Protein sequencing

Sequencing Of Protein with All Methods

1. Protein Sequencing

2. Introduction to Protein Sequencing

3. What is Protein Any of a class of nitrogenous organic compounds which have large molecules composed of one or more long chains of amino acids and are an essential part of all living organisms, especially as structural components of body tissues such as muscle, hair, etc., and as enzymes and antibodies."a protein found in wheat"

4. What is sequence a particular order in which related things follow each other. a set of related events, movements, or items that follow each other in a particular order.

5. Protein Sequencing Protein sequencing  is the practical process of determining the  amino acid sequence  of all or part of a  protein  or  peptide . This may serve to identify the protein or characterize its  post-translational modifications .

6. Typically, partial sequencing of a protein provides sufficient information (one or more sequence tags) to identify it with reference to databases of protein sequences derived from the conceptual  translation  of  genes .

7. The two major direct methods of protein sequencing are  mass spectrometry  and  Edman degradation  using a  protein sequenator  (sequencer). Mass spectrometry methods are now the most widely used for protein sequencing and identification but Edman degradation remains a valuable tool for characterizing a protein's  N -terminus.

8. Why we do Protein sequencing?? Determining amino acid composition. It is often desirable to know the unordered amino acid composition of a protein prior to attempting to find the ordered sequence, as this knowledge can be used to facilitate the discovery of errors in the sequencing process or to distinguish between ambiguous results

9. . Knowledge of the frequency of certain amino acids may also be used to choose which  protease  to use for digestion of the protein. The disincorporation of low levels of non-standard amino acids (e.g. norleucine) into proteins may also be determined.  A generalized method often referred to as  amino acid analysis  for determining amino acid frequency is as follows: Hydrolyse a known quantity of protein into its constituent amino acids. Separate and quantify the amino acids in some way.

10. Hydrolysis Hydrolysis  is done by heating a sample of the protein in 6 M  hydrochloric acid  to 100–110 °C for 24 hours or longer. Proteins with many bulky  hydrophobic  groups may require longer heating periods. However, these conditions are so vigorous that some amino acids ( serine ,  threonine ,  tyrosine ,  tryptophan ,  glutamine , and  cysteine ) are degraded. To circumvent this problem,

11. Biochemistry Online suggests heating separate samples for different times, analysing each resulting solution, and extrapolating back to zero hydrolysis time. Rastall suggests a variety of reagents to prevent or reduce degradation, such as  thiol   reagents  or  phenol  to protect tryptophan and tyrosine from attack by chlorine, and pre- oxidising cysteine. He also suggests measuring the quantity of  ammonia  evolved to determine the extent of  amide hydrolysis .

12. Separation and quantitation The amino acids can be separated by  ion-exchange chromatography  then derivatized to facilitate their detection. More commonly, the amino acids are derivatized then resolved by  reversed phase HPLC .

13. An example of the ion-exchange chromatography is given by the NTRC using sulfonated polystyrene as a matrix, adding the amino acids in acid solution and passing a buffer of steadily increasing  pH  through the column. Amino acids are eluted when the pH reaches their respective  isoelectric points . Once the amino acids have been separated, their respective quantities are determined by adding a reagent that will form a coloured derivative.

14. History Of Protein Sequencing

15. The advent of protein sequencing can be traced to two almost parallel discoveries by Frederick Sanger and Pehr Edman . In 1950, Pehr Edman published a paper demonstrating a label-cleavage method for protein sequencing which was later termed “Edman degradation”.

16. Pehr Edman began his work in the Northrop- Kunitz laboratory at the Princeton branch of the Rockefeller Institute of Medical Research in 1947 where he attempted to find a method to decode the amino acid sequence of a protein using chemicals; specifically he had early success with fluorodinitrobenzene (FDNB) and phenylisothiocyanate (PITC).

17. Throughout his year at Princeton, Edman was able to conduct enough experiments to understand that it was feasible to use reagents like FDNB and PITC to determine amino acid sequence. Edman returned to Sweden in 1947 and after two more years of work he was able to publish his paper that would describe the first successful method to sequence proteins [1] This ground breaking paper described a method to determine the amino acid sequence of a protein and would come to be known as the Edman Degradation .

18. F.SANGER Around the same time Fred Sanger was developing his own labeling and separation method which led to the sequencing of insulin. For this work, Sanger was awarded the 1958 Nobel Prize for Chemistry.

19. Plus and minus in the 1970’s Fast-forward once again to the 1970’s and we find Fred Sanger still at the forefront of nucleic acid sequencing. In 1975 whilst at the Laboratory of Molecular Biology in Cambridge, Fred Sanger developed the “plus and minus” method for DNA sequencing (Sanger and Coulson , 1975). Again there was competition in the field with Maxam and Gilbert working on degradation sequencing ( Maxam and Glibert , 1977) however, their method was ultimately to falter due to the ease and quality of the Sanger method.

20. plus and minus method A primer is extended by a polymerase to generate a population of newly synthesized deoxyribonucleotides of assorted lengths; the unused dNTP s are removed, and polymerization continues in four pairs of plus and minus reaction mixtures; the minus mixtures have three NTPs and the plus mixtures have only one. After a second polymerization, the mixtures are fractionated by gel electrophoresis , and each plus and minus pair is compared to indicate the length of the new polydeoxyribonucleotide (by the mobilities of the bands) and the position at which polymerization had terminated as a result of the absence of the missing dNTP

21. Five years earlier, Frederick Sanger had demonstrated a method to determine the amino acid residue located on the N-terminal end of a polypeptide chain by using the reagent fluorodinitrobenzene. While it was thought, that at most, this method could only provide the sequences found on the N-terminal, Sanger was able to take the method one step further.

22. By using several proteolytic enzymes, partial hydrolysis and early version of chromatography, Sanger was able to cleave the protein into fragments and piece together the residues like a jigsaw puzzle. It wasn’t until 1955 that Sanger was able to present the complete sequence of insulin which led to him being awarded a Nobel Prize in Chemistry in 1958.

23. Other scientist Emile Zuckerkandl and Linus Pauling, whose work in the mid1960s advanced the use of nucleotide and protein sequences to explore evolution In the 1970s,Carl Woese used ribosomal RNA sequences to define archaebacteria as a group of living organisms distinct from other bacteria and eukaryotes

24. Methods Of Protein Sequencing

25. Protein sequencing Technique to find out the sequence of amino acids in a protein Sequencing methods 1-N-terminal sequencing (Edman degradation) 2-C-terminal sequencing 3-Prediction from DNA sequence

26. Edman degradation N-terminal sequencing

27. STEPS Protein purification Protein denaturation Protein digestion N-terminal labeling Separation of labeled amino acid by chromatography Detection through mass spectrometry Data analysis

28. Protein isolation(purification) 1-SDS-PAGE (sodium dodecyl sulfate-poly acryl amide gel) 2-Two dimensional gels Protein of interest is immobilized by being absorbed onto a chemically modified glass or by electro blotting onto a porous polyvinylidene fluoride (PVDF) membrane.

29. by heating a sample of the protein in 6 Molar HCL up to 100-110 degrees Celsius for 24 hours or longer It may degrade some amino acids To avoid this Thiol reagents or phenol are used Performic acid for intra chain or inter chain S-S bonds Protein hydrolysis(denaturation)

30. Protein digestion Use Endoproteinase Lys-C, CNBr, Pepsin or trypsin to digest proteins into a population of peptides Other enzymes include Glu-C and chymotrypsin Add enzyme at 1:20 enzyme: protein ratio incubate at room temperature for 6-9hrs For better results use mixture of enzymes

31. N-terminal labeling The Edman reagent, phenylisothiocyanate (PTC), is added to the adsorbed peptide, together with a mildly basic buffer solution of 12% trimethylamine This reacts with the amine group of the N-terminal amino acid The terminal amino acid can then be selectively detached by the addition of anhydrous acid The derivative then isomerises to give a substituted phenylthiohydantoin which can be washed off and identified by chromatography, and the cycle can be repeated

32. CHROMATOGRAPHY Chromatography is a technique in which molecules are separated based on volatility and bond characteristics when subjected to a carrier Derivatives of amino acid can be separated by 1-HPLC 2-Gas chromatography In gas chromatography (GC), the mobile phase is an inert gas such as helium

33. MASS SPECTROMETERY Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios Separated amino acid derivatives are analyzed by mass spectrometer

34. MS procedure A sample is loaded onto the MS instrument, and undergoes vaporization The components of the sample are ionized by one of a variety of methods (e.g., by impacting them with an electron beam ), which results in the formation of charged particles ( ions ) The ions are separated according to their mass-to-charge ratio in an analyzer by electromagnetic fields The ions are detected, usually by a quantitative method The ion signal is processed into mass spectra

35. Mass spectrometer

36. first strategy for identifying an unknown compound is to compare its experimental mass spectrum against a library of mass spectra Standard solutions of amino acids are also used and the resulting pattern is compared with standard spectrum. MS data analysis

37. Limitations of Edman degradation Need Pure Samples of Peptides Requires 40-60 min / Amino Acid Can’t Analyze N-Terminally Modified Peptides Advantages Most Reliable Sequencing Technique

38. C terminal sequence

39. Definition: The  C-terminus  (also known as the  carboxyl-terminus ,  carboxyl-terminus ,  C-terminal tail ,  C-terminal end , or  COOH-terminus ) is the end of an  amino acid  chain ( protein  or polypeptide ), terminated by a free  carboxyl group  (-COOH). C terminal

40. C-terminal retention signals Proteins are naturally synthesized starting from the N-terminus and ending at the C-terminus. While the  N-terminus  of a protein often contains targeting signals, the C-terminus can contain retention signals for protein sorting. The most common  ER retention  signal is the amino acid sequence -KDEL (Lys-Asp- Glu - Leu ) or -HDEL (His-Asp- Glu - Leu ) at the C-terminus. This keeps the protein in the  endoplasmic reticulum  and prevents it from entering the  secretory pathway .

41. C-terminal modifications The C-terminus of proteins can be modified  post translationally , most commonly by the addition of a  lipid  anchor to the C-terminus that allows the protein to be inserted into a membrane without having a  trans membrane domain . Another form of C-terminal modification is the addition of a phosphoglycan ,  glycosylphosphatidylinositol  (GPI), as a membrane anchor. The GPI anchor is attached to the C-terminus after proteolytic cleavage of a C-terminal propeptied . The most prominent example for this type of modification is the  prion  protein.

42. C-terminal domain: The C-terminal domain of some proteins has specialized functions. In humans, the CTD of  RNA polymerase II  typically consists of up to 52 repeats of the sequence Tyr- Ser -Pro- Thr - Ser -Pro-Ser. [1]  This allows other proteins to bind to the C-terminal domain of RNA polymerase in order to activate polymerase activity. These domains then involved in the  initiation  of DNA transcription.

43. C terminal sequencing technique Top Down sequencing by MALDI ISD is used to sequence the c terminal of amino acid chain. MALDI MS: “matrix-assisted laser desorption/ionization mass spectrometry” through which the c-terminal can be analyzed. This method is used when the N-terminal is blocked and there is only C-terminal available. The technique can fragment and sequence both the N- and C-terminal in the same mass spectrum.

44. Admen degradation is only used for N-terminal sequencing. The most common method is to add carboxy peptidases to a solution of the protein. Take a sample at regular at regular intervals and determine the terminal amino acid by analyzing a plot amino acid concentration and time.

45. A peptide mixture is generated by cleavage of the protein with cyanogen bromide and is incubated with carboxy peptidase Y. The enzyme is only able to act on the C-terminal fragment, because this is the only peptide without a homoserine lactone residue at its C terminus. The resulting fragments, forming a peptide ladder, are analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The entire protocol, including the CNBr cleavage, takes 21 h and can be applied to proteins purified either by SDS-PAGE or by 2D PAGE or in solution. Use of peptidase:

46. Top down sequencing: Top-down  proteomics  is a method of  protein  identification that uses an  ion trapping   mass spectrometer  to store an isolated protein ion for mass measurement and  tandem mass spectrometry  analysis. Top-down proteomics is capable of identifying and quantitating unique proteoforms through the analysis of intact protein.  Top-down proteomics interrogates protein structure through measurement of an intact mass followed by direct ion dissociation in the gas phase.

47.   Fragmentation  for tandem mass spectrometry is accomplished by  electron-capture dissociation  or  electron-transfer dissociation . Effective fractionation is critical for sample handling before mass-spectrometry-based proteomics. Proteome analysis routinely involves digesting intact proteins followed by inferred protein identification using mass spectrometry. The main advantages of the top-down approach include the ability to detect degradation products, sequence variants, and combinations of post-translational modifications.

48. MALDI MS top down sequencing: 0.5-1 ml  salt-free protein solution placed on a  MALDI-plate, covered with the MALDI Matrix solution, is analyzed in the in-source decay mode on an UltrafleXtreme mass spectrometer. The generated mass spectrum (a complex mass spectrum, exhibiting mainly c- and y ions) is further analyzed with the software Bio. tools or is processed via a Mascot search. A .pdf result file with sequence coverage of the target sequence would be the result output.

49. UltrafleXtreme mass spectrometer The UTX is used for a variety of MALDI applications, including mass spectrometry imaging (MSI), protein identification, peptide fingerprinting, and structure identification for a wide spectrum of biomoles (including lipids, polymers, glycans ).

50. MALDI ISD

51. Peptide Sequencing by Mass Spectrometry

52. Introduction MS/MS plays important role in protein identification (fast and sensitive) Derivation of peptide sequence an important task in proteomics Derivation without help from a protein database (“de novo sequencing”), especially important in identification of unknown protein

53. Basic lab experimental steps 1. Proteins digested w/ an enzyme to produce peptides 2. Peptides charged (ionized) and separated according to their different m/z ratios 3. Each peptide fragmented into ions and m/z values of fragment ions are measured Steps 2 and 3 performed within a tandem mass spectrometer.

54. Mass spectrum Proteins consist of 20 different types of a. a. with different masses (except for one pair Leu and Ile) Different peptides produce different spectra Use the spectrum of a peptide to determine its sequence

55. Objectives Describe the steps of a typical peptide analysis by MS (proteomic experiment) Explain peptide ionization, fragmentation, identification

56. Why are peptides, and not proteins, sequenced? Solubility under the same conditions Sensitivity of MS much higher for peptides MS efficiency

57. MS Peptide Experiment

58. Choice of Enzyme Cleaving agent/Proteases Specificity A. HIGHLY SPECIFIC Trypsin Arg-X, Lys-X Endoproteinase Glu-C Glu-X Endoproteinase Lys-C Lys-X Endoproteinase Arg-C Arg-X Endoproteinase Asp-N X-Asp B. NONSPECIFIC Chymotrypsin Phe-X, Tyr-X, Trp-X, Leu-X Thermolysin X-Phe, X-Leu, X-Ile, X-Met, X-Val, X-Ala

59. ESI Liquid flow Q or Ion Trap analyzer ESI is a solution technique that gives a continuous stream of ions, best for quadrupoles, ion traps, etc . + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + MALDI 3 nS LASER PULSE Sample (solid) on target at high voltage/ high vacuum MALDI is a solid-state technique that gives ions in pulses, best suited to time-of-flight MS . TOF analyzer Atmosphere Low vac. High vac. High vacuum

60. ….MALDI or Electrospray ? MALDI is limited to solid state, ESI to liquid ESI is better for the analysis of complex mixture as it is directly interfaced to a separation techniques (i.e. HPLC or CE) MALDI is more “flexible” (MW from 200 to 400,000 Da)

61. Q2 Collision Cell Q3 I II III Correlative sequence database searching Theoretical Acquired Protein identification Peptides 1D, 2D, 3D peptide separation 200 400 600 800 1000 1200 m/z 200 400 600 800 1000 1200 m/z 200 400 600 800 1000 1200 m/z 12 14 16 Time (min) Tandem mass spectrum Protein Identification Strategy Q1 * * Protein mixture

62. Breaking Protein into Peptides and Peptides into Fragment Ions Proteases, e.g. trypsin, break protein into peptides MS/MS breaks the peptides down into fragment ions and measures the mass of each piece MS measure m/z ratio of an ion

63. Peptide fragmentation Amino acids differ in their side chains Predominant fragmentation Weakest bonds

64. Tendency of peptides to fragment at Asp (D) Mass Spectrometry in Proteomics Ruedi Aebersold* and David R. Goodlett 269 Chem. Rev. 2001, 101, 269-295 C-terminal side of Asp

65. Protein Identification by MS Artificial spectra built Artificially trypsinated Database of sequences (i.e. SwissProt) Spot removed from gel Fragmented using trypsin Spectrum of fragments generated MATCH Library

66. Conclusions MS of peptides enables high throughput identification and characterization of proteins in biological systems “de novo sequencing” can be used to identify unknown proteins not found in protein databases

67. Prediction From DNA Sequence

68. The rapid increase of publicly available sequences and protein structures means that an increasing amount of information can be obtained for any protein sequence through its relatedness to others. If a set of homologous proteins can be found and aligned, the information content at each position in the alignment profile is far greater than in any single member of the family, and any structural or functional prediction algorithm should utilize this collective information. Profile information of this type is extremely sensitive to the quality of the multiple alignment, and distant homologues should only be included in the alignment if they can be aligned with confidence.

69. Figure 17.3b-3 RNA PROCESSING Nuclear envelope DNA Pre-mRNA mRNA TRANSCRIPTION TRANSLATION Ribosome Polypeptide

70. DNA template strand TRANSCRIPTION mRNA TRANSLATION Protein Amino acid Codon Trp Phe Gly 5  5  Ser U U U U U 3  3  5  3  G G G G C C T C A A A A A A A T T T T T G G G G C C C G G DNA molecule Gene 1 Gene 2 Gene 3 C C

71. Protein structure prediction  is the inference of the three-dimensional structure of a  protein  from its  amino acid  sequence—that is, the prediction of its  folding  and its  secondary  and  tertiary structure  from its  primary structure . Structure prediction is fundamentally different from the inverse problem of  protein design .

72. Protein structure prediction is one of the most important goals pursued by  bioinformatics  and  theoretical chemistry ; it is highly important in  medicine  (for example, in  drug design ) and  biotechnology  (for example, in the design of novel  enzymes ).

73. Protein structure and terminology Proteins are chains of amino acids joined together by peptide bonds. Many conformations of this chain are possible due to the rotation of the chain about each Cα atom. It is these conformational changes that are responsible for differences in the three dimensional structure of proteins. Each amino acid in the chain is polar, i.e. it has separated positive and negative charged regions with a free C=O group, which can act as hydrogen bond accepto r and an NH group, which can act as hydrogen bond donor . These groups can therefore interact in the protein structure. The 20 amino acids can be classified according to the chemistry of the side chain which also plays an important structural role.  Glycine  takes on a special position, as it has the smallest side chain, only one Hydrogen atom, and therefore can increase the local flexibility in the protein structure.  Cysteine  on the other hand can react with another cysteine residue and thereby form a cross link stabilizing the whole structure.

74. The protein structure can be considered as a sequence of secondary structure elements, such as α helices and β sheets, which together constitute the overall three-dimensional configuration of the protein chain. In these secondary structures regular patterns of H bonds are formed between neighboring amino acids, and the amino acids have similar Φ and Ψ angles. Bond angles for ψ and ω The formation of these structures neutralizes the polar groups on each amino acid. The secondary structures are tightly packed in the protein core in a hydrophobic environment. Each amino acid side group has a limited volume to occupy and a limited number of possible interactions with other nearby side chains, a situation that must be taken into account in molecular modeling and alignments.  [

75. α Helix The α helix is the most abundant type of secondary structure in proteins. The α helix has 3.6 amino acids per turn with an H bond formed between every fourth residue; the average length is 10 amino acids (3 turns) or 10 Å but varies from 5 to 40 (1.5 to 11 turns). The alignment of the H bonds creates a dipole moment for the helix with a resulting partial positive charge at the amino end of the helix. Because this region has free NH2 groups, it will interact with negatively charged groups such as phosphates. The most common location of α helices is at the surface of protein cores, where they provide an interface with the aqueous environment. The inner-facing side of the helix tends to have longer helices, forming a bend.

76. hydrophobic amino acids and the outer-facing side hydrophilic amino acids. Thus, every third of four amino acids along the chain will tend to be hydrophobic, a pattern that can be quite readily detected. In the leucine zipper motif, a repeating pattern of leucines on the facing sides of two adjacent helices is highly predictive of the motif.

77. β sheet β sheets are formed by H bonds between an average of 5–10 consecutive amino acids in one portion of the chain with another 5–10 farther down the chain. The interacting regions may be adjacent, with a short loop in between, or far apart, with other structures in between. Every chain may run in the same direction to form a parallel sheet, every other chain may run in the reverse chemical direction to form an anti parallel sheet, or the chains may be parallel and anti parallel to form a mixed sheet.

78. The pattern of H bonding is different in the parallel and anti parallel configurations. Each amino acid in the interior strands of the sheet forms two H bonds with neighboring amino acids, whereas each amino acid on the outside strands forms only one bond with an interior strand . Looking across the sheet at right angles to the strands, more distant strands are rotated slightly counterclockwise to form a left-handed twist. The Cα atoms alternate above and below the sheet in a pleated structure, and the R side groups of the amino acids alternate above and below the pleats.

79. Loop Loops are regions of a protein chain that are (1) between α helices and β sheets, (2) of various lengths and three-dimensional configurations, and (3) on the surface of the structure. Hairpin loops that represent a complete turn in the polypeptide chain joining two antiparallel β strands may be as short as two amino acids in length.

80. Loops interact with the surrounding aqueous environment and other proteins. Because amino acids in loops are not constrained by space and environment as are amino acids in the core region, and do not have an effect on the arrangement of secondary structures in the core, more substitutions, insertions, and deletions may occur. Thus, in a sequence alignment, the presence of these features may be an indication of a loop.

81. The positions of  introns  in genomic DNA sometimes correspond to the locations of loops in the encoded protein [ . Loops also tend to have charged and polar amino acids and are frequently a component of active sites. A detailed examination of loop structures has shown that they fall into distinct families .

82. Coils A region of secondary structure that is not a α helix, a β sheet, or a recognizable turn is commonly referred to as a coil. 

83. Applications of Protein Sequencing

84. In Functional genomics: functional genomics  is a field of molecular biology that attempts to make use of the vast wealth of data produced by genomic and transcriptomic projects (such as genome sequencing projects and RNA sequencing) to describe gene (and protein) functions and interactions. Unlike genomics, functional genomics focuses on the dynamic aspects such as

85. gene transcription, translation, regulation of gene expression and protein–protein interactions, as opposed to the static aspects of the genomic information such as DNA sequence or structures.

86. The goal of functional genomics is to understand the relationship between an organism's genome and its phenotype. The term functional genomics is often used broadly to refer to the many possible approaches to understanding the properties and function of the entirety of an organism's genes and gene products.

87. The promise of functional genomics is to expand and synthesize genomic and proteomic knowledge into an understanding of the dynamic properties of an organism at cellular and/or organismal levels. This would provide a more complete picture of how biological function arises from the information encoded in an organism's genome. The possibility of understanding how a particular mutation leads to a given phenotype has important implications for human genetic diseases, as answering these questions could point scientists in the direction of a treatment or cure.

88. Prediction of protein function from protein sequence and structure The sequence of a genome contains the plans of the possible life of an organism, but implementation of genetic information depends on the functions of the proteins and nucleic acids that it encodes. Many individual proteins of known sequence and structure present challenges to the understanding of their function.

89. In particular, a number of genes responsible for diseases have been identified but their specific functions are unknown. Whole-genome sequencing projects are a major source of proteins of unknown function. Annotation of a genome involves assignment of functions to gene products, in most cases on the basis of amino-acid sequence alone.

90. 3D structure can aid the assignment of function, motivating the challenge of structural genomics projects to make structural information available for novel uncharacterized proteins. Structure-based identification of homologues often succeeds where sequence-alone-based methods fail, because in many cases evolution retains the folding pattern long after sequence similarity becomes undetectable.

91. Nevertheless, prediction of protein function from sequence and structure is a difficult problem, because homologous proteins often have different functions. Many methods of function prediction rely on identifying similarity in sequence and/or structure between a protein of unknown function and one or more well-understood proteins. Alternative methods include inferring conservation patterns in members of a functionally uncharacterized family for which many sequences and structures are known.

92. In Proteomics Proteomics  is the large-scale study of proteomes. A  proteome  is a set of proteins produced in an organism, system, or biological context.  The proteome is not constant; it differs from cell to cell and changes over time. To some degree, the proteome reflects the underlying  transcriptome . However, protein activity (often assessed by the reaction rate of the processes in which the protein is involved) is also modulated by many factors in addition to the expression level of the relevant gene.

93. Protein sequencing denotes the process of finding the amino acid sequence, or primary structure of a protein. Sequencing plays a very vital role in  Proteomics  as the information obtained can be used to deduce function, structure, and location which in turn aids in identifying new or novel proteins as well as understanding of cellular processes. Better understanding of these processes allows for creation of drugs that target specific metabolic pathways among other things.

94. In Bioinformatics What is bioinformatics?  In recent years, molecular biology has witnessed an information revolution as a result of the development of rapid DNA sequencing techniques and the corresponding progress in computer-based technologies, which are allowing us to cope with this information deluge in increasingly efficient ways. The term that was coined to encompass computer applications in biological sciences is bioinformatics.

95. The term bioinformatics is now used to mean rather different things, from artificial intelligence and robotics to genome analysis. The term was originally applied to the computational manipulation and analysis of biological  sequence  data (DNA and/or protein), but now tends also to be used to embrace the manipulation and analysis of 3D structural data.

96. Identifying protein-coding genes in genomic sequences The vast majority of the biology of a newly sequenced genome is inferred from the set of encoded proteins. Predicting this set is therefore invariably the first step after the completion of the genome DNA sequence.  The genome sequence is an organism's blueprint: the set of instructions dictating its biological traits. The unfolding of these instructions is initiated by the transcription of the DNA into RNA sequences. According to the standard model, the majority of RNA sequences originate from protein-coding genes; that is, they are processed into messenger RNAs (mRNAs) which, after their export to the cytosol , are translated into proteins.

97. To Determine the protein folding Protein folding is the process by which a protein structure assumes its functional shape or conformation. Protein folding is the physical process by which a protein chain acquires its native 3-dimensional structure, a conformation that is usually biologically functional, in an expeditious and reproducible manner. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil. Each protein exists as an unfolded polypeptide or random coil when translated from a sequence of mRNA to a linear chain of amino acids. 

98. All protein molecules are heterogeneous unbranched chains of amino acids. By coiling and folding into a specific three-dimensional shape they are able to perform their biological function. Proteins are formed from long chains of amino acids; they exist in an array of different structures which often dictate their functions. Proteins follow energetically favorable pathways to form stable, orderly, structures; this is known as the proteins’ native structure. Most proteins can only perform their various functions when they are folded. Scientists believe that the instructions for folding a protein are encoded in the sequence. Researchers and scientists can easily determine the sequence of a protein, but have not cracked the code that governs folding .

99. In Drugs production What is Protein Drug A type of  drug s made of  protein . These  drug s usually have large molecular weight with  protein characteristics. structure of an unusual class of proteins called beta-peptides. Eventually, these peptides could become the basis for drugs that are cheaper to manufacture than existing protein-based pharmaceuticals and last longer in the body. A  drug's  efficiency may be affected by the degree to which it  binds  to the  proteins  within blood plasma. The less  bound  a  drug  is, the more efficiently it can traverse cell membranes or diffuse. Common blood  proteins  that drugs bind  to are human serum albumin, lipoprotein, glycoprotein, α, β‚ and γ globulins