The effect of oxidation or alkylation on the separation of wool keratin proteins by two-dimensional gel electrophoresis.

Comparative two-dimensional electrophoretic (2-DE) studies were performed over a time-course to examine the effect of oxidation or alkylation on the separation of wool keratin proteins. The effect of oxidation was followed by treating scoured wool fibres with increasing levels of hydrogen peroxide, ranging from 0-12 g/L, using conditions mimicking the industrial wool bleaching process. Peroxide treatment was found to have only a minor effect on the 2-DE separation of the intermediate filament protein (IFP) class. Conversely, peroxide treatment of the 24-28 kDa high sulphur protein (HSP) class, which contains up to 40 cysteine residues per protein, resulted in the gradual disappearance of the major HSP spots correlated with the appearance of a few discrete spots at lower isoelectric point (pI). This suggested that only a few specific cysteine residues were being oxidized to cysteic acid by treatment with hydrogen peroxide. Peroxide treatment also appeared to have affected a discrete number of cysteine residues among proteins in the high glycine-tyrosine protein (HGTP) class, reducing the intensity of the high pI spots, while correspondingly increasing the intensity of those at lower pI. In a separate study, wool proteins were alkylated with iodoacetamide (1 M, pH 8) for periods ranging from 10 min to 48 h. In contrast to treatment with peroxide, the pI values of the HSP spots were unaffected by alkylation, irrespective of the length of this treatment. Alkylation resulted in a shift to lower pI and a loss of resolution of individual spots in the Type I and II IFP trains, to the extent that after 24 h alkylation individual spots in these trains merged. In addition after 1 h the intensity of the high pI Type II IFPs decreased until they were no longer visible on the 2-DE map after 24 h. Similarly as alkylation time increased, the major, high pI HGTP spots decreased in intensity. In unison with their decrease, some of the lower pI spots increased in intensity, while new spots appeared at more acidic pIs. Mass spectral studies indicated that cysteine alkylation was relatively fast, with 70-95% of the cysteines in the keratin proteins being alkylated within the first 10 min, while in the case of the HGTPs there was evidence for noncysteine alkylation occurring within this period. Alkylation of proteins for periods of up to 6 h prior to electrofocusing is being promoted as a better alternative to the current 2-DE protocol of the inclusion of a reductant in the immobilized pH gradient rehydration solution. This study has clearly demonstrated that long alkylation times do not suit all protein types or classes.

The high sulphur proteins of wool: Towards an understanding of sheep breed diversity.

High sulphur proteins (HSPs) form part of the matrix surrounding the intermediate filaments in the cortical cells of the wool fibre. There are three known families of HSPs, comprising in excess of 40 components and their molecular weights range from 10-30 kDa. Here we report the use of the increased resolving power of isoelectric focusing in the first dimension of two-dimensional electrophoresis and modern gel comparison software to investigate the nature of within- and between-breed variations amongst the proteins of three breeds of sheep: Merino, Romney and Corriedale. In agreement with past studies we observed very little variation in the intermediate filament protein content in wool, both between and within these three sheep breeds. Instead, most of the observed variation occurred among the HSPs, along with some minor variation among the high glycine-tyrosine proteins. Breed-specific differences were observed in the HSP patterns in the wool proteome maps. Merino sheep were found to exhibit the simplest HSP expression patterns, with eight major spots linked to form four pairs. In contrast, the Romney and Corriedale HSP patterns exhibited more spots at lower isoelectric point values (around 4.8), while some of the lower molecular weight HSPs were less prevalent in Romney sheep and absent from the Corriedales.

Problems associated with the identification of proteins in homologous families: the wool keratin family as a case study.

The keratin proteins from wool can be divided into two classes: the intermediate filament proteins (IFPs) and the matrix proteins. Using peptide mass spectral fingerprinting it was possible to match spots to the known theoretical sequences of some IFPs in web-based databases, as enzyme digestion generated sufficient numbers of peptides from each spot to achieve this. In contrast, it was more difficult to obtain good matches for some of the lower molecular weight matrix proteins. Relatively few peaks were generated from tryptic digests of high-sulfur proteins because of their lower molecular weight and the absence of basic residues in the first two-thirds of the sequence. Their high sequence homology also means that generally only a few of these peptides could be considered to be unique identifiers for each protein. Nevertheless, it was still possible to uniquely identify some of these proteins, while the presence of two peptides in the matrix-assisted laser desorption/ionization time-of-flight mass spectrum allowed classification of other protein spots as being members of this family. Only one major peptide peak was generated by the high-glycine tyrosine proteins (HGTPs) and there were relatively few sequences available in web-based databases, limiting their identification to one HGTP family.