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Custom Peptide - Technical Corner Sequence design Indicated below are a few empirical rules designed to increase our chances to prepare your products under optimal conditions. These rules are derived from the basic properties of the monomers (figure 5 and table 1).

Click here to enlarge Fig. 5

Peptide length:
As a general rule, the yields of a peptide synthesis decreases with increasing length. Most peptides of less than 25 aa can be obtained with satisfactory yields.
Hydrophobic residues:
Less than 50% of total aa and less than 4-5 in a row.
ß-Strands formation between peptide chains during elongation may occur when stretches of hydrophobic residues are present. The latter will often result in poor yields.
Reactive amino-acids:
avoid multiple cys, met and trp residues.
One illustration is shown in figure 6 where a slight local change in peptide structure e.g., 1 internal aa, can have significant incidence on hydrophobicity of the molecule as a whole.

Click here to enlarge Fig. 6

Peptide Solubilization
For difficult-to-solubilize peptides, the following basic rules may help.
- Neutral peptide: one may try H2O:DMF mixes.
- Basic peptide (presence of lys, arg,...): one may try 1% acetic acid
- Acidic peptide (presence of glu, asp,...): one may try 1% ammonium hydroxide
In case of aggregation, one may briefly sonicate the solution.

Storage & Handling
For maximum stability, peptides should be stored lyophilized at –20°C.

Amino acids basic properties
Table 1. Name, Abbreviations, Chemical classification, Relative Abundance, and Acid-Base properties of each of the natural amino acidsAmino acid.

Click here to have complete table, with molecule diagram.

(1) Analysis of 9 proteins: insulin (bovine), lysozyme (chicken), mannitol permease & lactose permease (E. coli), hemoglobin & cytochrome c (human), glucagon (pig), ferridoxin (spinach), wool (sheep).
(2) Isotopic average of neutral amino acyl residu in a peptide bond.
(3) In peptides in aqueous solution. N-terminus: pKa=7.8 & C-terminus: pKa=3.7.

MALDI-TOF mass spectrometry
Matrix-Assisted Laser Desorption Ionization-Time Of Fly (MALDI-TOF) is one of the latest and most gentle ionization approach in Mass Spectrometry. This approach uses UV laser pulses to generate and sputter ions into the gas phase from high molecular weight species such as peptides that are embedded within an organic matrix compound. The resulting ions are then accelerated in an electric field and down a flight tube and collected in a detector. The lag time between ionization and detector impact (i.e. Time Of Fly) is measured and correlates directly to mass/charge (m/z) ratio.
Some phenomenon should be kept in mind when using and interpreting MALDI-TOF MS. The extend of ionization and ejection in the gas phase of a given peptide will determine peak intensity. The latter is sequence-, mass- and sample-dependent (Fig. 8a). Some higher MW peptides may give small intensity Molecular Ion peak with this approach (Fig. 7). An alternative approach is a higher-energy ionization techniques such as Electrospray Ionization (Fig. 7). Another inherent phenomenon is the slight broadening of MH+ peaks resulting from very small local differences of absorbed energy during the initial UV-light laser pulses between similar peptide ions which in turn results in slight differences in Times of Flight and hence a <<Gaussian-like>> spectrum. Routine calibration procedures afford average mass error of ± 0.1%.

Click here to enlarge Fig. 7

Data acquired in a MALDI TOF spectrophotometer provides <<average isotopic>> mass for the singly protonated peptide MH+. The detailed mechanism for ionization is still uncertain and the process can also generate cationized species, e.g. [MNa+] (Fig 8).

Click here to enlarge Fig. 8