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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