Ch 3 Text 5

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  1. __ is a Powerful Technique for the Identification of Peptides and Proteins
    ·         Studying biological processes in their native state is advantageous, but difficult due to limits of techniques
    o   __ enables the highly precise and sensitive measurement of the atomic composition of a particular molecule, or __, without prior knowledge of its identityà can be used to detect identity
    Mass Spectrometry

    Mass spec

  2. §  Analyte molecules are converted into__ and __ is measured
    §  Three essential components: __, __, and __
    • gaseous, charged forms
    • mass to charge of ion
    • the ion source, the mass analyzer, and the detector
  3. · Ion source: __
    converts analyte into gas-phase ions (techniques like matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) enabled this
  4. o   MALDI: analyte evaporated to __in presence of volatile, aromatic compound (the matrix) that can absorb light at specific wavelengths. A laser pulse tuned to one of these wavelengths __ and __, converting some of the analyte into the gas phase. Later gaseous collisions enable the __, ionizing the analyte.
    • dryness 
    • excites and vaporizes the matrix
    • intermolecular transfer of charge
  5. In ESI, a solution of the __is passed through an electrically charged nozzle. Droplets of the __emerge from the nozzle into a chamber of very low pressure, __ the solvent and __, which enters the __
    • analyte 
    • analyte 
    • evaporating
    • yielding the ionized analyte
    • mass analyzer
  6. · Mass analyzer: distinguishes ion on basis of __
    o   __: ions are accelerated through an elongated chamber under a fixed electrostatic potential
    o   The sequential action of the __ and __ enables the highly sensitive measurement of the mass of potentially massive ions
    · In the __, a family of ions with the same mass but different net charge is formed from a single analyte and appear as peaks in the mass spec
    mass-to-charge ratio

    Time-of-flight (TOF) mass analyzer

    ion source and the mass analyzer 

    ionization process
  7. §  Peptides can be sequenced by __ as an alternative to __
    · Ions that have been analyzed by a mass spec, the __, can be broken into smaller peptide chains by __with atoms of an inert gas like He or Ar. The __ can be passed through a second mass analyzer called __
    o   Product ions can be formed such that __--> family of ions detected (fragments)
    • mass spec as alternative to Edman degradation
    • precursor ions
    • bombardment 
    • product ions
    • tandem mass spec
    • individual amino acid residues are cleaved from the precursor ion
  8.  §  Individual proteins can be identified by mass spec
    · When a protein is cleaved by chemical or enzymatic methods, a specific and predictable family of peptide fragments is formed; and, the identity of the individual peptides formed from the cleavage is a __ for that particular protein
    · Protein cleavageà __--> rapid identification and quantitation of signatures
    • distinctive signature
    • chromatographic separation and mass spec-->
  9. Peptides can be Synthesized by Automated Solid-Phase Methods
    Peptides of defined sequence can be synthesized to assist in biochemical analysis. These tools are valuable because __(4)__
    • o   Synthetic peptides can serve as antigens to stimulate the formation of specific antibodies: peptides can be synthesized that match the translation of part of the gene’s nucleic acid sequence, and antibodies can be generated that target these peptidesà then can isolate the intact protein or localize it within the cell
    • o   Synthetic peptides can be used to isolate receptors for many hormones and other signaling molecules
    • o   Synthetic peptides can serve as drugs
    • o   Studying synthetic peptides can help define the rules governing the three-dimensional structure of protein
  10. ·         The __group of one amino acid is linked to the __group of another to construct a peptide. However, a unique product is formed only if a __. So, we need to block some groups and activate others to prevent unwanted reactions


    single amino group and a single carboxyl group are available for reaction
  11. o   First, the carboxyl-terminal amino acid is attached to an __ by its __, protecting it from further peptide-bond-forming reactions. The __ of this amino acid is blocked with a protecting group like __ and then removed with __
    • insoluble resin b its carboxyl group
    • alpha-amino group 


    trifluoroacetic acid
  12. o   The next amino acid (in the protected t-Boc form) and __are added together, leaving only the __ and __ free to form a peptide bond
    §  DCC reacts with the __ of the incoming amino acid, activating it for the peptide-bond-forming reaction. After the peptide bond forms, excess reagents and dicyclohexylurea are washed away, leaving the desired dipeptide product attached to the beads. 
    • dicyclohexylcarbodiimide (DCC) 
    • incoming amino acid and the amino group of the resin-bound amino acid
    • carboxyl group
  13. o   At the end of the synthesis, the peptide is released from the beads by addition of __, which cleaves the __ anchor without disrupting peptide bonds; and, protecting groups on potentially reactive side chains are removed at this time
    • HF
    • carboxyl ester
  14. ·         An advantage of this __ is that the desired product at each stage is bound to beads that can be filtered and washedà no need to purify
    ·         Synthetic peptides can be linked to create even longer molecules with the use of __ methods
    • solid-phase method
    • peptide-ligation
  15. ·         Structure (active and binding sites) determine function
    ·      __ reveals the 3D structure in atomic detail and provides the clearest visualization of the precise 3D positions of most atoms within a protein
    o   Best resolution for the determination of molecular structures because their wavelength approximately corresponds to that of a covalent bond.
    X-ray crystallography
  16. ·         Three components: __ 

    ·         First requires the prep of a protein or complex in __, in which all protein molecules are oriented in a fixed, repeated arrangement with respect to one another, which can be done by slowly adding __ or another salt to a concentrated solution of protein to reduce its solubility 
    • protein crystal, source of x-rays, and detector
    • crystal form
    • ammonium sulfate
  17. o   Protein crystallization can be quite challenging:__ is required and it is often difficult to predict which experimental conditions will yield the most effective-crystals. Methods for screening many different crystallization conditions using a small amount of protein sample have been developed
    a concentrated solution of highly pure material
  18. ·         After a pure crystal of protein has been obtained, a source of __ is required; __ has more intense wavelengths and is created through acceleration of electrons in circular orbits at speeds close to the speed of light
    o   A __ forms when beams are directed at __; and, these are detected by electronic detector. 

    synchrotron radiation

    scattering pattern

    protein crystals
  19. ·         The basic principle for this technique: ??
    Electrons scatter x-rays (amplitude of wave scattered is proportional to number of electrons)

    scattered waves recombine (scattered waves reinforce one another)

    the way in which the scattered waves recombine depends only on the atomic arrangement
  20. §  The crystal is rotated so the beam can strike in several directions. This results in an __ containing __; and, they are analyzed
    · Each reflection is formed from a __. Each wave has a __—the timing of its crests and troughs relative to those of other waves
    xray photograph containing reflections

    from a wave with an amplitude proportional to the square root of the observed intensity of the spot.

  21. o   The next step is to __
    §  Image formed by applying a mathematic relation called a __ to the measured amplitudes and calculated phases of every observed reflection--> __(3D representation of where the electrons are most densely localized and used to determine the positions of the atoms in the crystallized molecule
    reconstruct an image of the protein from the observed reflections

    Fourier transform

    electron density map
  22. §  Critical is the resolution, which is determined by the number of __ used in the Fourier transform. The ultimate resolution of an x-ray analysis is determined by the degree of perfection of the crystal
    scattered intensities
  23. ·         NMR spectroscopy reveals structure of __
    o   Certain atomic nuclei are intrinsically magnetic and only a limited number of isotopes display this property, called __
    §  Protons, for example, can take either of two orientations, or __when an external magnetic field is applied
    • proteins in solution
    • spin
    • spin states (alpha and beta)
  24. · The energy difference between these states is proportional to the __
    · Alpha state: __
    A spinning proton in the alpha state can be raised to an __by applying a pulse of __, provided that the frequency corresponds to the energy difference between the alpha and beta states 
    • strength of the imposed magnetic field
    • slightly lower energy because it is aligned with this applied field
    • excited state (beta state) 
    • electromagnetic radiation
  25. o   These properties can be used to examine the __ of the hydrogen nucleus. The flow of electrons around a magnetic nucleus generates a __ that __
    §  __in different environments will change states
    §   A __ is obtained by keeping the magnetic field constant and varying the frequency of the electromagnetic radiation
    §  The different frequencies, called __, are expressed in fractional units δ relative to the shifts of a standard compound
    • chemical surroundings
    • small local magnetic field that opposes the applied field
    • Nuclei 
    • resonance spectrum
    • chemical shifts
  26. o   More info can be obtained through examining how spins affect their neighbors
    §  Especially revealing is a 2D spectrum obtained by __, which graphically displays pairs of protons that are in close proximity. The basis for this technique is the __, an interaction between nuclei that is proportional to the inverse sixth power of the distance between them 
    • nuclear Overhauser enhancement spectroscopy (NOESY)
    • nuclear Overhauser effect
  27. · Magnetization is transferred from an __ to an __ if the two nuclei are less than about __ apart
    o   Diagonal peaks mean that the __
    • excited nucleus to an unexcited one
    • five angstroms
    • pairs of protons are less than five angstroms apart
  28. o   In practice, a family of related structures is generated by NMR spectroscopy for three reasons

    What is the first?
    § First, not enough constraints may be experimentally accessible to fully specify the structure
  29. o   In practice, a family of related structures is generated by NMR spectroscopy for three reasons

    What is the second?
     Distances obtained from analysis of the NOESY spectrum are only approximate
  30. o   In practice, a family of related structures is generated by NMR spectroscopy for three reasons

    What is the third?
     The experimental observations are made not on single molecules but on a large number of molecules in solution that may have slightly different structures at any given moment
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Ch 3 Text 5
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