Chapter7-PrimarySequence - Chemistry 233 Fundamentals of...

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Chemistry 233 Fundamentals of Biochemistry Primary Sequence Determination
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Covalent Structures of Proteins Sequence determination techniques: Composition (how many Asp, Ala, etc) N-terminal and C-terminal reagents cleavage of peptides (smaller fragments are easier to sequence) sequencing: Edman method or mass spectrometry
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Proteins come in all sorts of sizes and shapes What methods can we use on all proteins for sequence determination? Primary structure (sequence) of bovine insulin
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Composition of a Protein Gives pretty much all the amino acids in the protein, but no sequence information! The steps in the procedure: i) hydrolyze all amide bonds with 6 N HCl at 100-110 o C for 12-36 hrs in sealed tube ii) run the mixture on an ion exchange column (usually cation exchange at low pH) or reverse-phase (C18-hydrophobic) HPLC (high performance liquid chromatography)
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amino acids are derivatized for visualization integrate peaks to get relative numbers of each amino acid separation of an amino acid mixture on HPLC.
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Cautions: all glutamine and asparagines residues are converted to the acids, glutamate and aspartate (why?). How could we be able to tell how many asparagines/glutamines are present? serine, threonine and tyrosine are degraded under the hydrolysis conditions; acid conditions also degrade Trp. Must do a time course hydrolysis (we will assume that we CAN measure Ser, Thr, Tyr and Trp in this manner)
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Acid hydrolysis time course
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Cautions, continued Amino acid analysis only gives the empirical composition need the MW of peptide (or protein) to determine the real composition. Or add known amount of standards to the experiment to be able to quantitate the amino acids exactly by integrating the peaks.
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Example: both the peptides Asp-Ser-Gly and (Asp-Ser-Gly) n will give the same composition, but clearly are different peptides Solution: the MW of the tripeptide is 259. Suppose the MW of your sample is 1036; 1036/259 = 4. So the composition must be 4 Asp, 4 Ser, 4 Gly the MW of the sample does not need to be an integral multiple of the empirical MW
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For example: composition: 7 Gly + 10 Leu + 13 Asp MW =
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