chapter 5: protein sequencing Flashcards
primary structure of protein
linear sequence of amino acids that contain disulfide (S-S) bridges
Frederick Sanger
-won the nobel prize for sequencing bovine insulin
-won nobel prize for DNA sequencing
Steps to study a proteins primary structure
- Separate into individual polypeptide chains by cleaving disulfide bonds
- End terminus analysis (C and N)
- Sequencing
- Cleaving disulfide bonds: Method 1
Oxidize with performic acid
- Cleaving disulfide bonds: Method 2
- Reduce with XS Thiol/mercaptoethanol
- Cap thiols with iodoacetic acid
*can reverse
- End terminus analysis (C)
*Carboxypeptidases and exopeptidases are exoproteases that cleave at the C-Terminus
-Exopeptidases: cleave protein peptide bonds from end inwards
- Carboxypeptidase B: cleave at Lysine and Arginine only
-Carboxypeptidase A: cleave everything else
- end terminus analysis (N pt.1)
-Fluorodinitrobenzene (DNFB)
-Dabsyl Chloride
Dansyl Chloride
1.Undergo NAS to get one of the three above to join the polypeptide on the N-term. due to reacting with a weak base (OH-)
2. reacts with a strong acid to get acid catalyzed hydrolysis to break all peptide bonds
3. have your N-terminus
*disadvantage: destroy protein sequence, except for N terminus
*Lysine give a false positive bc of its NH2 side chain
- End terminus analysis (N pt.2: Edman Reagent/Degregation)
Edman reagent= PITC (phenyl isothiocyanate) * look at structure
- Uses a weak base (OH-) to add PITC to the polypeptide chain on the N terminus-PTC polypeptide
- use anhydrous F3CCOOH (trifluooroacetic acid, TFA) to break polypeptide bond- Thiazoline derivative
- uses a mild acid (h+) to rearrange
*TFA rsn is self cleaving
*leaves remaining polypeptide in aq soln. for another round of rsn with PITC
*Rxn is 98% efficient but after 50-100 cycle is error prone
*100 AA is the upper limit
*if chain has more than 100 AA then need to fragment into more segements
Fragmentation Methods: Enzymatic Fragmentation with Proteases
- Trypsin: only cuts lysine and arginine
- Chymotrypsin: cuts after bulky aromatic side chains (Phe, Trp, Tyr)
- Elastase: cuts after neutral aa
*if R group next to it is proline the proteases won’t be able to cut
Fragmentation Methods: Cyanogen Bromide (CNBr)
-CNBr cleaves on the side of Methionine
-will cleave regardless of what R group is next to it
- Determination of protein seq by Mass Spectrometry: MALDI-TOF
Matrix Assisted Laser Desorption Ionization- Time of Flight
-use trypsin digestion to determine protein sequence by Peptide Mass Fingerprinting (PMF)
-use vaporization technique to get protein fragments into a gas phase (ESI also can do this)
-Time of Flight (TOF) MS then uses a long tube where each ion has the same electrostatic force applied
* smallest fragments accelerates faster and hit detector 1st and larger molecules hit the detector giving a fingerprint fragment masses
- Determination of protein seq by Mass Spectrometry: Electrospray Ionization (ESI)
Can also use electrospray ionization (ESI) instead of MALDI to get protein into ions gas phase
- Determination of protein seq by Mass Spectrometry: Tandem Mass Spectrometry
-sep and seq mixtures of proteins
-purifies and fragments proteins
-useful for studying proteomics
invaraint
aa do not change
-must have it for function
conserved regions
aa have same polarity
-polarity is imp for function
hypervariable region
aa of varied polarity
-polarity not imp
Cell disruption: osmotic lysis
hypotonic lysis
Cell disruption: Mechanical disruption
-high-speed blender
-french press
-sonication
Cell disruption: Chemical
-detergents (SDS)
-enzyme (lysozyme)
Liquid Chromatography
-protein is dissolved in a liquid (mobile phase) and is percolated (eluted) through a column that contains a porous solid matrix (stationary phase)
*smallest elution volume
Ion exchange Chromotography (IEC)
-sep proteins by charge
-CM (-): cation exchanger
-DEAE (+): anion exchanger
*zero and like charge proteins elute 1st together (use pH line)
IEC: desorption
-increasing salt conc. (NaCl)
-altering the pH
Gel filtration chromatography
-size exclusion
-molecular exclusion
-molecular sieve
-separates by size based on percolation through cross-linked gel
-largest elute first, smallest last
-Can be used to determine MW by plotting log(MW) vs. elution volume (Ve)
-get whole MW of protein
Affinity Chromatography (AC)
-separates based on attraction of the molecule of interest to a particular ligand
-specific and selective capture
-uses His Tagging because it can bind to N or C term and binds Ni2+ tightly (adsorbed)
Affinity Chromatography: elution
-changing salt conc. or pH
-adding excess of free ligand/ligand annalog
HPLC
-High pressure/performance Liquid Chromatography
-uses high pressure to push a liquid mobile phase solution through a column
-Normal phase: polar (hydrophilic) silica beads on column
*hydrophobic elutes 1st
-Reverse phase: nonpolar (hydrophobic) silica beads on column
*hydrophilic elutes first
Protein solubility is affected by…
- pH (least soluble when pH=pI)
- solvents (acetone)
- high tempreature
-if proteins have the same charge they are soluble
Separation by solubility: Salting IN
-at low concentrations, adding slats increases the solubility of proteins
*salts get in between the proteins
Separation by solubility: Salting OUT
-at high salt concentration, salt lowers the solubility of proteins because it pulls water away from them
Separation by solubility: Best salt
ammonium sulfate (NH4)2SO4
-kosmotropic ion=stabilizing ion
Dialysis
-uses osmosis to change ions in solution
-used to change buffers during protein purification
Gel electrophoresis
-separate DNA/RNA based on size
-pH=9
-anions go to the anode
-largest moves the least
PAGE
Polyacrylamide Gel Electrophoresis
Coomassie Blue
-stains and visualizes protein on gel
-protein structure looks like a Y
- SO3, H5C2, R, NH, OC2H5
-Binds proteins through pi-stacking and ionic bonding
SDS
-stands for sodium dodecyl sulfate (detergent)
-gives all proteins the same negative charge
-separates based on size (largest moves least)
SDS-PAGE
-log(MW) vs relative migration = straight line
-gives subunit MW
Paper Electrophoresis/ Chromatography
-used to separate biomolecules
-Uses Ninhydrin to stain for aa
*know how Ninhydrin (Powder and liquid form) and Ruhemann’s purple look
Isoelectric Focusing (IEF)
Separates proteins by their isoelectric points (pI=pH)
2D gel electrophoresis
- Run IEF gel in one dimension to separate by pI
- Runs SDS-PAGE to separate by mass
- can be used to determine all proteins expressed in a cell= PROTEOMICS
ELISA
*look at notes
Western blotting
-transfer of protein form gel to membrane
-detect with antibody targeted to protein of interest (immunoblotting)
