Proteomic Analysis of Human Tendon and Ligament: Solubilization and Analysis of Insoluble Extracellular Matrix in Connective Tissues

Connective tissues such as tendon,
ligament and cartilage are mostly
composed of extracellular matrix (ECM). These tissues are insoluble,
mainly due to the highly cross-linked ECM proteins such as collagens.
Difficulties obtaining suitable samples for mass spectrometric analysis
render the application of modern proteomic technologies difficult.
Complete solubilization of them would not only elucidate protein composition
of normal tissues but also reveal pathophysiology of pathological
tissues. Here we report complete solubilization of human Achilles
tendon and yellow ligament, which is achieved by chemical digestion
combined with successive protease treatment including elastase. The
digestion mixture was subjected to liquid chromatography–mass
spectrometry. The low specificity of elastase was overcome by accurate
mass analysis achieved using FT-ICR-MS. In addition to the detailed
proteome of both tissues, we also quantitatively determine the major
protein composition of samples, by measuring peak area of some characteristic
peptides detected in tissue samples and in purified proteins. As a
result, differences between human Achilles tendon and yellow ligament
were elucidated at molecular level.