Tyramide signal amplification for antibody-overlay lectin microarray: a strategy to improve the sensitivity of targeted glycan profiling.
Antibody-overlay lectin microarray (ALM) has been used for targeted glycan profiling to identify disease-related protein glycoforms. In this context, high sensitivity is desired because it allows for the identification of disease-related glycoforms that are often present at low concentrations. We describe a new tyramide signal amplification (TSA) for the antibody-overlay lectin microarray procedure for sensitive profiling of glycosylation patterns. We demonstrate that TSA increased the sensitivity of the microarray over 100 times for glycan profiling using the model protein prostate specific antigen (PSA). The glycan profile of PSA enriched from LNCAP cells, obtained at a subnanogram level with the aid of TSA, was consistent with the previous reports. We also established the glycan profile of prostate specific membrane antigen (PSMA) using the TSA and ALM. Thus, the TSA for antibody-overlay lectin microarray is a sensitive, rapid, comprehensive, and high-throughput method for targeted glycan profiling and can potentially be used for the identification of disease-related protein glycoforms.
Department of Pathology, Johns Hopkins University, Baltimore, Maryland 21231, USA.
Danni L
DL
Meany
Laszlo
L
Hackler
Hui
H
Zhang
Daniel W
DW
Chan
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D037102:Lectins / Q000378:metabolism*; D008297:Male; D046228:Microarray Analysis / Q000379:methods* / Q000592:standards*; D011134:Polysaccharides / Q000032:analysis*; D017430:Prostate-Specific Antigen / Q000032:analysis; D012680:Sensitivity and Specificity
D000906:Antibodies; D037102:Lectins; D011134:Polysaccharides; D017430:Prostate-Specific Antigen