Practical 4 & 5: HUMAN PLASMA ENRICHMENT BY GLYCOPROTEIN AND REMOVAL

OF ALBUMIN

Objectives
1. To isolate glycoproteins from Human plasma
2. To improve serum component analysis by rapidly removing abundant albumin protein
from serum samples

Introduction

Post-translational modification refers to the covalent and generally enzymatic

modifications of proteins during or after protein biosynthesis. The PTM increases the
functional diversity of the proteome by covalent addition of functional groups or proteins,
proteolytic cleavage of regulatory subunits or degradation of entire proteins. These
modifications include phosphorylation, glycosyation, ubiquitination, methylation lipidation snd
proteolysis and influence almost all aspects of normal cell biology and pathogenesis.

PTM can occur on the amino acid side chains or at the protein’s C or N terminal. Many
eukaryotic proteins also have carbohydrate molecules attached to them in a process called
glycosylation which can promote protein folding and improve stability as well as serving
regulatory functions. Over the past decades, scientist has discovered that the human
proteome is more complex than the human genome. This is because it is estimated that the
total number of protein in human proteome is around 1 million and this means that a single
gene can encode for multiple protein.

In this experiment, two methods of enrichment were conducted which are glycoprotein
enrichment and albumin depletion. Both share the same purpose to ease the protein analysis
but the underlying concept is different. In glycosylation enrichment, the low abundance protein
is enhanced thus during analysis the proteins will can be seen clearer. While in albumin
depletion, the high abundance protein is removed thus the protein of interest with low
abundance can be analyse without interference.
Methods

A. Preparation of sample

Firstly, the binding/wash and elution buffer was equilibrated to room temperature. The
sample containing 1 to 1.5 mg of total protein was diluted with 5x binding/wash buffer stock
solution using total protein of 4 : 1. The total volume dilution was not exceeded 800µl.

B. Enrichment of glycoproteins

The binding/was buffer was prepared by diluting 460µl of 5x binding/wash buffer with
1840µl of ultrapure water. A column was then inserted into a collection tube. A bottle of
ConA Lectin Resin was gently swirl in order to obtain a homogenous suspension. A cut
pipette tip was used to transfer 200µl of 50% resin slurry to the column. It is centrifuged
for 1 minute at 1000xg and the storage buffer was discarded. The collection tube was
reused throughout the steps. The column was placed in the collection tube and 200µl of
1x binding/wash buffer was added to the resin. The top of the column was closed and
centrifuged the column for 1 minute at 1000xg. Yhe rinse was discarded and the step was
repeated two times. The bottom cap was placed on column and sample from earlier step
was added to the resin. The top cap was closed and the column was incubated for 10
minutes in room temperature with end to end mixing using a rotator or by rocking back and
forth on a rocking platform. The top cap was removed and then followed by the bottom
cap from column. The column was placed in the collection tube and the top was replaced.
The column was centrifuged for 1 minute at 1000xg and the flow through was collected
into an Eppendorf tube. The column was reinserted and 200µl of 1x binding/wash buffer
was added to the resin. The column was capped, centrifuged for 1 minutes at 1000xg and
the flow through was collected in the previous tube. The step was repeated. The bottom
cap was placed on the column and 200µl of 1x binding/wash buffer was added to the resin.
The column was capped and incubated at room temperature for 5 minutes while mixing
by a rotator. The top and bottom cap was removed from the column. The column was
placed in the collection tube and the cap was replaced. The column was centrifuged for 1
minutes at 1000xg abd the rinse was collected again in the same tube. The bottom cap on
the column was removed and 200µl of elution buffer was added to resin and the column
was capped again. The column was incubated for 5 minutes at room temperature while
mixing using an incubator. The top cap was removed first before removing the bottom cap
from the column. The column was placed in a new collection tube. The top cap was
removed from the column and centrifuged for 1 minute at 1000xg. The collection tube was
carefully set aside and labelled as ‘bound’. The top cap was removed. The last three step
was repeated another time. The elute was collected in the same collection tube containing
the eluate from the first elution. The eluted glycoprotein was stored on ice for intermediate
use or froze for later analysis.

C. Acetone precipitation

The required volume of acetone was cooled to -20°C. The protein sample was placed in
acetoe compatible tube. The cold acetone was added four times with sample volume. The
tube was votexed and incubated for 60 minutes at -20°C. The tube was centrifuged for 10
minutes at 13000 to 15000xg. The supernatant was disposed carefully so that the protein
pellet was not dislodge.

D. Albumin depletion using Piece™ Albumin Depletion Kit

The resin bottle was shaken to resuspend the resin. By using a half-cut micropipette tip, a
volume of 400 𝜇l of slurry was trasnfered into a spin column and cap the column loosely.
Twist off the bottom closure of the spin column and placed into 1.5 ml collection tube.
Excess liquid was removed by centrifugation at 12000xg for 1 minute. Flow through was
discarded and the spin column was placed back into the same collection tube. A volume
of 200 𝜇l of Binding/Wash buffer was added to the spin column. Centrifuged at 12000xg
for 1 minute and discarded the flow through and placed the spin column into a new
collection tube. A volume of 5 to 50 𝜇l of albumin containing sample was applied to resin
and incubated for 1 to 2 minutes at room temperature. The mixture was centrifuged at
1200xg for 1 minutes and the flow through was reapplied to the spin column and incubated
for 1 – 2 minutes at room temperature to ensured maximal albumin binding and this step
was repeated twice. The resin was washed by adding 50 𝜇 of Binding / Wash Buffer to
each 200 𝜇l of resin used to released the unbound proteins. Centrifuged at 12000xg for 1
minute and the flow through was retained. The spin-column was placed in new collection
tube. The washing and centrifugation steps was repeated for three to four times. The
retained fraction was analysed by SDS-PAGE analysis.

E. Gel electrophoresis and staining

The same tube from C was used and the sample protein was reduced with 10x reducing
agent. Distilled water and 4x sample was added into the tube to make up the 15µl volume
and incubated at 70°C for 10 minutes. A concentration of 1x running buffer was prepared
(50ml of 20x NuPAGE-Mes SDS Running buffer was added to 950ml deionized/distilled
water). A pre-cast gel was placed in the tank and buffer was poured. The inner chamber
was filled (200ml) and outer chamber (600ml) with 1x running buffer. A volume of 500ml
antioxidant was added to the inner chamber. The benchmark blue marker and protein
sample was load into the designated wells. Electrophoresis was run at 200v for 60
minutes. The gel was removed and placed in a container. The gel was rinsed twice with
100ml of distilled water. A volume of 40ml safety blue coommassie blue stain was added
and microwaved at high for 1 minute and agitated for 5 minutes
Results

1 3 5 6 8 9

kDa

100

50

20

Figure 1 shown the SDS-PAGE electrophoresis gel loaded with Human plasma
samples that is enriched with glycoprotein and albumin removal.

Lane 1: BenchMark™ protein marker

Lane 3: Total human plasma
Lane 5: bound ConA
Lane 6: unbound ConA
Lane 8: albumin depleted (1st collection)
Lane 9: albumin depleted (2nd collection)

From Figure 1, the albumin in total human plasma is approximately 64 kDA (shown in
a red circle) which coherent with the theoretical value. The albumin can be seen as a
thick band in total human plasma before any enrichment. Lane 5 and Lane 6 is
corresponding to human plasma with glycoprotein enrichment. Lane 5 is unseen while
Lane 6 has a faint band. This is due to lack of time during sample preparation where
acetone preparation step was skipped. Lane 8 and Lane 9 correspond to human
plasma sample enriched by removal of albumin. Lane 9 shown a faint band as
compared to Lane 8.
Discussion

Figure 1 shown the SDS-PAGE gel of human serum albumin with glycoprotein
enhancement and albumin depletion. The albumin depletion samples in lane 8 and 9, the
albumin at approximately 64 kDa are observed to in a thin band as compared to the albumin
in the total human serum where it can be observed as one thick band. This method separates
the high abundance proteins, non diagnostic proteins from the low abundance proteins as
albumin is the most abundant protein in the human serum with 30 – 50 mg/ml concentration
and constitute half of the plasma. Lane 8 exhibit clearer band compared to lane 9, as the
concentration of the protein will decrease with increase number of collection. This
enhancement helps in further analysis of proteins by increase detection of weaker spot in 2D
analysis as proved by Hamrita et.al, (2012). The enhancement step / prefactionation were
done prior of isoelectric focusing and gel electrophoresis to enhance the resolution of proteins
of interest. In this method, the unbound proteins flow through will be collected. This method
often used in detecting low molecular weight biomarkers.

From Figure 1, lane 5 and lane 6 shown samples from glycoprotein enrichment. But,
both lane has unsatisfactory results. Lane 5 is unobservable while lane 6 shown a faint band.
This method enhances a low molecular weight protein to ease detection and further
downstream analysis such as mass spectrometer. The unsatisfactory results are due to
skipping the acetone precipitation steps because of time constrains. This step serves to
remove interfering substances and to concentrate the proteins. Other technical error while
preparing the samples may also contribute to this results. In this method, both bound and
unbound fraction was collected for the purpose of comparison later. The unbound fraction
should have more band compared to the unbound fraction as this method based on specific
binding. The proteins are enriched with Concanavalin (ConA Lectin) which will bind to the
specific carbohydrates residue. In the experiment conducted by Fraser et.al, (1972) shown
ConA recognize alpha-mannose which are very common in N-linked glycans.

Conclusion

The glycoproteins from human plasma was unsuccessfully isolated and the serum component
analysis was improved by rapidly removing the albumin protein from the serum samples.

References

Bechr Hamrita,. (2012). Enrichment methods for revealing lower molecular weight
biomarkers in human breast plasma proteome. African Journal Of Biochemistry Research,
6(1), 1 - 7. http://dx.doi.org/10.5897/ajbr11.075

Fountoulakis, M., Juranville, J., Jiang, L., Avila, D., R�der, D., & Jakob, P. et al. (2004).
Depletion of the high-abundance plasma proteins. Amino Acids, 27(3-4), 249-259.
http://dx.doi.org/10.1007/s00726-004-0141-1

R. Fraser, A., J.Hemperly, J., L. Wang, J., & M. Edelman, G. (1972). Monovalent derivatives
of concanavalin A. Proc.Nat.Acad.Sci., 73(3), 790 - 794.