Using Antibody Arrays to Measure Protein Abundance and Glycosylation: Considerations for Optimal Performance

Abstract

Antibody arrays provide a valuable method of obtaining multiple protein measurements from low volumes of biological samples. Antibody arrays can be designed to target not only core protein abundances (relative or absolute abundances, depending on the availability of standards for calibration), but also protein post-translational modifications, provided antibodies or other affinity reagents are available to detect the modifications. Glycosylation is a common modification that has important and diverse functions, both in normal and disease biology. The methods for measuring glycan levels on multiple, specific proteins using antibody arrays and glycan-binding reagents have made significant progress. Here we describe practical approaches to develop, optimize and use antibody array assays to determine both protein abundances and glycosylation states. We cover the use of low-volume arrays to reduce sample consumption and a new way to improve the binding strength of particular glycan-binding reagents through multimerization. These methods could be useful for a wide range of biological studies in which glycosylation may be changing or affect protein function.

Probing the O-Glycoproteome of Gastric Cancer Cell Lines for Biomarker Discovery*

“…Several O-glycoproteome studies of serum have been undertaken in the past using PNA (35,52), and these have provided insight into the general presence of O-glycoproteins and O-glycosites in serum. Our interest was to isolate the small subfraction of circulating O-glycoproteins with aberrant STn O-glycosylation, which are predicted to exist from studies with the CA72-4 assay (53, 54) and lectin arrays (55)(56)(57)(58). High levels of STn are detectable in sera of patients with different cancer types (59 -61) and this is associated with lower survival (62,63).…”

The Interaction of the Gut Microbiota with the Mucus Barrier in Health and Disease in Human

“…More specific reagents need to be used for screening and here the choice is much larger. Ideally an array approach is required and this is the current mode of study, with array technology using lectins [ 371 , 372 , 373 ] and antibodies [ 212 , 374 , 375 , 376 ]. This approach is in addition to the glycoarray platforms discussed earlier.…”

Associated measurement of fucosylated levels of AFP, DCP, and GPC3 for early diagnosis in hepatocellular carcinoma

“…As an excellent protein research method, protein microarray has been extensively applied to measure AFP. [9][10][11] For instance, in our previous research, we have shown that the AFP serum level can be detected faithfully and correctly by protein microarray with a sensitivity of 91.67% and a specificity of 93.24%. 12 However, the serum level of AFP could also be increased in benign liver diseases, such as LC and hepatitis, and up to 40% of HCC patients' AFP levels can be normal.…”