Ch 3 Text 5

__ 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

§  Analyte molecules are converted into__ and __ is measured
§  Three essential components: __, __, and __

· Ion source: __

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.

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 __

· 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

§  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)

 §  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

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)__

·         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

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 __

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. 

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

·         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

·         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.

·         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 

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

·         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. 

·         The basic principle for this technique: ??

§  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

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

§  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

·         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

· 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 

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

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 

· Magnetization is transferred from an __ to an __ if the two nuclei are less than about __ apart
o   Diagonal peaks mean that the __

o   In practice, a family of related structures is generated by NMR spectroscopy for three reasons

What is the first?

o   In practice, a family of related structures is generated by NMR spectroscopy for three reasons

What is the second?

o   In practice, a family of related structures is generated by NMR spectroscopy for three reasons

What is the third?