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of this approach is 100% sequence coverage of protein and improved detection of post-translational modifications [9,10]. It is disadvantaged for the reason it requires high field magnetic fields. The most popular and widely used approach for identifying proteins and determining details of their sequence and posttranslational modifications is the bottom-up approach [6,11]. In bottom-up approach proteins of interest are digested with proteolytic enzyme like trypsin and then analyzed by mass spectrometry. In First step masses of peptides are determined followed by fractionation of these peptide ions for further downstream analysis. This approach is useful for identifying proteins because tryptic peptides solubilize and separate readily than the parent proteins. The disadvantage with this approach is that only a small fraction of tryptic peptides are normally detected and as a result some information gets deleted that would be crucial in construction of what are called ‘fragmentation ladders’. Whatever the method or methods utilized to generate the data, a large set of data is generated that needs to be further analyzed to derive meaningful information. Second step of mass spectrometry based protein characterization is data analysis and interpretation. The First step here is deducing amino acid sequence from large datasets generated to be followed by peptide identity. Although many software tools exist for such analysis nevertheless they are deemed to be in need of more accuracy and consistency to reduce results from data redundancy. Of late there has been an emphasis on integrating information from bioinformatics with results that come from proteomics experiments. Three major protein databases SWISS-PROT, TrEMBL and NCBI have been successful in achieving the goals of ability to store, allow searching and retrieving information generated from proteomics [12]. A statistical analysis of the results is an important consideration to ensure confidence in the outcomes [7]. Sedimentation Sedimentation as the term implies is ability of suspended particles to settle during course of motion as a consequence of effect of different operational forces in sedimenting solution. Proteins like other macromolecules can also undergo sedimentation and this ability of proteins to settle in solution has been exploited in their characterization through use of analytical ultracentrifugation (AUC). In analytical ultracentrifugation protein solutions to be studied are subjected to a high gravitational field and resulting changes in concentration distribution are monitored in real time using various optical methods. The optical systems that are currently available for analytical ultracentrifuges include absorbance, fluorescence and interference [13,14]. Although analytical ultracentrifugation places few restrictions on the sample and nature of solvent, there are few fundamental requirements; (i) That sample has a differentiable or distinguishing optical property, (ii) It sediments or floats at an gravitational field that is achievable experimentally and (iii) that it is chemically compatible with the sample cell [13]. The molecular weights that are suitable for AUC vary from between hundred Daltons like peptides, oligosaccharides to million Daltons like viruses and cellular organelles. Sedimentation during ultracentrifugation can be considered as an outcome of three forces [2,13-16]. The centrifugal force that a protein molecule experiences because of spinning is M p ω 2 r, where M p is mass of protein, ω is rotor speed in radians/sec (ω=2π*rpm/60) and r is the distance from centre of rotor. A counter force M s ω 2 r exerted on protein molecule is generated by mass M s of the solvent displaced as the protein molecule sediments. The mass of solvent that is displaced equals Mp * v * ρ, where v is partial specific volume (in cm 3 /g) of the particle and ρ (in g/cm 3 ) is density of the solvent. Therefore the effective buoyant mass M b of protein molecule equals M p (1- v ρ). The net force (M p -M s )ω 2 r or M p (1- v ρ)ω 2 r contributes to the overall viscous drag of protein molecule undergoing sedimentation. This gets balanced by a frictional force. This frictional force is represented by fv, where f is the frictional coefficient and v is the velocity. A net outcome will be molecule moving at a velocity that is bare enough to make the total force equivalent to zero. M p (1- v ρ) ω 2 r – fv = 0 (4) Multiplying equation 4 by Avogadro’s number (NA) to put entities on molar basis and rearranging to place molecular parameters on one side of equation and experimentally measured ones on other we get Mp( 1− vρ ) v NA* = = s 2 (5) f ω r This velocity divided by centrifugal field strength is called sedimentation coefficient, s. Units of s are ‘second’ and values of 10 -13 are commonly encountered, the quantity of 1*10 -13 sec has been called 1 Svedberg. Svedberg is named after T. Svedberg a pioneer in sedimentation analysis. Sedimentation coefficient is directly proportional to molecular weight and inversely proportional to frictional coefficient and is experimentally measurable as ratio of velocity to field strength. It can be presumed that sedimentation coefficient will increase with increasing molecular weight; however it also depends on friction f, which in turn depends on size, shape and hydration of a protein molecule. Application: As explained above analytical ultracentrifugation (AUC) relies on properties of mass and fundamental laws of gravitation, it has therefore diverse applicability. Also during analytical centrifugation protein samples can be characterized in their native state and under physiologically and biologically relevant solution conditions. AUC can deliver information about two aspects of solution behavior; hydrodynamic and thermodynamic [2,13-15,17]. Information about hydrodynamic properties like size and shape of protein molecules is deduced from sedimentation velocity while as information about thermodynamic properties like molar mass, stoichiometry and association constant comes from sedimentation equilibrium. Sedimentation equilibrium is a more effective method for determination of molecular mass as well as one of the effective methods for characterization of macromolecular interactions. This is for reasons that sedimentation equilibrium estimations are not dependent upon macromolecular shape and the reaction kinetics are not part of data analysis [15]. In sedimentation OMICS Group eBooks 06
equilibrium an equilibrium concentration distribution of protein macromolecules is obtained where the flux of sedimenting protein molecules is balanced by flux of their diffusion. At this stage the chemical potential of solution is constant, resulting in a concentration gradient that is defined by equilibrium concentration gradient c(r) [13,15]. The study of interacting systems by sedimentation equilibrium is made possible by this fact that there is chemical equilibrium at sedimentation equilibrium and therefore different components in system like protein and a ligand are related by laws of mass action [15]. Sedimentation equilibrium experiments are usually conducted at low rotor speeds so that a balance is created between flux of sedimenting molecules and their diffusion. Extensive use of AUC in study of interactions of proteins with nucleic acids [18-21], ligands [22-24] and receptors [22-24] has been made for studying both affinity as well as stoichiometry. AUC has also been employed in studying self-association phenomenon in various regulatory proteins and receptors [25-29]. Surface plasmon resonance Surface Plasmon Resonance (SPR) is an optical technique that helps in detecting changes in refractive indices coming from mass transfer on SPR active surfaces. Reliance on the intrinsic optical properties of protein thus makes SPR a labelfree biosensing technology. A typical biosensing experiment involves studying interaction between a ligand in solution (called ‘analyte’ in SPR terminology) with an immobilized interacting partner (called ‘ligand’) on an SPR active surface [30,31]. An SPR active surface usually is composed of an SPR-active metal coated glass slide that forms one wall of a thin flow cell, the refractive index changes in which are induced by introduction of analyte solution. SPR occurs when light is shone through the glass slide and onto the metal surface at angles and wavelengths near the so-called “surface plasmon resonance” condition. The incident light couples to oscillations of conducting electrons, the plasmons, at the metal surface and these oscillations in turn create an electromagnetic field commonly referred to as ‘evanescent wave’ [30,31]. This wave extends from the metal surface into the sample solution on the back side of the surface relative to incident light, i.e. the non-illuminated surface. The intensity of reflected light decreases at sharply defined angle on occurrence of resonance; this angle is called SPR angle and is dependent on refractive index, penetrable by evanescent wave close to metal surface. When other parameters like wavelength of light source or metal of the film are kept constant, the SPR angle shifts are dependent only on changes in refractive index of thin layer adjacent to metal surface. A gradual increase of material resulting from biomolecular interaction at the surface will cause a successive increase of SPR angle and this can be monitored in real time [31-33]. The SPR angle shifts obtained from different protein solutions have been found to be linear within a wide range of surface concentration. In SPR instruments output is given in terms of resonance signal measured in resonance units (RU); 1000 RU corresponds to 0.1° shift in SPR angle and for an average protein this is equivalent to surface concentration change of around 1ng/mm 2 [30,34]. SPR Biosensor chips and Instrumentation: The most common metal in sensor chips consists of gold and the commercially available chips are made up of glass substrate on to which a 50 nm thick gold film is deposited. A monolayer of a long-chain hydroxyalkanethiol that covers the gold film serves as a barrier to prevent analyte from coming in contact with gold [30,31,34]. This also serves as attachment point for carboxymethylated dextran chains that create a hydrophilic surface to which proteins are covalently coupled. Interacting partners of SPR active surfaces run through a system of flow channels which are formed when a microfluidic cartridge comes in contact with sensor surface [30,31,33]. Although a significantly diverse number of commercial biosensor instruments are available, the BIAcore systems are the ones that have been very frequently used. The recent versions of BIAcore instrumentation like BIAcore T100, BIAcore A100 and BIAcore X100 also offer thorough method and evaluation software [30]. Application: The major and most important application of SPR has been in analyzing interactions in biomolecules. With huge volume of data coming from various genome sequencing projects and emphasis being on defining functions of annotated genes and hypothetical proteins, SPR has proved to be a robust technique in achieving the same. As mentioned above a typical SPR experiment involves immobilization of one partner from the interacting pair with other being injected across the surface over it. In interaction experiments involving DNA, it is the DNA molecule that is preferably immobilized for reasons it is conformationally flexible as compared to protein hence doesn’t affect outcome of the interaction [30]. The DNA molecule to be immobilized is tagged with some molecule through which it can be immobilized on sensor chip. For example a biotin labeled DNA molecule can be immobilized on a sensor chip which is coupled with streptavidin [30]. In case of protein-protein interactions one of the interacting proteins can be immobilized by linking to an affinity tag like a poly-histidine tag or a whole protein like GST or MBP [34-37]. The tagged proteins can be immobilized on a sensor chip that is coupled to antibody specific to the tag on protein. The results of interaction analyses experiments are represented in form of a ‘sensogram’ i.e. a plot of response units (RU) versus time (s). Data obtained is usually analyzed by using software supplied by manufacturer and data analysis is usually performed to elicit information about binding constants and kinetic parameters. To extract correct kinetic parameters from sensogram, the curve must be analyzed in terms of a defined mathematical model [30]. For a simplest case the protein that is injected across the surface after an infinite time arrives at an association equilibrium producing a signal R eq , therefore resonance signal R at time t during this process following injection at t=0 when R=R 0 , should obey the expression: ( obs ) ( ) = + ( − ) − −k t R t R [1 ] 0 Req R0 e (6) The dissociation of bound protein can be expressed by: ( ) ( eq ) ( −koff t) R t = R [1 ] 0 + R −R0 −e (7) OMICS Group eBooks 07
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Tyrosine Nitrated Proteins: Biochem
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[58-61]. Protein sulfhydryls [62] a
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Oligoclonality of the antibody resp
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The Recent Methodologies of Protein
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are due to altering protein activit
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Figure 6: At Domain Level, Protein
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Brownian Dynamics (BD) method Prote
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protein demonstrate the lowest ener
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Figure 12: Steps in Protein Prepara
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FlexServ The flexibility of protein
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Advances in Protein Thermodynamics
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3. Fluorescence correlation spectro
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Figure 3: Molecular Chaperone (Top
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Figure 7: A Potherse is Consists of
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Figure 12: Residue Clusters with Mu
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Figure 16: Various Features at the
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Figure 21: Stability of protein str
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Advances in Protein Chemistry Chapt
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MMPs are believed to remodel the EC
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MMPs7 (Matrilysin, PUMP 1) A big ro
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Proteins and Peptides- Reemergence
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