Practical Protein Chemistry - A. Darbre 1989

Peptide Mapping of Proteins
Peptide Mapping in Practice
Peptide Mapping by High-Performance Liquid Chromatography

Three approaches for analytical peptide mapping by High-Performance Liquid Chromatography (HPLC) are known: reversed-phase HPLC (RP-HPLC), Ion-exchange chromatography, and Gel filtration chromatography.

Class="center">

FIG. 7.3. Separation of tryptic Peptides of mollusk and rabbit actins on thin-layer silica gel G plates. Tryptic peptides of mollusk (a) and rabbit (b) actins; unique peptides are indicated by arrows; Electrophoresis, pH 3.5; chromatography: chloroform — methanol — ammonia (2:2:1); c and d — tryptic peptides of mollusk (c) and rabbit (d) actins; unique peptides are marked by arrows, and the difference in spot position is indicated by an arrow with an asterisk; electrophoresis, pH 6.5; chromatography: propanol — ammonia (7:3). e — rabbit Actin hydrolysate, proteolysis and mapping in the presence of SDS; f — the same hydrolysate under standard conditions; some spots (circled) are less intense in the presence of SDS, whereas others (arrows) are more distinct; electrophoresis, pH 3.5; chromatography: propanol — ammonia (7:3). All samples were applied at 1 nmol in 2.5 µl (Table 7.1) [60]. (Reproduced with permission from Academic Press.)

7.3.3.1. Reversed-phase HPLC (RP-HPLC) using gradient elution [22, 57]. Currently, this is the most widely used and most efficient method for peptide separation. It relies on columns packed with small (3–10 µm) porous particles whose surface is coated with aliphatic or phenyl groups acting as a non-polar stationary phase. A mixture of small peptides obtained by enzymatic [8, 22, 49] or chemical [68] Protein Cleavage is applied to the Column in a solvent chosen so that all or at least the majority of components bind hydrophobically to the packing surface. A Mobile phase—an aqueous solution of an organic solvent with an increasing concentration gradient (by volume)—is then passed through the column. Peptides are eluted in extremely narrow zones; hydrophilic peptides that interact most weakly with the hydrophobic stationary phase emerge first. The method is particularly effective for peptides containing up to ~50 Amino Acids and is less suitable for the separation of larger fragments.

Prior to RP-HPLC analysis, the protein is hydrolyzed with Trypsin or another enzyme, typically in an ammonium bicarbonate buffer. The peptide mixture can then be treated with specific Reagents to enhance detection sensitivity following fractionation [8]. A disadvantage of pre-column derivatization is that the introduced fluorescent or UV-absorbing groups level out the structural differences between peptides, thereby reducing separation efficiency.

Hydrocarbon chains C₈ and C₁₈ are most commonly used as the stationary phase, although other Supports are commercially available [57].

The choice of mobile phase is crucial for the separation process; numerous solvent mixtures and chromatographic conditions have been proposed for specific Applications (for a more detailed Structure/133.html">Discussion, see [57]). Attempts have been made to use computational Methods to predict peptide retention times for a given solvent system and Column packing [5, 46, 71]; however, as a general rule, a qualitative comparison of elution profiles is usually sufficient to establish the degree of similarity between test samples.

Several solvent systems are widely used in practice. For instance, a 0.1% (v/v) (0.013 M) aqueous trifluoroacetic acid (TFA) solution serves to equilibrate the column [44]. After applying the peptide mixture, several column volumes of 0.1% TFA are passed through to ensure the binding of all or most mixture components. A linear gradient of acetonitrile concentration in 0.1% TFA is then applied to the column to elute individual peptides. Nearly all peptides investigated in this system elute by the time the acetonitrile concentration reaches 60%. To avoid potential refractive index changes due to incomplete solvent mixing, aqueous acetonitrile (60% acetonitrile + 40% Water) containing 0.1% TFA is used as the second solvent. All Solvents used must be of high purity, as even trace impurities can concentrate on the column and subsequently elute at the corresponding acetonitrile concentration. The optimal flow rate is 1 ml/min; slower elution does not improve resolution. Upon completion of the gradient program, the column is re-equilibrated with the starting buffer to prepare it for the next Sample application. Other buffer systems, such as ammonium acetate or sodium phosphate at neutral pH, can be used to achieve different separation selectivities.

FIG. 7.4. HPLC separation of peptides from tryptic digests (0.8 mg) of human Hemoglobin ß-chains by reversed-phase HPLC: a — normal hemoglobin; b — hemoglobin Cocody (pI 7.205); c — hemoglobin Avicenna (pI 7.225); d — hemoglobin Korle Bu (pI 7.210) [1]. (Reproduced with permission from Elsevier/North Holland, Biomedical Press.)

A Practical example of applying RP-HPLC to peptide mapping is the comparative analysis of normal and mutant hemoglobin molecules [1, 62]. In a Study of the ß-chains of several human Hemoglobins, their tryptic digests were separated on a C₁₈ column equilibrated with an ammonium acetate buffer using an acetonitrile gradient [1]. In each chromatogram, the peak of a single peptide was shifted relative to the fragment positions of normal hemoglobin (Fig. 7.4). Peptide fractions with abnormal retention times were collected and subjected to Amino Acid Composition and sequence analysis. In each case, a single amino acid residue differing from the corresponding normal hemoglobin residue was identified. The separation time was only 80 min.

7.3.3.2. Ion-exchange HPLC. The second method frequently used for analytical purposes is ion-exchange HPLC (IEX-HPLC), where peptide binding to the ionogenic groups of the support occurs via charge interactions. Elution is achieved by increasing the Ionic strength or altering the pH of the mobile phase, sometimes in combination with an increasing organic solvent content [38, 64]. This technique yields the best results with relatively small molecules, although certain capabilities exist for protein separation as well [57]. Ion-exchange HPLC relies on the same ionic interactions that operate in open columns of classical ion-exchange chromatography. The sample is applied to the column in a low-ionic-strength solution at a pH where both the stationary phase and the sample are ionized but possess opposite net charges. To elute the peptides, a solution with an increasing ionic strength gradient or a changing pH is passed through the column. The advantages of IEX-HPLC over conventional ion-exchange chromatography include minimal sample dilution during separation and highly sensitive detection methods. As in reversed-phase HPLC, the sample can be applied to the ion-exchange column in a large volume if necessary, since the peptides bind to the support before gradient elution begins.

7.3.3.3. Size-exclusion HPLC. The third method, size-exclusion HPLC, is less frequently used for analytical peptide mapping [66], primarily due to the lack of column types suitable for resolving complex mixtures of small peptides (MW < 5,000). However, columns capable of fractionating larger Peptides and Proteins are available and are employed during Protein Purification or Desalting stages.

7.3.3.4. Detection methods. Measurement of optical density, fluorescence, and radioactivity are standard techniques across all types of HPLC peptide mapping; refractive index measurement and electrochemical methods are used considerably less often [57].

Measurement of optical density at a specific wavelength remains the most common method for peptide detection in HPLC. The sole prerequisite for its successful application is the absence of absorbance by the solvent in the chosen region (especially for the second, gradient-forming component). The TFA–acetonitrile gradient system is particularly convenient in this regard, as it allows absorbance measurements down to 210 nm [44]. Detection can be performed simultaneously at multiple wavelengths, enabling the selective identification of peptides containing specific amino acid residues (Tyrosine, Tryptophan). Modern detectors offer capabilities to obtain precise profiles of individual chromatographic peaks, facilitating the assessment of compound homogeneity and identity.

When dealing with very small quantities of peptide fragments, they are derivatized with o-phthaldialdehyde or fluorescamine for subsequent fluorescence measurement [35]. The technique is based on focusing UV radiation of a specific wavelength to excite the fluorogenic reagent within a flow Cell, with the emitted fluorescence being collected by a lens positioned at a 90° angle to the light source. Detection sensitivity in this setup is enhanced by two orders of magnitude compared to absorbance measurement; however, derivatization of mixture components post-column requires the installation of an additional pump (Section 8.16.3.1).

Radioactive peptides are identified using flow-through detectors or, more commonly, by measuring radioactivity in fractions with appropriate gamma or liquid scintillation counters.