Enhanced Diagnostic Tools
Positively charged residues: K, R, H, N-terminus
Negatively charged residues: D, E, C-terminus
Hydrophobic uncharged residues: F, I, L, M, V, W, and Y
Uncharged residues: G, A, S, T, C, N, Q, P, acetyl, amide
N-terminal Glutamine (Q) will cyclize to pyroglutamate when exposed to the acidic conditions of cleavage.
Recommendation: synthesize with pyroglutamate instead of Q, remove the Q, substitute Q with another amino acid, or acetylate the N-terminus.; Any of these suggestions will result in a peptide of higher quality.
N-terminal Asparagine (N): Asparagine has a protecting group, which can be difficult to remove, when placed at the N-terminus.
Recommendation: remove the N or substitute N with another amino acid to the N-terminus.
If there is a nonstandard amino acid, including D-amino acids, at the C-terminus, the peptide should be amidated.
If there is a modification at the C-terminus (fluorescein, biotin, etc.), the modification must be attached via the side chain of a lysine. These peptides must also be amidated.
Length: as the sequence length increases, the purity of the peptide decreases. The result may be a peptide containing several deletion products. Coupling efficiencies are compromised after ~30 residues. Sequences less than 5 amino acids can be somewhat problematic during the cleavage and purification steps of production. Our baseline for accepting 3-5mer sequences is that the sequence should have at least one hydrophobic residue (L, I, W, V, F, Y, M). If the sequence does not have a hyrdophobic residue, but contains a modification that contributes to the hydrophobic nature (such as Flc, Dansyl, Dabsyl, Btn, Lissamine, etc), this will help in purification. We do not synthesize 2mers.
Multiple Prolines (P) in a sequence may undergo a cis/trans isomerization, resulting in an apparent lower purity product.
Adjacent Serines (S) in a sequence frequently result in product that is low in purity and/or contain many deletions.
Multiple Aspartic Acids (D) in a sequence frequently result in the formation of aspartimide adducts, resulting in a product of lower purity.
Multiple modifications within a sequence often result in a product with a low yield and/or purity.
Multiple consecutive Glycines (G) (4 or more) tend to undergo hydrogen bonding (gel formation) in the peptide backbone. The hydrogen bonding may cause difficulty in dissolving and purifying the peptide.
Coupling efficiencies are greatly reduced after a phospho amino acid. Therefore, sequences containing phospho amino acids should have no more than 10 amino acid couplings after the phospho amino acid. Synthesis is performed from the C-term to the N-term. This means that there should be no more than 10 residues after (towards the N-terminus) the phospho amino acid.
Count the number of charged residues in the peptide, including the uncapped N and C termini
Typically you want at least 1 charge for every 5 residues. Fewer charges may result in an insoluble product.
c. Even if a peptide has enough charges, make sure there are not long stretches (more than 5 amino acids) of uncharged residues.
d. Sequences containing long stretches of charged amino acids or peptides that are short and hydrophilic may not be retained well on the HPLC column, resulting in a product that is difficult to purify.
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