Ahead of the game: Proteomics studies of keratins in hair find broken proteins

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  • Published: Mar 1, 2012
  • Author: Steve Down
  • Channels: Proteomics & Genomics / Proteomics
thumbnail image: Ahead of the game: Proteomics studies of keratins in hair find broken proteins

Keratins connected

A comprehensive proteomics study of the proteins in human hair by two complementary approaches has provided a broad picture of the keratins and keratin-associated proteins and also revealed a set of broken keratins. 

The structure of human hair has never been examined more closely than it is nowadays, being the focus of cosmetic companies aiming to produce new hair care treatments. At the protein level, a number of keratins and keratin-associated proteins (KAPs) have been identified but their growth in mature hair, their modifications, and they way they might be involved in the quality of hair is still unclear.

Scientists in France from L'Oreal Research and Innovation, Aulnay-sous-Bois, and the University of Strasbourg wanted to take a closer look at the proteins in human hair and took a proteomics approach. Nukhet Cavusoglu and colleagues noted that there have been a limited number of proteomics studies to date, possibly due to the difficulty of extracting and solubilising the proteins in a way that is amenable to HPLC or gel electrophoresis.

Moreover, many of the proteins have very similar peptide sequences, which makes it more difficult to find unique peptide fragments after enzyme digestion that can be used for unambiguous identification.

The inner cortex of the hair strand consists of structures known as keratin intermediate filaments, which are made up of two kinds of keratins. There are nine type I keratins, which are acidic, and six type II keratins, which are neutral or basic. These are surrounded by an amorphous matrix of more than 100 very similar KAPs spread across 26 families.

Within these hair sub-structures, the proteins are connected by various bonds such as hydrogen bonds, disulphide bonds, amide bonds and electrostatic interactions which help to maintain the rigidity of the hair itself and render it resistant to external influences like hair treatments or UV exposure.


Mud and gels

Cavusoglu used two complementary proteomics techniques to examine the protein structures, working with untreated hair from volunteers. In the first instance, soluble and insoluble hair extracts were obtained corresponding to proteins from the cortex and the outer cuticle layer, respectively.

Both extracts were digested with the standard enzyme trypsin and the resultant peptides were separated into 35 fractions by strong cation exchange chromatography. Each of these fractions was analysed by liquid chromatography-tandem mass spectrometry in the global approach known as MudPIT (multidimensional protein identification technology).

In the second approach, the soluble proteins were extracted for separation by 2D gel electrophoresis. The gel spots were removed and the proteins were digested for liquid chromatography-tandem mass spectrometry analysis. Gel-based analysis of keratins is known to be difficult due to poor separation, but the researchers improved the migration characteristics and increased protein loadings to give a new protein distribution with a rich group of spots.

Broken keratins

The MudPIT method identified eight of the nine type I keratins and five of the six type II keratins, as well as 21 KAPs. In addition, three different post-translational modifications (PTMs) to the keratins were identified - cysteine oxidation, histidine methylation and lysine dimethylation. However, all of the PTMs could not be assigned to a specific protein because of the structural similarities between the keratins. Some of the modified peptides detected could have originated from more than one protein.

The cysteine oxidation was interesting because it is often associated with the chemical treatment of hair. In this case, all of the hair samples were declared to be untreated, so the researchers postulated that the oxidation might have occurred as a result of UV light exposure.

In the gel-based studies, the most comprehensive 2D map of human hair proteins to date was obtained, due to the modified conditions employed. Here, seven type I and four type II keratins were identified, with just nine KAPs. Although the number of KAPs is smaller than the MudPIT approach, the results show that it is still possible to study them by gel-based proteomics methods.

Some of the proteins identified from the gels were reanalysed by tandem mass spectrometry on a high-resolution instrument, measuring the intensities of the peptide ions. This led to the surprising identification of cleaved type II keratins. They displayed a preferential cleavage site "between the middle of coil 1B and the middle of coil 2."

The origin of these broken proteins in untreated hair is unclear. It might occur naturally in the hair or could be a consequence of the extraction procedure. However, the findings point to a weakness in the helical keratin segments. The fragmented proteins were only observable following gel separation, confirming the importance of the two-pronged proteomics approach.

The results show that proteomics can glean useful information about the arrangements of proteins within the hair matrix but "improvements will need to be brought to the qualitative and quantitative aspects of keratins and KAPs in order to understand their prevalence and to study whether a relation exists with the macromolecular aspect of hair quality or shape."

Related Links

Analytical Biochemistry 2012, 421, 43-55: "Proteomic tools for the investigation of human hair structural proteins and evidence of weakness sites on hair keratin coil segments"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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