Abstract
AbstractAlpha-1-acid glycoprotein (AGP) is an acute phase glycoprotein in blood, which is primarily synthetized in the liver and whose biological role is not completely understood. It consists of 45% carbohydrates that are present in the form of five N-linked complex glycans. AGP N-glycosylation was shown to be changed in many different diseases, and some changes appear to be disease-specific; thus, it has a great diagnostic and prognostic potential. However, AGP glycosylation was mainly analyzed in small cohorts and without detailed site-specific glycan information. Here, we developed a cost-effective method for a high-throughput and site-specific N-glycosylation LC-MS analysis of AGP which can be applied on large cohorts, aid in search for novel disease biomarkers, and enable better understanding of AGP’s role and function in health and disease. The method does not require isolation of AGP with antibodies and affinity chromatography, but AGP is enriched by acid precipitation from 5 μl of bloodplasma in a 96-well format. After trypsinization, AGP glycopeptides are purified using a hydrophilic interaction chromatography-based solid-phase extraction and analyzed by reversed-phase-liquid chromatography-electrospray ionization-MS. We used our method to show for the first time that AGP N-glycan profile is stable in healthy individuals (14 individuals in three time points), which is a requirement for evaluation of its diagnostic potential. Furthermore, we tested our method on a population including individuals with registered hyperglycemia in critical illness (59 cases and 49 controls), which represents a significantly increased risk of developing type 2 diabetes. Individuals at higher risk of diabetes presented increased N-glycan branching on AGP’s second glycosylation site and lower sialylation of N-glycans on AGP’s third and AGP1’s fourth glycosylation site. Although this should be confirmed on a larger prospective cohort, it indicates that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes.
Highlights
Cost-effective method for high-throughput detailed acid glycoprotein (AGP) glycoprofiling is presented. Site-specific AGP N-glycan profile can be obtained from 5 μl of blood plasma. AGP N-glycan profile is stable in a healthy individual. AGP glycoprofile could help identify individuals who are at risk of type 2 diabetes
Individuals at higher risk of diabetes presented increased N-glycan branching on AGP’s second glycosylation site and lower sialylation of N-glycans on AGP’s third and AGP1’s fourth glycosylation site. This should be confirmed on a larger prospective cohort, it indicates that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes
Because identifying the exact glycoproteins that contribute to those differences would probably help to develop stratification methods which could reliably distinguish individuals who are at risk of type 2 diabetes development, we have chosen this population for the pilot study of our method
Summary
The study contains three sets of experiments: method development, intraindividual temporal stability study, and pilot study with individuals with and without hyperglycemia during critical illness. For all participants, fasting blood samples for N-glycan profiling were collected in tubes containing EDTA anticoagulant, plasma was separated immediately (at 1370g for 10 min) and stored at −20 ◦C until analysis. Amino acid sequence and glycan composition were confirmed in the MS/MS spectra of the most abundant AGP glycopeptides (supplemental Fig. S2, showing representative annotated fragmentation spectra for each glycosylation site identified). For the glycopeptides which did not have MS/MS data at all (mostly due to low intensity, nine out of 96 reported), the annotation was done according to following criteria: same retention time window (coelution) as confirmed AGP glycopeptides, m/z value matched to internal database, presence of more than one charge states (depending on the size of the glycopeptide), and delta m/z representing mass difference of one or more monosaccharides from the glycopeptide thoroughly confirmed as previously described (supplemental Fig. S4, supplemental Table S4). Normalization serves the purpose of removing the variation in signal intensity between samples and allows for their comparison
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