Proteomics applications

welcome to the proteomics course we started
discussing about some case study two dimensional electrophoresis applications let us start with the first case study towards
a proteomic definition of coartem action in plasmodium falciparum malaria a study by makanga
et al in 2005 so as you know each year hundreds of millions of new malaria infection cases
result in over 1 million deaths worldwide but due to the lack of effective vaccine and
widespread resistance to the anti-malarial drug still lot of deaths are happening and
the malaria problem is still posing challenges for its control the anti-malarial therapy of chloroquine and
primaquine these have not been able to control the mortality rate because of the anti-malarial
drug resistance development so therefore there is an urgent need for identifying new drug
targets as well as understanding the course of action of these drugs by applying various
type of high throughput techniques so in this paper authors have discussed how
two different drugs which are effective for the anti-malarial can be studied for looking
at the proteome changes in the plasmodium falciparum parasite so coartem is a combination
of artemisinin-derived artemether with lumefantrine how these two drugs behave and how the proteome
changes occur due to the action of these two drugs were studied in this paper applied proteomic
approaches the two-dimensional electrophoresis to study the proteomic alternation of each
of these drugs so these drugs are applied as a drug of choice
for all cases of non-severe malaria worldwide the artemisinin drug action is mediated specifically
through its endoperoxidase moiety however the more detailed mechanism of action of these
drugs are still unknown so the purpose of this study was to investigate the action of
two active components of new anti-malarial coartem artemether and lumefantrine on human
malaria parasite plasmodium falciparum and author tried to look for the alterations in
parasite proteome which were induced by each of these drugs to obtain insight of the proteomic alteration
they separated the proteins on the two-dimensional electrophoresis gels and compared the response
of the proteomic alteration based on these two drugs and then they identified certain
proteins which were either commonly expressed due to these drugs or they were differentially
expressed due to these drugs so certain proteins were found to be commonly up regulated due
to both of these drugs and certain proteins have different patterns but before looking at the proteomic alterations
author first determined ic10 20 50 and ic 90 values for both the drugs arn and lum we
will use the abbreviations now for artemisinin and lumefantrine an effect of these concentration
of drugs on parasite growth over 24 hours was characterized as you can see the growth
curve in the slide so synchronised ring stage parasite cultures were harvested over 24-hour
period after the exposure to the arm and lum the parasite growth was determined by using
hypoxanthine uptake assay after establishing the culture conditions
and the drug concentration then author look for the proteomic alterations so first of
all they did fractionation of the p falciparum proteome synchronised parasites which were
isolated from the host erythrocytes washed those initially and then solubilised that
in a tris buffer recipe the tris-insoluble fraction was further subjected to extraction
in the urea-based lysis buffer once protein section was done then authors
used ipg strip off ph3 to 10 range for the first dimension separation of protein in the
linear ipg strips after ief was done they equilibrated these ipg strips and then applied
that on 125% of the vertical sdl gel after the second dimension separation based on the
molecular weight then these just were stained with the silver or coomassie brilliant blue
stains so by employing two-dimensional electrophoresis
and comparing the gel images by using the pd-quest software authors are able to see
that there is a differential proteomic response which is drug specific so quantitative analysis
of alternate protein expression levels following exposure to the arm and lum were analysed
and then those protein spots which were differentially expressed and it showed a significant wear
further subjected to the mass spectrometry analysis so the comparative analysis of 2-d gels from
untreated and the drug-treated parasite protein fractions provided direct and distinct alterations
in parasite proteome following artemisinin or lumefantrine drugs certain proteins were
identified few of those showed common response due to both drugs; however there are certain
proteins which showed opposite trends due to each of these drugs protein such as membrane
associated calcium binding protein was up regulated in both the drugs aspartic proteinase was up regulated in both
the cases heat shock proteins such as hsp60 70 and 90 those were up regulated due to both
the drug treatments there are certain protein such as enolase fructose bisphosphate aldolase
and phosphoglycerate kinase these proteins were down regulated in artemether treatment
and up regulated in the lumefantrine treatment so interestingly the arm treatment regulated
in more than three fold down regulation of the glycolytic enzymes such as enolase phosphoglycerate
kinase fructose bisphosphate aldolase and glyceraldehyde-3-phosphate dehydrogenase the expression of the same enzymes were also
up regulated more than threefold due to the lumefantrine treatment; however there are
certain proteins such as the stress responsive proteins like heat shock proteins which were
commonly induced due to either of these drug treatments which looks like a general stress
response as compared to very unique response to the given drugs so from this study the major findings were
that authors successfully investigated alterations of parasite proteome induced by two component
of coartem artemether and lumefantrine by using proteomic approach they investigated
specific and non-specific effects of two anti-malarial drugs in pharmacological relevant conditions
expression of certain proteins were quite interesting including a membrane-bound calcium
binding protein which was up regulated due to artemether and lumefantrine treatment the study also established a relationship
between the pharmacologically relevant concentration and time of exposure for the two components
of coartem so in this study authors aim for identification of serum markers by depicting
the progression of prostate cancer by using difference in the electrophoresis technique so prostate cancer is recognized as a significant
problem in older male population the prostate cancer screening rely heavily upon testing
for the higher level of prostate specific antigen also known as psa within the peripheral
circulations so psa is very sensitive marker but there are a lot of discussion on reliability
and the specificity of psa for the prostate cancer reason being that the level of psa
is also high in benign prostatic hyperplasia or prostatitis so therefore there is a lot of discussion
whether one should rely on only psa for detection of the prostate cancer so this study aims
to identify some new markers in the prostate cancer by studying the serum proteome analysis so as you are aware and in fact we have discussed
the protein preparation from the serum earlier so each of the sample poses a lot of technical
challenges and serum is one among them where presence of highly abundant proteins such
as albumin and immunoglobulin they result into the masking of low abundant proteins
so to eliminate those high abundant proteins authors used multiple affinity removal system
from the agilent technologies and they removed most highly abundant proteins from serum sample
including albumin igg antitrypsin iga transferrin and heptoglobins after the abundant proteins
were depleted for the serum sample then authors move for the protein extraction and further
analysis so the differential proteomic analysis was
performed in two different cohorts of histologically confirmed prostate cancer with different rates
of the disease so they used the patients with two different
grading system based on the gleason grading so the gleason grading system that is used
to help and evaluate the prognosis of men with prostate cancer so depleted serum samples
obtained from the patients with gleason score 5 and gleason score 7 were used for comparison
and further analysis as you can see in the slide these samples were first labelled with
cy3 cy5 and also the internal reference pools were made which were labelled with cy2 dyes
these samples were then further mixed the depleted cancer serum from first cohort
of gleason score 5 and second cohort of gleason score 7 those were mixed separated in the
first dimension and followed by proteins that separated in the second dimension now when authors analysed these diced images
they found that 63 protein spots were differentially expressed between the gleason score 5 and
7 cohorts and 13 of these proteins were statistically significant among these two populations so
as you know analysis of the gels is always challenging especially if you are looking
at the conventional 2-d gels where you have separate gels obtained from each of these
groups but analysis in dye gel is more automated if your remember our previous discussion in
the dye gel analysis this analysis is more automated and more straightforward
but still you have to go through individual spots and you have to look for how significant
those changes are and you have to look at the 3-d views of those spots to ensure that
it is reproducible among various control and treatment groups there are different level
of analysis performed which we have talked earlier but this just shows you the final
output that 63 spots after all the analysis steps were considered significant after 2-d dye gel image analysis authors those
spot and subjected for the mass spectrometry identification of proteins so the proteins
excised from the gels were analysed by using finnigan ltq mass spectrometer and data from
these ms/ms experiments were analysed by using bioworks browser by using c-quest program after mass spectrometry was done the identity
of these proteins were established authors also tried analysis of these dye gels by using
two different software packages the decyder and progenesis just so that they are very
confident that all the proteins which they are going to be analysed for mass spec those
are very reproducible so among the proteins which are common in both decyder and progenesis
and the identity of those proteins were further established by using mass spec those proteins included pigment epithelium
derived factor which was down regulated in both the cases in decyder as well as progenesis
analysis zinc alpha-2 glycoprotein it was up related from both the software analysis
ficolin 3 was down regulated and apolipoprotein a-ii was up regulated in both software analysis
so these spots were having similar or uniform trend regardless of what software they analysed
and then author these proteins could be interesting for further validation so these are only few
proteins which i have shown here there is a detailed list of the proteins which one
can study in the original manuscripts for validation authors employed various techniques
including western blots enzyme-linked immunosorbent assays or elisa and also immunohistochemistry
so the pigment epithelium derive factor pedf and zinc alpha-2 glycoprotein also known as
zag those proteins were further validated by the elisa technique so the pedf levels
were quantified by using elisa kit and results demonstrative as you can see in the slide
that the statistically significant decrease in the pedf in the gleason score 7 depleted
serum group whereas the results for zinc alpha-2 glycoprotein
elisa analysis which is shown in red in the bottom panel that indicated a 14 fold increase
in the zinc alpha-2 glycoprotein absorbents in the gleason score 7 group so these studies
this elisa validation confirm their findings from the 2-d dye experiments authors also employed immunohistochemistry
or ihc for validating the pigment epithelium derived factor pedf and zinc alpha-2 glycoprotein
so that they are very confident that the proteins which they have identified from the proteomic
profiling those are real and has also tested those on the independent tissue samples so from this paper the major conclusions were
that there are markers which are reflective of the pathological grade and stage could
be beneficial for the identification of appropriate treatment strategies authors confirm that
differential expression of pedf and zag can be performed by using various techniques such
as western blot elisa and immunohistochemistry based on their studies and the follow-up experiment
they concluded that pedf could be a potential marker of early-stage prostate cancer prediction
however more studies and follow-up required on the large number of patients before it
can establish as a good biomarker let us now discuss few application of silac
briefly the silac method is very promising for any cell line so this method can be applied for initial
lines whether it is hela cell c127 hek293 or different type of cell lines people have
shown however the media formulation and the growth optimisation is required individually
for each cell line silac applications have been demonstrated in different applications
such as cell signaling studying the induced protein complexes studying temporal dynamics
identification of kinase substrates studying differential membrane proteomics so there
are various applications we will have a look on some applications now so ong et al in 2002 published a paper in
molecular cell proteomics which was the first silac application demonstrated where they
used relative quantitation of changes in protein expression during the time course of myoblast
differentiation in mouse cells researchers have reported various unique metabolic
labelling strategies for example by using tyrosine the identification of tyrosine kinase
substrates using 13 c-tyrosine labeling is also performed by using methionine the in
vivo methylation sites by methyl silac there are numerous studies based on the global
protein expression profiling using silac method i am just highlighting some of the very earlier
studies which set a path for performing these proteins expression profiling so study by
everly et al in 2004 analysed the expression level of more than 440 proteins in the microsomal
fractions of prostate cancer cells with very metastatic potential another study by gu et
al investigated the early stage of apoptosis by inducing the p53 up regulated modulator
of apoptosis silac has also been used for functional assays
to study the protein protein interactions a study by blagoev et al used the differential
labelling of proteins in egf is stimulated versus unstimulated cells a study by de hoog
et al with quantification of proteins interacting in an attachment-dependent manner with focal
adhesion proteins these are just few example of studying the functional assays and performing
protein interactions using silac the identification of proteins which are enriched
in specific cellular structures a study by foster et al used the first functional proteome
analysis of rafts and they showed the specific detection of proteins depleted from the rafts
by cholesterol disrupting drugs silac has been widely used for multiplexed
analysis to compare the cellular states anderson et al shown the quantitative analysis of proteome
of human nucleoli blagoev et al performed a temporal analysis of the phosphotyrosine-dependent
signaling networks to compare the proteome of three-cell populations kratchmarova et
al analysed the divergent growth factors in mesenchymal stem cell differentiation these
are just few examples of multiplexed analysis now if you look into literature there are
many studies which have used the silac method for comparison of cellular states silac method has also been used to study the
protein turnover study by pratt et al used the rate of breakdown of individual proteins
by analysis of the mass shift in tryptic peptide fragments the analysis of the abundant proteins
in glucose-limited yeast cells which are grown in the aerobic chemostat cultures at steady
state was performed by using silac method silac has been used for identification and
quantitation of protein post-translational modification study by ibarrola et al identified
and quantitated phosphorylation sites another study by bailiff et al also identified and
quantitated the phosphorylation sites so there are many studies which have used silac method
for studying post-translational modifications so interestingly more silac method has been
used in different organism in bacteria in yeast these were the more commonly used silac
methods due to the growth in the cell culture but there are some studies on the arabidopsis
in the plants as well as in the mice which has shown that silac can be applied to the
wide variety of organisms let us briefly look at the itraq applications
and i will show you in this animation in this animation we will look at one application
of itraq method a study performed by boyle et al in 2010 used the itraq method for identification
of candidate biomarkers in ovarian cancer serum serum samples of controlled and cancer patients
were first of all depleted by using multiple of affinity removal system to carry out immuno
depletion of the serum samples from normal controls and ovarian cancer samples this step
helped in removing the high abundance proteins leaving behind only the medium and low abundance
protein for itraq analysis the immuno depleted serum samples were then labelled with itraq
regions and further analysed in ms/ms the author detected a total of 220 unique
proteins of which 14 were found to be elevated in ovarian cancer serum samples as compared
to the healthy controls and for normal candidate biomarker were first-time reported these results
were further validated by the western immune blotting this just gives you an overview of
how itraq regions can be used for various types of applications including biomarker

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