abstract: Background  -Glutamyl transpeptidase 1 ( @entity4 ) is an N-glycosylated membrane protein that catabolizes extracellular glutathione and other   -glutamyl-containing substrates. In a variety of disease states, including @entity1 formation, the enzyme is shed from the surface of the cell and can be detected in serum. The structures of the N-glycans on @entity0 @entity4 ( @entity4 ) have been shown to be tissue-specific. Tumor-specific changes in the glycans have also been observed, suggesting that the N-glycans on @entity4 would be an important biomarker for detecting @entity1 and monitoring their progression during treatment. However, the large quantities of purified protein required to fully characterize the carbohydrate content poses a significant challenge for biomarker development. Herein, we investigated a new antibody-lectin sandwich array (ALSA) platform to determine whether this microanalytical technique could be applied to the characterization of N-glycan content of @entity4 in complex biological samples.ResultsOur data show that @entity4 can be isolated from detergent extracted membrane proteins by binding to the ALSA platform. Probing @entity4 with lectins enables characterization of the N-glycans. We probed @entity4 from normal @entity0 liver tissue, normal @entity0 kidney tissue, and @entity4 expressed in the @entity2 @entity3 . The lectin binding patterns obtained with the ALSA platform are consistent with the @entity4 N-glycan composition obtained from previous large-scale @entity4 N-glycan characterizations from these sources. We also validate the implementation of the @entity5 lectin, microvirin, in this platform and provide refined evidence for its efficacy in specifically recognizing high-mannose-type N-glycans, a class of carbohydrate modification that is distinctive of @entity4 expressed by many @entity1 .ConclusionUsing this microanalytical approach, we provide proof-of-concept for the implementation of ALSA in conducting high-throughput studies aimed at investigating disease-related changes in the glycosylation patterns on @entity4 with the goal of enhancing clinical diagnoses and targeted treatment regimens.
  