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so the proteome is very complex whether you want to perform your proteomic analysis for
the whole organism it means you want to know all the proteins present in a given organism
or in a tissue or in body fluids or in different type of cells proteomics can be global or
it can be very targeted or expression based so very highly reproducible samples is very
important for performing comparative proteomic analysis
if you want to know the difference in your sample as compared to the controls you need
to ensure that your sample preparation is very reproducible if you introduce some artifacts
to begin with then obviously you are not going to identify the reproducible biological changes
so let me give you 3 different terminology here for proteomic analysis one is global
proteome analysis expression proteome analysis and targeted proteome analysis
when i am talking about global proteome analysis it means your aim is to characterize all the
proteins present in the given sample expression proteome analysis it means you are mainly
interested to look for those changes which are due to any chemical or your treatment
those are induced either going up or down the protein amount is changing so the protein
expression analysis that is most commonly used for various type of clinical and different
studies
targeted proteomic analysis if you are very focused for a given organelle or a given sample
type often you would like to know what is happening in that particular proteome for
example mitochondrial proteome so one need to try different type of strategies when thinking
about performing a sample preparation what is your objective whether you want to do global
profiling or you want to do expression profiling
in either case you need to extract all the proteins present in that particular target
sample now when you are looking at targeted proteome analysis you just want to pre fractionator
your sample in such a way that only that particular component is isolated and then all the proteins
from that organelle or cell is being extracted so different types of strategies need to be
used to perform this type of proteomic analysis
now all of this sample processing involves solubilisation denaturation reduction and
treatment of sample proteins but you need to involve additional steps depending upon
the type of samples and your type of objective so that the protein quality the protein extract
can be improved and while you are doing this you have to be very cautious that when you
are performing various steps and sequential type of extraction you may also lose a small
fraction of the proteins
so one has to be careful when adding various additional steps during the sample preparation
now protein extraction protocols they need to ensure that most if not all the proteins
in a cell or its organelle are extracted the presence of interfering compounds should be
minimized so if you have optimized a very good protein extraction procedure that should
ensure that you have a very wide proteome coverage and that is ultimately going to determine
the success of your proteomic experiment
so first of all you may ask why to analyze serum or plasma for any proteomic applications
so as you know blood proteome is one of the most complicated components of the human proteome
the liquid portion of the blood is referred to as plasma and removal of fibrinogen as
well as other clotting factors from the plasma result into serum
so human serum or plasma proteins they mostly originate from a variety of tissue and blood
cells as a result of secretion or leakage from the neighbouring tissue or the blood
cells the rapid alteration in the expression pattern of various serum proteins due to response
of a diseased condition or an external stimulus is true reflection of physiological changes
occurring in an individual
so to get a feel about what are all the physiological changes happening in a patient due to a disease
people analyze serum or plasma proteome very oftenly because blood removal for various
type of tests is being performed in clinics so blood is very easily accessible sample
and performing the serum hot plasma proteome becomes very easy as compared to dissecting
out a tissue for further analysis
although sample removal is really but sample analysis the serum or proteome analysis is
not so easy there are major challenges in serum or plasma proteome analysis let us talk
about some of these challenges point wise the first point the dynamic range of the protein
concentration in serum there is a large diversity of proteins which provide a very dynamic environment
of almost 10 to the power 10 magnitude
so the concentration of serum proteins range more than 10 orders of magnitude if you want
to obtain the full spectrum of serum or plasma by applying any of the conventional proteomic
techniques it is very challenging because the typical dynamic range for any of these
techniques any these platforms will be much smaller which in 10 to the power 2 to 10 to
the power 4 so how to capture all the dynamic events which are happening in the serum
if the dynamic range of the proteins are very large and your detection techniques are not
able to capture that whole dynamic range so to avoid these issues people try to remove
some of the abundant proteins from very complex serum proteome so that overall the dynamic
range can be reduced and minimized so the second point the high abundance proteins there
are different high abundant proteins which are present in serum and plasma which makes
its analysis very very complicated
there are almost 22 highly abundant proteins present in serum which represent about 99
percent of total protein mass of serum or plasma these high abundance proteins prevent
the detection of very low abundant proteins and often these low abundant proteins could
be the target which you are probably looking for as a part of biomarker discovery so how
to get rid of the high abundant proteins i will describe some of the strategies which
can be used to remove high abundant proteins in next couple of slides
but first let us talk about which are other challenging factors for the serum or plasma
analysis the third point the presence of high salt and other interfering compounds as we
have talked about different type of interfering compounds so now you are familiar that salt
are one of the several component which could be very much interfering during your proteomic
applications now salts are present in the blood which are required for various function
such as the maintenance of osmotic balance acid base balance etc
few salt such as sodium chloride or potassium chloride are also added when you are processing
the serum sample during this whole sample manipulation due to the intrinsic salt present
in the blood as the less extrinsic start added during the sample processing the overall salt
component becomes very high and that is that creates problem for various type of proteomic
applications
so high salt and interfering compounds should be removed now these presence of excessive
salt detergents or other contaminants can tremendously influence the electrophoretic
separation of proteins if your target technology is 2 dimensional electrophoresis or other
gel based method you have to really ensure that salt is very low in the serum or plasma
components it also affects the direct determination of proteins or peptides by mass spectrometry
based techniques
so regardless of whether you use gel based or gel free method we have to ensure that
the overall salt component is removed efficiently from the serum or plasma now let us talk about
fourth point other challenge so variations among the individuals and lack of reproducibility
these are some of the very major issues in clinical studies where you have inter and
intra individual variation
intra individual variation is obviously more expected but even within one person due to
the dye due to different type of medication the serum or plasma proteins can be changed
so how to avoid these inter and intra individual variations and as if you refer to the previous
section when we talked about how to minimize different type of these factors which are
going to ensure the success for clinical studies
probably you will be able to keep an eye on different type of extrinsic and intrinsic
influences which one should try to remove as much as one can during the clinical proteomic
analysis so the drastic heterogeneity or large biological variations such as gender age the
genetic factors dietary considerations environmental factors and drug treatments are going to affect
the reproducibility of your experiments
so if you are careful in designing experiment one can at least minimize these variations
and increase the reproducibility of the proteome analysis so i am giving you an overview here
for the serum sample preparation the various steps are shown in images such as withdrawal
of intravenous blood blood collection in the tube centrifugation step of whole blood how
to remove the serum from the whole blood serum can be transferred into the fresh tube
now for the serum proteome analysis different type of modifications can be performed such
as sonication can be used for disrupting the high abundant proteins and it also helps in
better resolution then depletion strategies to remove the abundant proteins precipitation
of these proteins by adding acetone and then drying out the protein pellet and reconstituting
for the suitable buffer for further proteomic applications
okay
so i hope in the animation you are able to understand how to go step by step to perform
the serum proteome analysis now as i promised previously i will talk to you about how to
remove the high abundant proteins which are present in the serum so as we talked there
are more than 20 abundant proteins which are present in the serum and albumin alone covers
approximately 50 percent of these abundant proteins
immunoglobulin g contains 15 to 25 percent of the abundant proteins then iga haptoglobin
transferrin and antitrypsin these are also major high abundant proteins so in the diagram
i have shown you the 6 high abundance serum proteins the different ways people have tried
to remove these abundant proteins molecular weight cut off and different type of chromatography
methods have been used
the affinity chromatography based methods are highly efficient for its specific removal
of these abundant proteins which are present in the serum because there is always that
fear with the molecular weight cut off whether along with albumin and other high body weight
proteins you will also get rid of your various high molecular weight protein which are non
abundant so the affinity based methods ensure the specific targeting of albumin igg and
other specific abundant proteins
the antibody affinity ligands are used for albumin igg and other abundant proteins and
it result into very specific depletion these resins can selectively bind to these proteins
and unbound proteins can be eluted in suitable buffer by applying the affinity resin based
fractionation method and different type of depletion strategies one can get rid off the
high abundant proteins as shown in this image of sds page gel
the left lane is loaded with the untreated serum and the right lane is loaded with the
treated serum as you can see some of the very high abundant proteins such as albumin igg
those are efficiently removed and which allowed for some low abundant proteins to appear on
the gel let us now move on to next chromatography
method which is ion exchange chromatography this is one of the most versatile chromatographic
separation method which relies on differences between number of charges and distribution
of charge groups in defined ph and solvent conditions
in ion exchange chromatography the proteins are separated based on charge difference the
proteins with overall negative charge will interact with positive charges or the vice
versa so by varying the amount of positive and negative amino acids and even ph can influence
the net charge on proteins
so in this slide some of the common ion exchange matrices are shown like carboxymethyl cm and
diethylaminoethyl deae so when a desired protein is positively charged the cation exchange
chromatography should be used when a desired protein is negatively charged the anion exchange
chromatography method should be used
so in ion exchange chromatography the column is packed with a resin whether its cation
or anion exchanger depending upon the charge of the protein that needs to be bound to the
column and purified
so proteins are adds up to the ion exchange column and then it can be dissolved by increasing
the salt or altering the ph of the buffer which can change the charge on protein so
various anionic buffers such as acetate and phosphate are used for cation exchange and
cationic buffers such as trischloride or ethanol amine are used for the anion exchange
now the buffered solution is changed so that the net ph of the protein of interest can