Practical Protein Chemistry - A. Darbre 1989

Peptide Mapping of Proteins
Introduction

W. J. GULLICK (Protein Chemistry Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, U.K.)

Peptide mapping generally serves two main purposes: determining the structural Homology of protein molecules isolated from different sources, and identifying structural alterations in individual Proteins resulting from normal or pathological METABOLISM. In the first case, these are:

1) Comparing proteins with the same function derived from various Tissues or organisms.

2) Comparing in vitro Translation products with their equivalent counterparts isolated from Cells.

3) Comparing the subunits of complex proteins.

Individual proteins studied via mapping can, in turn, be divided into two groups: proteins that mature into functional cellular forms through post-translational mechanisms (such as Limited proteolysis, glycosylation, or The addition of prosthetic groups), and proteins undergoing reversible structural modifications (such as phosphorylation or Acetylation) that can affect their activity.

Peptide mapping relies on comparing the PHYSICOCHEMICAL CHARACTERISTICS OF individual Peptides or their mixtures obtained through the fragmentation of a protein molecule. Any method yielding reproducible results can be used for fragmentation—specifically, the Cleavage of peptide bonds using Proteolytic Enzymes or chemical Reagents. The efficacy of peptide mapping in each specific case is determined by selecting optimal conditions for separating and detecting the fragment mixture, alongside a reliable interpretation of the results.

Three widely used peptide Separation Methods are described below. The first is SDS-Polyacrylamide gel Electrophoresis (SDS-PAGE), typically performed in a one-dimensional format. The resolution of peptide fragments is achieved through differences in their relative mobilities, which do not always reflect true molecular weights. Crucially, the electropherogram of fragments from one protein can be compared with the peptide electropherogram derived from another protein. This technique is known as Cleveland mapping (named after the first author of the article describing it) [12]. Polypeptides with molecular weights ranging from 10³ to 10⁶ can be separated using SDS-PAGE, though the most reliable results are obtained when analyzing proteins with true molecular weights between 10⁴ and 10⁶; therefore, the method is generally not applied to proteins with a molecular weight <10⁴.

The second method is two-dimensional mapping, commonly known as peptide fingerprinting, which was introduced by Ingram [36, 37] to localize and subsequently identify the single amino acid residue distinguishing The polypeptide chains of normal and sickle-Cell Hemoglobins. The protein is typically hydrolyzed using Trypsin or a-Chymotrypsin (Ingram performed Hydrolysis in 12 M HCl at 37 °C for 2–3 days), yielding a non-overlapping mixture of short peptides (10–20 Amino Acids long). This mixture is subsequently separated via electrophoresis in the first dimension and liquid Chromatography In the second. Peptide maps provide a more efficient mapping approach than gel electrophoresis because they analyze A large number of components that form a distinct pattern of spots (figure) characteristic of each individual protein.

The third method developed for peptide mapping, similar to the previous one, begins with the fragmentation of a protein into low molecular weight peptides. These are subsequently separated in solution using reversed-phase High-Performance Liquid Chromatography (RP-HPLC) or ion-exchange HPLC (IE-HPLC). In the former approach, columns are packed with silica gel-based resin functionalized with hydrocarbon chains of varying lengths; in the latter, the support also consists of silica gel, but with ionizable groups on its surface. Peptide separation is driven by differences in their retention times on the columns. The primary advantages of this method include high Processing speed (the separation of a single hydrolysate can take <30 min), high sensitivity, and the recovery of peptides in a form suitable for downstream analyses (such as Amino Acid Composition and Sequence Determination). Peptide content is quantified by measuring the optical density of the eluate at one or more wavelengths, while radiolabeled preparations are monitored for radioactivity (either online or in fractionated samples). To enhance detection sensitivity, a fluorescent label can be introduced into the peptides.