Deer antler proteome: the value of parallel extraction methods

The antler model for tissue regeneration

Antlers are unique. Not only in their appearance, but also in their behaviour. They are the only mammalian tissue that is capable of repeated regeneration, growth, shedding and regrowth year after year with unerring regularity. New nerves, cartilage, skin and bone are generated and the antlers mature within 60 days or so, then when their work is done, they shed within about five days.

This behaviour has made the antler a good model to study the development of tissue regeneration and, possibly, lead to the design of therapeutic aids to tissue regrowth. Within the swathe of changes that take place during antler growth and shredding, the protein profile must be one of the most important. Nevertheless, there has only been one report on the proteome of deer antlers.

This research, published in 2004, confirmed the unique nature of the antler proteome. The bone-like tissue near the antler tips even had a different proteome composition from the pedicle, the attachment point on the skull. In addition, only 67% of the identified proteins were present in deer plasma.

However, this proteome was far from complete, with only 130 proteins identified in total. No growth factors were found, although proteins associated with growth signalling pathways were detected.

Better extraction the key to a wider proteome

This deficiency has prompted another research team to take a closer look at the antler proteome. Lihua Zhang and colleagues from the Dalian Institute of Chemical Physics and Graduate School of the Chinese Academy of Sciences, Beijing thought that they could expand the number of proteins identified by adopting different techniques.

In the first place, they applied a range of extraction protocols to the antler, to see if they could draw more proteins into solution. They argued that antler could be regarded more like bone than normal tissue, so different techniques might be appropriate.

So, five portions of the growing tip of a 15-day-old antler tissue were extracted with different solutions. Hydrochloric acid and sodium chloride solutions were used to extract acidic and basic proteins, respectively. The third solution comprised Tris and guanidine hydrochloride, a common extractant for soluble proteins.

The fourth solution was the same as the third, but with the addition of EDTA and it was aimed at non-collagenous proteins which are strongly associated with the bone matrix. Finally, a solution of Tris, CHAPS and dithiothreitol was supplemented with concentrated urea.

The value of the diverse media was illustrated by the number of proteins found in each extract. Solutions one through five yielded 153, 49, 161, 153 and 138, proteins, respectively, with only 11 found in every extract. The number of unique proteins was 78, 29, 61, 61 and 59, respectively, The total number of unique proteins was 416, more than 3-fold higher than the earlier antler proteome study.

The good results were aided in no small part by the use of liquid chromatography-tandem mass spectrometry for separation and identification, rather than 2D gel electrophoresis which was employed in the original study. The protein extracts were digested with trypsin and the peptides were passed through a series of coupled microcolumns to effect better separation and resolution.

The molecular masses of the proteins ranged from 2000 to 600,000 and the isoelectric points from 3.84 to 11.57, much broader than reported earlier.

Deer antler protein functions

More than 60% of the identified proteins were subcellular, representing most of the cell compartments. Many of them were membrane proteins, which are difficult to extract and analyse due to their hydrophobicity, but the extraction techniques employed here, particularly under acidic conditions, appeared to be suitable.

Fourteen proteins were involved in growth. They included alpha-tubulin and tuberin, which restrict cell growth, and macrophage migration inhibitory factor and chromodomain-helicase-DNA-binding protein 7 which regulate axon regeneration and multicellular growth. The presence of proteins that restrict and encourage growth illustrates the complicated growth mechanism at play. Other proteins are involved in ossification.

The increased proteome and the broad range of proteins identified illustrate the importance of an extensive extraction protocol and refined HPLC separation for mass spectrometry analysis.

The research team are continuing their work on deer antlers, examining the proteome of antlers at different stages of development in an attempt to shed more light on the growth and regeneration mechanisms involved in these unique mammalian features.

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