1. 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.