NPTEL

VIDEO COURSE PROTEOMICS

PROF. SANJEEVA SRIVASTAVA

LECTURE-09
Sample preparation for proteomics applications
ASSIGNMENT

You have a collection of serum samples and you want to perform an analysis of the
serum proteome. In order to perform this proteomic analysis you need to have a very
good protein sample. Please draft a protocol basing on the conditions mentioned bellow
and give a brief explanation when required.
1. The first step in workflow of sample preparation is to perform cell lysis. There are
two major types of cell lysis process; gentle and vigorous. Unfortunately you do
not have the equipment required for performing vigorous cell lysis, So you would
have to rely on gentle cell lysis method. Given the conditions above which cell
lysis method would you use?
2. With this protein extract you want to perform a gel-based proteomic experiment.
However, there are certain precautions need to be taken care of. One of them
being protection of the proteins from proteolysis. You suspect that your cell lysis
mixture might contain metalloproteins, which may be of interest to you. So which
protease inhibitors would you use in this scenario?
3. After the sample is treated with protease inhibitors you perform protein
fractionation. This step helps in simplifying the complex protein mixture by
isolating groups of proteins or fractions from the total proteome. Since you have
serum samples and it is a well-known fact that the sera are rich in high abundant
proteins. These high abundant proteins mask the low abundant proteins and
hence it would be impossible to study the effect of these on low abundant
proteins. Which fractionation method do you think would fit in here so that the
high abundant proteins can be depleted? Also comment on the adverse effects
the high abundant proteins have on your gel-based proteomics experiment.

DEPARTMENT OF BIOSCIENCES & BIOENGINEERING

INDIAN INSTITUTE OF TECHNOLOGY (IIT) BOMBAY, MUMBAI, INDIA
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NPTEL VIDEO COURSE PROTEOMICS

PROF. SANJEEVA SRIVASTAVA

4. The next step is the protein extraction. You would want to do your protein
extraction in aqueous buffer. Apart from the problems associated with high
abundant proteins, sera are also spiked with the problem of high salt
concentration. High salt content in your sample will lead to serious problem in
electrophoresis. Which extraction method do you think fits best in this case?
5. Final step in the sample preparation is sample solubilisation. Sample
solubilisation avoids problems associated with non-specific aggregation of
proteins. One can use either chaotrops or detergents for protein solubilisation.
Name two examples of each kind and discuss their pros and possible cons.

Answer:
1. Since the sample type provided in this case study here is serum sample, one can
use osmotic lysis which is a gentle cell lysis method. In this method one can
suspend the sample in a hypotonic medium.
2. One can use protease inhibitors for the protection of proteins released upon the
lysis of cells. However, in this study we suspect that we may have
metalloproteins in the sample mix. Hence we have two options for protease
inhibitors; EDTA and EGTA. Both of them effectively inhibit metalloprotease. But
EGTA has the ability to inhibit nucleases as well.
3. In the sample fraction step we have to consider a very crucial point that high
abundant proteins have to be removed. Otherwise, they will mask the low
abundant proteins on the electrophoretic gel. Therefore, we may never have a
chance to study the effects of those low abundant proteins in disease conditions.
Although there are various methods available for fractionation, the method of
choice in this study would be affinity chromatography.
4. Since the high concentration of salt results in a bad electrophoretic gel image for
eg. Streaking pattern in the gel image, therefore it is important to desalt the
serum sample. In addition to desalting, performing Trichloroacetic acid and
acetone treatment would yield better results.
DEPARTMENT OF BIOSCIENCES & BIOENGINEERING
INDIAN INSTITUTE OF TECHNOLOGY (IIT) BOMBAY, MUMBAI, INDIA
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NPTEL VIDEO COURSE PROTEOMICS

PROF. SANJEEVA SRIVASTAVA

5. Proteins have a tendency to form complexes with membranes, nucleic acids and
other proteins. To avoid this problem one needs to perform the solubilisation of
protein extracts. Solubilisation methods differ from sample to sample. As it is
already mentioned one can use chaotrops such as urea or thiourea or detergents
such as SDS and CHAPS.
Chaotrops:
Urea can be used as denaturant to solubilise and unfold most of the proteins to
fully random conformation. Urea helps in stabilization and all ionisable groups are
exposed to the solution. On the other hand, thiourea helps in the solubilisation of
membrane proteins.
Detergents:
One of the very commonly used detergents for performing protein solubilisation is
SDS. SDS is extremely effective in solubilising hydrophobic proteins. Samples
treated with SDS are not compatible with isoelectric focusing and hence are
suitable for conventional proteomic analysis.
CHAPS is a zwitter ionic detergent which helps in sample solubilisation. CHAPS
prevents non-specific aggregation of proteins through hydrophobic interactions.

DEPARTMENT OF BIOSCIENCES & BIOENGINEERING

INDIAN INSTITUTE OF TECHNOLOGY (IIT) BOMBAY, MUMBAI, INDIA
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