Isolation and Analysis of Glycosphingolipids, N-linked Glycans, and O-linked Glycans from a Single Sample of

Human Serum

Seth Burgess, Ashley Medeiros, David Ashline, and Vernon Reinhold

Department of Molecular, Cellular, and Biomedical Sciences

References • Ruhaak LR, Zauner G, Huhn C, Bruggink C, Deelder AM, Wuhrer M. Glycan labeling

strategies and their use in identification and quantification. Analytical and

Bioanalytical Chemistry. 2010;397(8):3457-3481. doi:10.1007/s00216-010-3532-z.

• Xhang XL. Roles of glycans and glycopeptides in immune system and immune-related

diseases. Current Medicinal Chemistry. 2006; 13(10):1141-7.

• Fredonia.edu/bio241/images/5.19_ER_and_Golgi.jpg

• University of British Columbia Department of Zoology. Vancouver, British Columbia.

http://www.zoology.ubc.ca/~berger/B200sample/unit_8_protein_processing/golgi/lec

t28.htm

• Brooklyn College Department of Biology. Brooklyn, New York.

http://academic.brooklyn.cuny.edu/biology/bio4fv/page/pm_mos.htm

Introduction Glycomics - The study of the glycome, or the

comprehensive collection of sugar elements

in an organism.

Glycan – A compound that contains an

oligomeric or polymeric number of

carbohydrate subunits.

In the cell, proteins are glycosylated in the

Golgi apparatus to result in a glycoprotein.

Based on the amino acid residue that the

sugar is attached to, this process creates

either an O-linked or an N-linked glycan.

N-linked glycan – Asparagine residue

O-linked glycan – Serine or Threonine residue

Glycolipids – Lipids with a carbohydrate

attached

Methods Glycolipids were extracted from the sample

using sonication and centrifugation, leaving the

glycoproteins in the human serum sample in the

pellet.

Proteolysis using the enzymes trypsin and

chymotrypsin were employed to solubilize the

peptides in the collected pellet sample.

N-Glycanase enzymes were used to release N-

linked glycans from the proteins to which they

were attached, and these glycans were purified

from the remaining O-linked glycans via C18

Solid Phase Extraction.

The N-glycans were reduced using borane-

ammonia reduction methods and purified via

graphitized carbon cleanup.

The O-linked glycans were then released via ion

exchange including the addition of sodium

hydroxide, sodium borohydride, and acetic acid.

C18 solid phase extraction was used to purify

the sample of any residual borane from the

borane-ammonia reduction of the N-glycans in

the previous step.

Permethylation was conducted on both the N-

and O-linked glycans to aid in later MALDI mass

spectrometric analysis of the glycans present in

the human serum sample under investigation.

Glycoprotein Processing During its synthesis in the rough endoplasmic reticulum, a

protein can be tagged with a mannose tree transferred

from a molecule called dolichol. This mannose tag

designates the protein for glycosylation in the Golgi

apparatus. Once it reaches the Golgi, the mannose tag is

removed and glycosylation of the protein can proceed to

form a functional glycoprotein.

Importance of Glycomic Discovery and Research

* Genomics is the past, proteomics is the present, glycomics is the future.

* Glycoproteins consist of the majority of compounds in immune response. Foreign antigens are also glycoproteins.

* Glycomic elements add stability to proteomic elements.

* Glycopeptides play a key role in cell signaling and development.

* Glycopeptides allow for specific epitopes that allow immune recognition responses to occur.

* Glycoproteins such as human serum in blood and blood plasma are involved in ensuring proper circulation.

* Glycomic discovery opens up opportunities for personal medicine, such as blocking viral attachment and immune system regulation.

Analysis of Results • N-linked glycans were more diverse and

generally more abundant than O-linked glycans. This means that the asparagine residue is either more easily glycosylated or more numerous in in human serum when compared to serine or threonine.

• N-glycans generally have a higher m/z ratio.

• Our ratios are well calibrated, as more than 90% of our structures deviate less than 2 mass units from the theoretical values.

• Our N and O-linked structures are found in the same profile, which means that there may have been some issues with our N-linked release.