Total amino acid composition of the peptide

The first step in determining the amino acid sequence of a peptide is to find out which amino acids are present, and how many of each.

This is achieved by hydrolysing the peptide, then subjecting the hydrolysate to high pressure liquid chromatography to identify (and quantify) the amino acids present.

Peptide hydrolysis and sample preparation

The peptide is subjected to complete acid hydrolysis by heating overnight at 105C in 10 mol/L hydrochloric acid, in a sealed tube. This hydrolyses all peptide bonds, releasing free amino acids. However, it also results in destruction of tryptophan, so if you suspect that your peptide contains tryptophan (e.g. from its absorption spectrum) then you may need to use alkaline or enzymic hydrolysis.

The hydrolysate is then neutralised, and reacted with o-phthaldialdehyde and mercaptoethanol to form fluorescent iso-indole derivatives, before being injected onto an hplc column.

High-pressure liquid chromatography

Chromatography depends on the partition of solutes between a stationary phase (usually a solid) and a mobile phase (usually a liquid, but sometimes a gas). Solutes that are more tightly adsorbed onto the stationary phase travel more slowly, and therefore in column chromatography are eluted from the column later than solutes that are less tightly adsorbed by the stationary phase. The time at which a compound is eluted from the column is its retention time. The separation of different solutes depends on their chemistry, the nature of the stationary phase and the composition of the mobile phase.

High pressure liquid chromatography (hplc) is an extremely sensitive column method, using very small volumes of sample, and columns that are typically some 150 mm long and 4 - 5 mm in diameter. The stationary phase is in the form of very uniform spheres, typically some 5 µm in diameter, packed into the column. The mobile phase is pumped through the column under high pressure, and 5 - 10 µL of the sample is injected into the system just above the column. Sometimes the mobile phase has a constant composition; at other times its composition is varied though the elution period by mixing two different solutions to provide a gradient of mobile phase composition.

In some hplc systems the sample is derivatised before chromatography (as in this case, where it is the OPT-derivatives of the amino acids that are subjected to hplc); in other cases the eluate is derivatised (post-column derivatisation) to form coloured or fluorescent derivatives. In some cases the detection system is such that no derivatisation is required before or after chromatography.

The eluate then passes through a detection system, which may be electrochemical, or may rely on absorption of light, of fluorescence. In the case case of OPT-derivatives of amino acids, the detection system is fluorimetric.

The hplc that is used in this simulation uses a reverse phase (C18) column, with a 12.5 mmol/L sodium phosphate buffer at pH 7.2, and a gradient of 10 - 50% acetonitrile. The eluate is monitored using a fluorescence detector with excitation at 340 nm and emission at 445 nm.

The figure below shows typical traces for a mixture of standard amino acids (left) and a peptide hydrolysate (right), so that it is possible to identify which amino acids are present in the peptide.

Fluorescence versus absorption spectrophotometry

In absorption spectrophotometry, light of s defined wavelength is shone through the sample, and some is absorbed.The absorption of light is determined by the concentration of solute and the path length. What is measured is the light that is transmitted through the solution. This means that at low concentrations of solute, sensitivity is low, since there is little difference between the intensity of the incident light (that shone into the sample) and that of the transmitted light, because little has been absorbed.

The light energy is used to excite electrons in the solute (this is why the wavelength is more or less specific for the compound under consideration). These electrons normally return to the resting state in a series of small jumps, losing the energy of excitation as heat, as shown on the left in the diagram below.

In some compounds, the energy of excitation is lost in a single quantum jump, as light, as shown on the right in the diagram below. Such compounds are said to be fluorescent - if they are excited with light of an appropriate wavelength, they will emit light of a longer wavelength (lower energy).

The light is emitted in all directions, and in a spectrophotofluorimeter what is measured is the light emitted at right angles to the incident light. This permits considerably greater sensitivity than absorption spectrophotometry, since, rather than looking for a small diminution in the intensity of the incident light, we are measuring light emitted without interference from the transmitted light. At least in theory, photomultipliers can detect a single photon.

Spectrophotofluorimetry also permits a higher degree of specificity than absorption spectrophotometry, since diffraction gratings can be used to select not only the wavelength of the incident light, but also that of the emitted light.