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