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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
be modified and it no longer binds to the ion exchange resin therefore the bound protein
can be eluted out as shown in this slide
so if you have negatively charged protein which gets eluted first will be present in
the initial fractions while the positively charged protein that bound to the column will
be eluted in the latter fraction or it will be vice versa so let me describe how an exchange
chromatography works is step by step in following animation
the charged stationary phase the colonization rephase consists of a positively or negatively
charged polymeric matrix which will bind molecules of the opposite charge commonly used ion exchangers
include negatively charged carboxymethyl cellulose or cm cellulose which is the cation exchanger
and positively charged dea cellulose which is an annoying exchange the protein mixture
and purified protein mixture which consists of proteins of different net charges are loaded
onto the column
the proteins having charges opposite to that of each stationary matrix will bind to it
while remaining proteins will be eluted mobile phase the proteins are eluted out of the columns
by using suitable mobile phase and then samples are collected by using different sample fractions
the solution leaving the column can be collected in suitably sized fractions for further analysis
after giving you a brief description of the components let me show you the process in
animation the column is packed with the suitable cation or anion exchange resin depending upon
the charge of the protein that needs to be bound to the column and purified the anion
exchange column is then loaded with the impure protein mixture consisting of various positively
and negatively charged proteins
the column is eluted with a buffered solution of suitable ph such that the negatively charged
molecules are removed from the column while the positively charged molecules remain bound
to the anion exchange resin the buffered solution is then changed such that the net ph of the
protein of interest is modified and no longer binds the ion exchange resin
therefore the bound protein also gets eluted out of the column in this manner the fraction
of appropriate size must be collected and analyzed for their protein contents
the negatively charged proteins which get eluted first will be present in the initial
fractions while the positively charged protein that bound to the column is eluted in the
latter fractions
once all the fractions are collected then the protein content can be analyzed by using
a spectrophotometer so analyse the contents these fractions for their protein content
by using a uv visible spectrophotometer at 280 nanometers
a graph of eluant volume versus protein concentration can then be plotted in this particular example
the negative charge large molecules coming first and then positively charged fractions
are coming later okay so now let us talk about tandem mass tag or tmt this method is similar
to the itraq which we just discussed tmt is also ms by ms based quantitative technique
which uses the isotopomer levels referred as tandem mass tags
it also provides the accurate quantification of peptides and proteins the tandem mass tags
have been developed by the proteome sciences and currently commercialized by thermo efficient
i have given you the reference for the original study on tandem mass tag in the slide so these
tender mass tags they are based on the similar principle of itarq here the possibility for
multiplexing is up to 6 possible labels
the tmt isobaric tagging technique can be used to perform absolute quantification by
adding a stable isotope labeled internal stereo peptides it can be done by comparing the peptides
from a target protein to a known amount of labeled standard peptide are spiked into a
sample in that way the absolute quantification can be obtained the nterminal amine and lysine
residues are labeled through the nhs group
there are family of chemical tags which are based on the common structures the series
of tmt tags available tmt0 tmt 2 plex tmt 6 plex so these tmts are an innovative set
of isobaric mass tags for labeling the proteins and peptides at amine functions and mixing
of up to 6 different protein samples are possible while duplex and 6 plex labels tmt differ
by the number of isotopic substitutions
the tmt 0 is a non isotropically substituted a structure that has been produced for only
method development during the ms by ms analysis the tmt tag give rise to 6 reporter ions from
126 to 131 dalton therefore it allows for the relative quantification the tmt 6 plex
each tag adds a mass of 229 daltons for labeled amine to the protein the tmt duplex and tmt
0 share the tmt complex structures
let us look at the tmt label structure in more detail the tmt0 tag is used for testing
and optimization of the sample preparation labelling fractionation and ms fragmentation
for peptide identification and reporter detection the modification is 224 daltons and ms by
ms reporter ion is 126 daltons
now let us look at tmt duplex the tmt duplex reagent allows for the comparison of 2 samples
126 and 127 these are 2 different ms by ms reporter ions available and modification is
225 daltons
let us now look at the tmt 6 plex reagent it allows the comparison of up to 6 conditions
the ms by ms reporter ions as you can see in the structure are from 126 127 128129 130
and 131 daltons so the tmt 6 plex regent allows comparison of up to 6 conditions it could
be useful for studying about time codes drug those responses replicates or looking for
multiple sample disease comparison
the modification is 229 daltons i am showing you one representative ms by ms spectrum of
tmt labeled peptide which is showing a reporter region the relative abundance of target protein
or peptide fragment in 6 different samples can be easily measured by comparing these
signature mass peaks which are generated by the different mass tags
let us now look at the comparison of itraq and tmt tags when itarq as we talked there
are 2 different type of regents available 4 plex and 8 plex in both there is a reporter
group a balancer group and then there is a protein reactive group same concept is also
in the tmt tags where we have a reporter group a balancer group and prg i have shown you
a comparison with a 6 plex tmt tag
but as we have seen earlier there is tmt duplex and tmt 0 tags are also available now in itraq
the reporter in the 4 plex consist of either from 114 to 117 dalton the balancer is between
28 to 31 dalton whereas in 8 plex it is from 113 to 121 and the balancer is from 182 to
192 in case of tmt the reporter groups having 126 to 132 daltons and the balancer consists
of 97 to 103 so tags are quite similar in the overall structure
the itraq analysis can be performed by using the software such as protein pilot and also
the mascot the tmt based analysis can be performed from software such as proteome discoverer
and mascot
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TMT In vitro labeli

180 Folder Collection
PP published on September 15, 2019
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