosslinking reaction between glucose and proteins in the blood isnon-enzymatic reaction. Earlier characterized of diabetes is diminished ability toregulate blood sugar, blood sugar rising or fluctuations, and protein glycationreactions speeding up. Human serum albumin (HSA), which has a longer half-life, ahighly abundance protein in the blood, can record glucose fluctuations in form ofprotein glycation and provide evidence for diagnosis of diabetes. For the earlydiagnosis of diabetes, this thesis presents a new research strategy: we tried to chooseHSA as the object of study, from different angles studied the reactions of each HSApeptide on glucose-modified. A group of typical peptides, which can reflect theglycated level of HSA, were screened as potential biomarkers for diabetes. Thesecharacteristic peptides also had been validated in clinical samples. The main contentsand results of the study show below.1. Study on nonenzymatic glycation of HSA and its products. First, HSA wasmodified with an equimolar mixture of12C6glucose and13C6glucose co-culturesystem in vitro. Glycated HSA molecular mass was accurately measured byhigh-resolution MALDI-TOF. HSA molecular mass increased,which demonstratedglycoprotein successfully modelled.After denaturation, reduction and specific Glu-C digestion, The digest glycatedHSA peptides were analyzed using HPLC-TOF for peptide chromatographicseparation and mass spectrometric detection. Since the reaction reagent of glucose labeling with isotopic13C, the glycated peptides will appear in pairs of Mass spectrumpeaks. Peakpair software further screened13C labeled peptides and glycation productswere preliminary analyzed. We set up two groups of experiment in vitro. First group，HSA was incubated with different concentrations of glucose(25mM，50mM，500mM)for10days. The other group, HSA was incubated with25mM glucose for10days,20days and30days. The result indicated that non-enzymatic glycation reaction showndose and time dependent manner. Eventually,30glycated peptides and glycationproducts information were obtained from the different concentrations of glucose fromtwo groups,26glycated peptides and products acquired from the group of differentincubating time. Further analysis revealed that glycation products of six peptidesgradually evolved from Amadori products to the stable advanced glycation endproducts. Furthermore, we used synthetic peptides as a model, and established methodvia neutral loss scan LC/MS/MS for identifying sequence of the non-enzymaticglycation peptides.2. Screening glucose-sensitive peptides and estimating glycation level of HSA.Usually, glycation level of proteins was characterized by measurement of glycatedproteins or glycated peptides. This work described a new strategy for HSA glycationresearch: instead of detecting just a peptide formation of glycation, testing the peptidewas not modified the amount of excess. Since it is easier to detecte the non-glycatedpeptides, this strategy helped to reflect the degree of glycation for protein. Due tonon-enzymatic glycation process in vivo is a dynamic reaction, the early stageglycation adducts were unstable, and the structure of advanced glycation end products(AGEs) were complexity and diversity, it is difficult to use a particular advancedglycation end product to characterize the level of glycation in diabetic patients blood.This study, using a new strategy contrast with conventional quantitation method, wefocus on the quantitation of the remaining glycated peptidesGlycated modification, formed another perspective to consider the level ofglycated peptides and proteins. First, screen easily ionized and zero miss cleavageHSA peptides by the Q-TOF MSMS as target peptides. Further the target peptides ofdifferent glycation level were detected by Mass spectrum. Data was aligned andnormalized by the MPP software, and target peptides were selected by in-house-developed software. Finally, extraction ions chromatographic peak areas ofthe target peptides were obtain. The values obtained for unmodified peptide peakarea/an internal standard peak area ratio were used to determine the degree ofmodification that occurred in various regions of glycated HSA.while using HSApeptide is not glycosylated as an internal calibration. In addition, the complex humanserum verified this relative quantitative strategy. After two rounds of experiments, anda peptide segment, DYLSVVLNQLCVLHE, which never been modified was servedas an internal calibration. Twelve glucose-sensitive peptides were screened, whichwould be used as putative biomarkers for early diagnosis of diabetes.3. Candidate peptides of biomarker were validated by clinical samples. First, thereference targeted proteomics research strategy don’t require synthetic criteriapeptides, but digest peptides of HSA could use as an alternative. Skyline softwareandpreviously screened peptides of HAS information were applied to establish MRMrelative quantitation method. Further,172cases of clinical serum with completeinformation of patients, which seperated into3groups including control group, DMgroup and IGT groups, were measured by this MRM approach. The values of targetpeptides peak areas and the internal standard peptide ratio can be used to estimate thedegree of HSA modification. Data was analyzed by statistical software SPSSstatistical exploration and one-way ANOVA analysis confirmed that the pre-screeningcharacteristic two peptides (VAHRFKDLGEE, VSKLVTDLTKVHTE) could be usedas biomarkers of type2diabetes mellitus with comparable to clinical blood glucoseassay. Other5glucose-sensitive peptides were selected which could be used asputative biomarker candidates of the diagnosis of diabetes In future, these candidatepeptides still need repeated verification and further research.This study, we developed a new method based on non-enzymatic glycationcharacteristic to estimate protein glycation level, which could be used to identify HSAglycosylated peptides and screen glucose-sensitive peptides as putative biomarkers fortype2diabetes mellitus, It provides very valuable applications of biomarkers for earlydiagnosis of diabetes and treatment.