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| retention times |
for Edman's reagent
HPLC chromatogram |
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mcs5109 Wed, 01 Oct 2008 03:34:20 GMT |
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| dansyl chorlide |
-reacts w/ N terminus
-acid hydrolysis breaks all peptide bonds
-dansyl has bright yellow fluoresecent shirt on detected by HPLA |
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mcs5109 Wed, 01 Oct 2008 03:30:38 GMT |
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| Gene duplication |
can occur through aberrant genetic recombination event (ortho, paralogs) |
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mcs5109 Tue, 30 Sep 2008 03:40:19 GMT |
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| Orthologous |
homologous proteins from different organisms |
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mcs5109 Tue, 30 Sep 2008 03:36:44 GMT |
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| Paralogous |
homologous proteins in same organism |
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mcs5109 Tue, 30 Sep 2008 03:36:44 GMT |
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| phylogeny |
-A phylogenetic tree can be constructed that indicates ancestral relationship between different species. |
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mcs5109 Tue, 30 Sep 2008 03:36:44 GMT |
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| Neutral Drift |
-random mutations occur which do not affect protein function
-random process is referred to as neutral drift
-Comparison of primary sequence of homologous proteins indicate which proteins are essential for their function
-ex: Pigeon cytochrome c can reduce wheat oxidase |
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mcs5109 Tue, 30 Sep 2008 03:36:44 GMT |
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| malaria resistance |
-SCA Mendelian genetics
-Heterozygotes have approx. 40% HbS. Such people have sickle cell trait and can lead normal life.
-heterozygote are resistant to malaria |
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mcs5109 Tue, 30 Sep 2008 03:32:58 GMT |
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| SCA mutation |
-Caused my mutant hemoglobin
-sickle cell hemoglobin HbS is two units more positive than HbA (normal).
-Caused by single mutation causing change of Glu (6th aa of B subunits) to Val (charge difference)
-under certain conditions (low O2 levels) HbS distort into sickle shapes
-block small capillaries causing necrosis of the tissue
-Conversion of charged glu to hydrophobic val forms patch that now can bind to hydrophobic portion of another deoxy Hb molecule. Polymerizes into fibers when deoxygenated. Causes rbc to become sickle shape |
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mcs5109 Tue, 30 Sep 2008 03:32:58 GMT |
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| Sickle Cell anemia |
-Hemoglobin has 4 subunits: a2b2
-in rbc's
-In Sickle cell anemia, rbc assume crescent-like shape in low oxygen tension. Are more rigid and hinders free passage
Hemolytic anemia caused by lysis of rbc |
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mcs5109 Tue, 30 Sep 2008 03:32:58 GMT |
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| Determining site of disulfide bond |
-Separate digested peptide with paper electrophoresis
-Treat samples with performic acid to cleave disulfides
-Run again on paper electrophoresis to which has two spots
-Remove bands and sequence to identify |
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mcs5109 Tue, 30 Sep 2008 03:32:58 GMT |
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| tandem mass spec |
-short polypeptides 9<25 aas) can be directly sequenced
-electrospray ionization
-2 MS's
-1st MS selects protein of interest
-peptide ion then crashes into chemically inert cell (He)
-peptide bonds break and MW of charged fragments measured
-computerized |
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mcs5109 Tue, 30 Sep 2008 03:20:54 GMT |
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| MALDI-TOF |
-Matrix-assited laser desorption/ionization-time of flight mass spec
Peptide is embedded in crystalline matrix of some low molecular weight compound.
Peptide is irradiated with laser pulse which ejects peptides into gas phase |
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mcs5109 Tue, 30 Sep 2008 03:20:16 GMT |
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| electrospray ionization |
-macromolecule H2O sol'n sprayed from high voltage
-fine, highly charged droplets remain
-H2O evaporates
-now have gas-phase macromolecule ions
-go through MS |
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mcs5109 Tue, 30 Sep 2008 03:20:16 GMT |
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| Mass Spec |
-Can be used to accurately measure molecular weight
-Use a spray of protein, remove water and then have proteins
-3 techniques to get gas-phase ions required for MS:
-electrospray ionization
-MALDI
-tandem |
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mcs5109 Tue, 30 Sep 2008 03:20:16 GMT |
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| Peptide cleavage: Endopeptidases |
Trypsin
Chymotrypsin
Elastase |
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mcs5109 Tue, 30 Sep 2008 02:51:09 GMT |
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| Elastase |
cleaves on carboxy side of small neutral residues: Ala, Gly, Ser, Val |
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mcs5109 Tue, 30 Sep 2008 02:51:09 GMT |
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| iodoacetate |
-ICH2COO-
-after DTT to acetylate |
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mcs5109 Tue, 30 Sep 2008 02:47:24 GMT |
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| to cleave SS bonds b/w cys residues... |
-Can be directly oxidized by performic acid. But this will also oxidize methionine residues.
-Can be reduced and then acetylated
-DTT reduces
-performic acid hydrolyzes |
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mcs5109 Tue, 30 Sep 2008 02:47:24 GMT |
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| Hydrazinolysis |
-can be used to determine C terminal amino acid
-treated w/ hydrazine (H2N-NH2)
-all peptide bonds cleaved
-yield hydrazine resideues except for C terminus aa
-only free aa is C terminus |
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mcs5109 Wed, 01 Oct 2008 03:54:59 GMT |
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| C-terminus analysis |
-Carboxypeptidase
-Hydrazinolysis |
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mcs5109 Tue, 30 Sep 2008 02:41:40 GMT |
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| Carboxypeptidase |
-exopeptidase that cleaves from C terminus
-Different enzymes have different specificities
Because of differences in specificities, cannot be used for sequencing.
-if 2nd aa cleaved same time as 1st, both aas releases simultaneously
-Carboxypeptidase also not active on Pro residues |
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mcs5109 Tue, 30 Sep 2008 02:41:40 GMT |
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| N terminus methods |
-dansyl chloride
-Edman degradation |
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mcs5109 Tue, 30 Sep 2008 02:41:40 GMT |
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| Edman degradation |
-Phenylisothiocyanate
-Removes one aa at a time
-only takes off N-terminal aa
-IDs 20-30 amino acids from the N terminus
-compare retention times on HPLC
-Edman can be repeated automatically to determine aa sequence |
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mcs5109 Wed, 01 Oct 2008 03:33:05 GMT |
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| Identification of N-terminus |
-Dansyl chloride reacts with N terminus
-Acid hydrolysis to break all peptide bonds
-Dansyl amino acid has fluoresence and detected by HPLC |
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mcs5109 Tue, 30 Sep 2008 02:20:49 GMT |
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| Cyanogen bromide |
cleaves at Met |
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mcs5109 Tue, 30 Sep 2008 02:20:49 GMT |
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| Chymotrypsin |
cleaves on C-side of bulky hydrophobic residues Phe, Tyr, Trp |
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mcs5109 Tue, 30 Sep 2008 02:47:50 GMT |
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| Trypsin |
cleaves on C-side of positively charged residues Arg or Lys |
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mcs5109 Tue, 30 Sep 2008 02:48:06 GMT |
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| DTT |
-reduces disulfide bonds |
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mcs5109 Tue, 30 Sep 2008 02:10:14 GMT |
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| Oxidize dissulfide |
with performic to cysteic acid |
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mcs5109 Tue, 30 Sep 2008 02:10:14 GMT |
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| pH, area, guanidinium hydrochloride, or high salt |
-ways to separate protein subunits if multimeric |
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mcs5109 Tue, 30 Sep 2008 02:10:14 GMT |
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