Alterations in glycosylation have been associated with a number of diseases. Generally, these changes are observed through glycan analysis of complex protein mixtures or through the analysis of a few specific individual proteins. Indeed, in most cases, glycan analysis is done on released glycan pools without any connection to the protein carrier. When analysis is done with the protein carrier included, this generally requires large amounts of protein and is only feasible for a small number of proteins, thus structural glycan information is often limited. In this application we propose a novel method that will allow for the glycan analysis of 100?s if not 1000?s of individual proteins found in complex protein mixtures such as serum. This method will involve three steps to achieve this goal: (i) the capture of specific proteins using an antibody array, (ii) treatment of captured protein with highly active recombinant PNGase F, and (iii) the glycan analysis of the specific captured proteins on a spot by spot basis by MALDI Imaging Mass Spectrometry. In this R21 application, we will first optimize the detection of N-linked glycan on immuno-captured proteins using specific well-known proteins. Subsequently, we will perform N-linked glycan analysis of over 30 serum proteins that we believe can act as biomarkers of liver cancer in both control patient and patients with liver cancer. This number is also chosen as it will allow us to create a microarray of four 32 spot quadrants, which will be used for both analysis and controls for signal and protein information. In the R33 application we will expand this platform to include over 100 capture antibodies and us this method to determine glycan changes in the serum of patients with hepatocellular carcinoma (HCC). At this end of this R21 application, we will have validated the basic methodology that will allow for the complete structure N-linked glycan information to be obtained from 100?s to 1000?s of glycoproteins in a complex solution at one time.

Public Health Relevance

It is know well understood that N-linked glycosylation is altered in many cancers. However, measuring these changes are often difficult and require copious levels of protein and significant amounts of time. In this application we propose a novel method that will allow for the glycan analysis of 100?s if not 1000?s of individual proteins found in complex protein mixtures such as serum.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA225474-02
Application #
9702774
Study Section
Special Emphasis Panel (ZCA1)
Program Officer
Rinaudo, Jo Ann S
Project Start
2018-06-01
Project End
2020-05-31
Budget Start
2019-06-01
Budget End
2020-05-31
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Medical University of South Carolina
Department
Pharmacology
Type
Schools of Medicine
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29407